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Leishmania Parasitophorous Vacuoles Interaction with the Macrophage Endoplasmic Reticulum

Permanent Link: http://ufdc.ufl.edu/UFE0041994/00001

Material Information

Title: Leishmania Parasitophorous Vacuoles Interaction with the Macrophage Endoplasmic Reticulum
Physical Description: 1 online resource (192 p.)
Language: english
Creator: Ndjamen, Blaise
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: calnexin, d12, endoplasmic, host, leishmania, macrophage, parasites, parasitophorous, phagocytosis, phlebotomines, reticulum, retrograde, ricin, sec22b, snares, syntaxin18, vacuoles, zymosan
Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Leishmania parasites infect approximately 2 million people every year and cause leishmaniasis. Leishmania are transmitted by sand flies to human tissues; where parasites preferentially enter macrophages by phagocytosis. It is well established that Leishmania reside and replicate in membrane-bound structures called parasitophorous vacuoles (PVs), which interact with endocytic compartments such as lysosomes. The extent of Leishmania PV (LPV)s interaction with host endoplasmic reticulum (hER) is poorly understood. This study presents a comprehensive assessment of hER contributions to LPV biogenesis and maturation, and Leishmania development within macrophages. Macrophages expressing either calnexin or ER-associated SNAREs tagged with fluorescence proteins were infected with parasites or ZymosanA. Samples were processed through immuno-fluorescence assays and analyzed by fluorescence microscopy to assess the recruitment of hER molecules to LPVs. We found that more than 90% of PVs harboring L. pifanoi or L. donovani parasites recruited calnexin and SNAREs to their PV membrane throughout the course of infection. Electron microscopy analysis of infected macrophages expressing Sec22b/YFP confirmed that LAMP-1 and Sec22b are recruited to LPVs. Unlike PVs, no more than 20% of Zymosan phagosomes recruited calnexin or Sec22b to their phagosomal membrane. Similarly, phagosomes containing dead parasites recruited lower level of ER molecules after infection. To further gain insight into hER-LPV interactions, the intracellular trafficking pathway of ricin was exploited. We established that Raw264.7 macrophages are a suitable system to study ricin trafficking in eukaryotic cells. Ricin was targeted into LPV compartments via hER, and this process could be blocked by BrefeldinA. Ricin accumulation in LPVs and hER was not immediate, suggesting that molecules in the hER are continuously delivered to LPVs by vesicular transport. Finally, we analyzed the Leishmania infection in Raw246.7 cells transiently expressing either dominant negative or over-expression constructs of Sec22b, Syntaxin 18, or D12. Our data suggest that hER membrane-associated SNAREs are essential for both Leishmania replication and PV development, but not for their entry in macrophages. Altogether, this study demonstrated that Leishmania PVs are hybrid compartments composed of both endocytic and host ER components; hER are essential for a successful Leishmania survival and replication, and PV development in macrophages.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Blaise Ndjamen.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Kima, Peter E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041994:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041994/00001

Material Information

Title: Leishmania Parasitophorous Vacuoles Interaction with the Macrophage Endoplasmic Reticulum
Physical Description: 1 online resource (192 p.)
Language: english
Creator: Ndjamen, Blaise
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: calnexin, d12, endoplasmic, host, leishmania, macrophage, parasites, parasitophorous, phagocytosis, phlebotomines, reticulum, retrograde, ricin, sec22b, snares, syntaxin18, vacuoles, zymosan
Microbiology and Cell Science -- Dissertations, Academic -- UF
Genre: Microbiology and Cell Science thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Leishmania parasites infect approximately 2 million people every year and cause leishmaniasis. Leishmania are transmitted by sand flies to human tissues; where parasites preferentially enter macrophages by phagocytosis. It is well established that Leishmania reside and replicate in membrane-bound structures called parasitophorous vacuoles (PVs), which interact with endocytic compartments such as lysosomes. The extent of Leishmania PV (LPV)s interaction with host endoplasmic reticulum (hER) is poorly understood. This study presents a comprehensive assessment of hER contributions to LPV biogenesis and maturation, and Leishmania development within macrophages. Macrophages expressing either calnexin or ER-associated SNAREs tagged with fluorescence proteins were infected with parasites or ZymosanA. Samples were processed through immuno-fluorescence assays and analyzed by fluorescence microscopy to assess the recruitment of hER molecules to LPVs. We found that more than 90% of PVs harboring L. pifanoi or L. donovani parasites recruited calnexin and SNAREs to their PV membrane throughout the course of infection. Electron microscopy analysis of infected macrophages expressing Sec22b/YFP confirmed that LAMP-1 and Sec22b are recruited to LPVs. Unlike PVs, no more than 20% of Zymosan phagosomes recruited calnexin or Sec22b to their phagosomal membrane. Similarly, phagosomes containing dead parasites recruited lower level of ER molecules after infection. To further gain insight into hER-LPV interactions, the intracellular trafficking pathway of ricin was exploited. We established that Raw264.7 macrophages are a suitable system to study ricin trafficking in eukaryotic cells. Ricin was targeted into LPV compartments via hER, and this process could be blocked by BrefeldinA. Ricin accumulation in LPVs and hER was not immediate, suggesting that molecules in the hER are continuously delivered to LPVs by vesicular transport. Finally, we analyzed the Leishmania infection in Raw246.7 cells transiently expressing either dominant negative or over-expression constructs of Sec22b, Syntaxin 18, or D12. Our data suggest that hER membrane-associated SNAREs are essential for both Leishmania replication and PV development, but not for their entry in macrophages. Altogether, this study demonstrated that Leishmania PVs are hybrid compartments composed of both endocytic and host ER components; hER are essential for a successful Leishmania survival and replication, and PV development in macrophages.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Blaise Ndjamen.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Kima, Peter E.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041994:00001


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1 LEISHMANIA PARASITOPHOROUS VACUOLESINTERACTION WITH THE MACROPHAGE ENDOPLASMIC RETICULUM By BLAISE NDJAMEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Blaise Ndjamen

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3 To my mom, daughter and wife

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4 ACKNOWLEDGMENTS First of all, I would like to thank Dr. Pe ter E. Kima, who prov ided the opportunity to work in his lab and perform interesting research first as an intern and then as a PhD student. He introduced me into the Leishmania research world and provided critical advice that has guided my research. He gav e me an extra-ordinary opportunity do an internship in his laboratory in 2005, which st imulated my interest to pursue this terminal degree in the Department of Micr obiology and Cell Science. I am also very grateful for his guidance and unfailing support through out t he course of my program. I would also like to thank the members of my PhD co mmittee; Drs. Zhonglin Mou, Howard M. Johnson, Nemat O. Keyhani and Daniel L. Pu rich for their valuable advice, time and commitment to helping me. I would like to a cknowledge Dr. Kiyotaka Hatsuzawa from Fukushima Medical University in Japan, who graciously provided us with the SNAREs DNA constructs used in this project. I am ve ry grateful to Drs. Madeline E. Rasche and Valrie de Crcy-Lagard, my firs t-year rotation advisors, for given me the opportunity to carry out research in their Labs and for hel ping me to develop as a scientist. I would also like to thank Dr. Basma El Yacoubi for her invaluable assistance in my first-year lab rotation project in Dr. Valrie de Crcy-Lagards lab. I would also like to thank Drs. K.T. Shanmugan, James F. Preston, and Joseph Larkin III for their advice and encouragement, and Dr. Kelly Rice for letti ng used her equipment to perform my fluorimetric assays. I would lik e to express to past and present members of Dr. Kimas lab Waltraud Dunn, Mr. Jonathan Canton, Kelly Johnson, Drs. Eumin Cho, Aaron Ruhland and Fred Bonilla for their great companionship, and who has assisted me performing the some the EM experiments. Waltrauld Dunn former Dr. Kima Lab technician, and undergraduate students Kelly Johns on, Paul Heyliger Fonseca, Adjoua

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5 Walker, Jessica Mayor, Megan Wicks, and Alyssa Moore. The ICBR Electron Microscopy Core staff: Dr. Byung-Ho Kang, Ms. Donna S. Williams and Karen for processing our EM samples, and letting me use their confocal microscopy. I would like to the staff and colleag ues in the Department who have always been supportive of me in the Department. Finally, I would like thank my family both in the United States of America and in Cameroon, who have always been a source of comfort, encouraged, support and love. They have supported me in this endeavor, and my relationships with them have become stronger dur ing this time. My wife, Ann Lee, and daughter, Kwayi have supported me in this endeavor, and my relationships with them have become stronger during this time. Nobody deserves as much praise for making me the person I am today than my mother Kwayi Esther. I dedi cate this thesis to her. Now I have to translate it in the West-Cameroon Bamile ke language, Medumba, for her to better appreciate.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES .............................................................................................................. 10 LIST OF FIGURES ............................................................................................................ 11 LIST OF ABBREVIATIONS .............................................................................................. 14 ABSTRACT ........................................................................................................................ 17 C H APT ER 1 GENERAL INTRODUCTION...................................................................................... 19 Introduction ................................................................................................................. 19 Leishmaniasis ............................................................................................................. 19 Lei shmania Life Cycle .......................................................................................... 20 Leishmania Structure ........................................................................................... 21 Leishmania Genetics ........................................................................................... 22 Leishmania Parasites and Vectors ...................................................................... 25 Leishmania sis Clinical Symptoms ....................................................................... 25 Post -kalaazar dermal leishmaniasis (PKDL) ............................................... 27 Cutaneous leishmaniasis (CL) ...................................................................... 27 Diffuse cutaneous leishmaniasis (DCL) ........................................................ 28 Mucocutaneous leishmaniasis (MCL) or espundia ...................................... 28 Diagnosis of leishmaniasis ............................................................................ 29 Treatment ...................................................................................................... 29 Vaccines ........................................................................................................ 32 Leishmania versus Mammalian Host Defense ................................................... 34 Leishmania versus the Host Adaptative Immune Responses ............................ 42 Resolution of Infection and the Th1 response .............................................. 44 Non heali ng L. major infection: Th2 dependent or defective Th1 response ..................................................................................................... 45 Cutaneous ( L. mexicana / L. amazonensis) infections and IL4 depe ndent .................................................................................................. 49 Non -curing visceral Leishmania sis and Th2 independent ........................... 51 Phagocytosis and Leishmania ............................................................................. 52 Inert particle phagocytos is ............................................................................ 52 ER -mediated phagocytosis ........................................................................... 53 Phagosome maturation ................................................................................. 55 Leishmania phagocytosis .............................................................................. 57 Membranes lining Leishmania p rimary and secondary PVs ........................ 58 Endoplasmic Reticulum .............................................................................................. 58

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7 Endoplasmic Reticulum Structure ....................................................................... 58 Endoplasmic Reticulum Functions ...................................................................... 60 Endoplasmic reticulum and ERAD ................................................................ 60 The ER -Golgi intermediate compartment (ERGIC) ...................................... 60 ER -Golgi transport and SNAREs .................................................................. 61 Ricin Trafficking in Eukaryotic Cells and the ER ................................................. 64 Project Rational and Design ....................................................................................... 79 2 MATERIAL AND METHODS ...................................................................................... 82 Material ....................................................................................................................... 82 Chemicals and Reagents ..................................................................................... 82 Biochemical Kits ................................................................................................... 82 Plasmids ............................................................................................................... 82 Antibodies ............................................................................................................. 83 Cell Lines and Maintenance ................................................................................ 83 Parasites ........................................................................................................ 83 Macrophages ................................................................................................. 83 Bacteria .......................................................................................................... 84 Molecular Cloning ....................................................................................................... 85 RNA Extraction and Purification .......................................................................... 85 DNA Extraction and Purification .......................................................................... 86 One -step RT -PCR and DNA amplification ................................................... 86 PCR and calnexin gene amplification ........................................................... 86 Agarose gel elect rophoresis ......................................................................... 86 Mini prep plasmid DNA isolation ................................................................... 86 Endotoxinfree maxi -prep DNA isolation ..................................................... 87 DNA Cloning ......................................................................................................... 88 Restriction enzyme digestion of DNA (vect or and insert) ............................ 88 Dephosphorylation of digested DNA ............................................................ 88 DNA ligation ................................................................................................... 88 DNA and RNA Analysis ....................................................................................... 89 DNA and RNA quantity and quality measurement ....................................... 89 DNA sequencing ............................................................................................ 89 DNA agarose Gels ........................................................................................ 89 Preparation of E.coli DH5 ............................................... 90 Transform ation of DH ...................................................................... 90 Screening of bacterial colonies ..................................................................... 91 Vectors Construction and Expression ................................................................. 92 Transfection (Nucleofection) of Raw264.7 Macrophages .................................. 93 I nfection of Raw 264.7 Macrophages with Leishmania Parasites ...................... 93 Cell counting (macrophages and parasites) ................................................. 93 Plating Raw 264.7 macrophages on glass cover slips ................................ 94 Incubation of Raw264.7 macrophages with Leishmania parasites ............. 94 Immunofluorescence Labeling and Imaging ....................................................... 95 Immuno Electron Microscopy .............................................................................. 95 Ricin Experiment .................................................................................................. 96

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8 Ricin pulse -chase experiment ....................................................................... 96 Targeting strategy of ric in into Leishmania parasitophorous vacuoles ....... 97 Statistical Analysis ............................................................................................... 98 3 HOST ENDOPLASMIC RETICULUM CONTRIBUTIONS TO THE LEISHMANIA PARASITOPHOROUS VACUOLE MEMBRANE ...................................................... 99 Introduction ................................................................................................................. 99 Results ...................................................................................................................... 101 Recruitment of Calnexin and LAMP -1 to the Leishmania PV Membrane ........ 101 Calnexin/GFP expression in Raw 264.7 ..................................................... 101 Recruitment of calnexin/GFP and LAMP 1 to L. pifanoi PV membranes 102 Enumeration of calnexin/GFP and LAMP 1 recruitment to L. pifanoi PVs ........................................................................................................... 102 Recruitment of calnexin/GFP and LAMP 1 to L donovani PV membranes .............................................................................................. 103 Recruitment of Host ER Membrane-Associated SNAREs the LPV Membrane ................................................................................................ 104 Enumeration of SNARE/YFP recruitment to L. pifanoi and L. amazonensis PV membranes .................................................................. 106 Recruitment of Sec22b/YFP to L. donovani PV membranes .................... 106 Electron Micros copy (EM) Analysis ................................................................... 106 Distribution of GFP/gold particles in control samples ................................ 107 Distribution of Sec22b/GFP/gold particles in macrophages ...................... 107 Discussion and Conclusion ...................................................................................... 108 4 CONTRIBUTIONS OF HOST ER TO LEISHMANIA PV LUMEN AND VESICULAR TRANSPO RT ...................................................................................... 123 Introduction ............................................................................................................... 123 Results ...................................................................................................................... 123 Ricin trafficking in Raw264.7 macrophages ...................................................... 123 Targeting Ricin into Leishmania PVs ................................................................ 124 Enumeration of ricin accumulation into Leishmania PVs ................................. 125 Discussion and Conclusion ...................................................................................... 125 5 EFFECT OF HOST ENDOPLASMIC RETICULUM ON LEISHMANIA DEVELOPMENT IN MAMMALIAN CELLS .............................................................. 133 Introduction ............................................................................................................... 133 Results ...................................................................................................................... 133 Effect of Host ER SNAREs on L. amazonensis Entry in Raw264.7 ................. 133 Effect of Host ER SNAREs on L. amazonensis Replication ............................ 135 Effect of Host ER SNAREs on L. amazonensis PV size .................................. 137 Discussion and Conclusion ...................................................................................... 138 6 OVERALL CONCLUSION AND PERSPECTIVES .................................................. 148

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9 Overall Conclusion .................................................................................................... 148 Perspectives ............................................................................................................. 151 APPENDIX: ANALYSIS OF THE CALNEXIN GENE AND THE PCMV/ER/GFP VECTOR ................................................................................................................... 154 LIST OF REFERENCES ................................................................................................. 158

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10 LIST OF TABLES Table page 2 -1 List of primers ......................................................................................................... 98

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11 LIST OF FIGURES Figure page 1 -1 World map of leishmani asis. .................................................................................. 66 1 -2 Life cycle of Leishmania parasites. ........................................................................ 67 1 -3 Schematic representation of the main intracellular organelles ............................. 68 1 -4 Clinical symptoms of leishmaniasis. ...................................................................... 68 1 -5 Leishmania taxonomy ............................................................................................ 69 1 -6 Leishmania major : healing and non-healing immunological responses. .............. 70 1 -7 Sc hematic simplified view of the endocytic (phagocytic) pathway. ...................... 72 1 -8 Different sub-compartments of the endoplasmic reticulum. ................................. 73 1 -9 Schematic representation of the endoplasmic reticulum quality control .............. 74 1 -10 Distinct membrane trafficking steps that can be controlled by a Rab GTPase .... 75 1 -11 Structure of the (neuronal) SNAREs. ................................................................... 76 1 -12 The SNARE conformational cycle during vesicle docking and fusion. ................. 77 1 -13 Pathway of ricin uptake by cells and the mechanism toxic activity of the A chain in the cytoplasm ............................................................................................ 78 2 -1 Design of ricin targeting experiment ...................................................................... 98 3 -1 Distribution of GFP and calnexin/GFP in Raw264.7 macrophages. .................. 112 3 -2 The recruitment of calnexin/GFP to L. pifanoi PVs in Raw264.7 ....................... 113 3 -3 The recruitment of calnexin/GFP to L. donovani PVs in Raw264.7 .................. 113 3 -4 Proportion of calnexin and LAMP 1 recruitment to L. pifanoi PVs. ................... 114 3 -5 Proportion of calnexin and LAMP 1 recruitment to L. donovani PVs. ................ 115 3 -6 Distribution of YFP and Se c22b/YFP in Raw264.7 macrophages. .................... 116 3 -7 The recruitm ent of Sec22b/YFP to L. pifanoi PVs in Raw264.7 macrophages. ...................................................................................................... 117 3 -8 The recruitment of Sec22b/YFP to L. donovani PVs in Raw264.7 .................... 117

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12 3 -9 D12/YFP expressing Raw264.7 macrophages infected with L. amazonensis Murine Raw264.7 macrophages 3 -10 Syntaxin18/YFP expressing Raw264.7 macrophages infected with L. amazonensis ....................................................................................................... 118 3 -11 Recruitment of Sec22b to Leishmania PVs. ........................................................ 119 3 -12 Endoplasmic reticulum membrane associated SNAREs recruitment to L. amazonensis parasitoph orous vacuoles ............................................................. 120 3 -13 Anti -GFP labeling of macrophages expressing pmVenus (YFP). ...................... 121 3 -14 Immuno EM analysis of ER components recruitment to Leishmania PVs. ........ 122 4 -1 Trafficking of ricin in non infected R aw 264.7 macrophages. ............................. 128 4 -2 Ricin accumulates in Leishmania PVs during infection in Raw 264.7 ................ 129 4 -3 Ricin accumulates in Leishmania donovani PVs during infection in Raw 264.7 130 4 -4 The pr oportion of ricin positive Leishmania PVs. ................................................ 131 4 -5 The proportion of ricin positive Leishmania PVs. ................................................ 132 5 -1 Effect of ER membrane associated SNAREs on Leishmania parasites entry into macrophages. ................................................................................................ 141 5 -2 Effect of ER membrane associated SNAREs on Leishmania parasites load in newly infected macrophages. .............................................................................. 142 5 -3 Represen tative images of the controls used. Non-transfected Raw264.7 ......... 143 5 -4 Representative images of the effect of host ER SNAREs on L. amazonensis replication and PV development. ......................................................................... 144 5 -5 Effect of Sec22b on parasite load in L. amazonensis PV within Raw264.7 macrophages. ....................................................................................................... 145 5 -6 Effect of D12 on parasite load in L. amazonensis PV with Raw264.7 ............... 146 5 -7 Effect of Syntaxin18 on parasite load in L. amazonensi s PV within Raw264.7 147 6 -1 Model of the macrophage endoplasmic reticulum contributions to Leishmania PV biogenesis and maturation. ............................................................................ 153 A-1 pCMV/myc/ER/GFP Map. Adapted from Invitrogen Inc. (www.invitrogen.com) 154 A-2 Schematic design of the pCMV/GFP/calnexin vector construction .................... 155

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13 A-3 0.8% agarose gel electrophoresis. ...................................................................... 156 A-4 Experimental design to assess host ER recruitment to Leishmania PVs. ......... 157

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14 LIST OF ABBREVIATION S ATCC American Type Culture Collection BFA Brefeldin A BLAST Basic Local Alignment Search Tool BME Basal Medium Eagle Degrees Celsius % Percent CD+4 Cluster of differentiation 4 CD+8 Cluster of differentiation 8 cm centimeter CO2 Carbon dioxide Da Dalton (atomic mass unit) DAPI 4',6-diamidino -2 phenylindole DMEM Dulbecco's modi DMSO Dimethyl sul foxide DNA Deoxyribonucleic acid EEA-1 Early Endosome Antigen 1 protein ER Endoplasmic reticulum ERS-24 Endoplasmic Reticulum SNARE 24 g Gram GFP Green fluorescent protein GM -130 Golgi matrix protein h Hour HEPES 4 -(2 hydroxyethyl) -1 piperazineethanesulf onic acid ICBR Interdisciplinary Center for Biotechnology Research

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15 IFA Immunofluorescence assay kDa Kilo Dalton kV kilovolt L Liter L. amazonensis Leishmania amazonensis L. donovani Leishmania donovani L. infantum Leishmania infantum L. mexicana Leishm ania mexicana L. pifanoi Leishmania pifanoi LAMP -1 Lysosomal associated membrane protein 1 LB Luria -Bertani broth LCM Legionella containing vacuole M Molar MD Maryland MES 2 -(N morpholino)ethanesulfonic acid mg Milligram MHC Major Histocompatibility Complex min Minute NaN3 Sodium azide NH4Cl Ammonium chloride nm Nanometer nM Nanomolar Ohm OD Optical density PBS Phosphate buffered saline

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16 pCMV cytomegalovirus plasmid PCR Polymerase chain reaction PFA Paraformaldehyde PH potential of Hydrogen PV Parasit ophorous vacuole PVM Parasitophorous vacuole membrane RCA II Ricinus communis agglutinin II RCF Relative centrifugal force RNA Ribonucleic Acid RPMI Roswell Park Memorial Institute medium RT -PCR Reverse transcription polymerase chain reaction SNARE Soluble N ethylmaleimide -sensitive factor attachment protein receptor SOC Super Optimal broth with Catabolite repression EM Electron Microscopy TGN Trans -Golgi network ug Microgram uL Microliter um Micrometer USDA United States Department of Agriculture YFP Yello w fluorescent protein w Weight WHO World Health Organization

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17 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy LEISH MANIA PARASITOPHOROUS VACUOLESINTERACTION WITH THE MACROPHAGE ENDOPLASMIC RETICULUM By Blaise Ndjamen August 2010 Chair: Peter E. Kima Major: Microbiology and Cell Science Leishmania parasites infect approximately 2 million people every year and cause l eishmaniasis Leishmania are transmitted by sand flies to human tissues; where parasites preferentially enter macrophages by phagocytosis. It is well established that Leishmania reside and replicate in membrane bound structures called parasitophorous vacuoles (PVs), which interact with endocytic compartments such as lysosomes The extent of Leishmania PV (LPV)s interaction with host endoplasmic reticulum (hER) is poorly understood. This study presents a comprehensive assessment of hER contributions to LPV biogenesis and maturation and Leishmania development within macrophages. Macrophages expressing either calnexin or ER associated SNAREs tagged with fluorescence proteins were infected with parasites or ZymosanA. Samples were processed through immuno -fluorescence assays and analyzed by fluorescence microscopy to assess the recruitment of hER molecules to LPVs. We found that more than 90% of PVs harboring L. pifanoi or L. donovani parasites recruited calnexin and SNAREs to their PV membrane throughout the course of infection. Electron microscopy analysis of infected macrophages expressing Sec22b/YFP confirmed that LAMP 1 and

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18 Sec22b are recruited to LPVs. Unlike PVs, no more than 20% of Zymosan phagosomes recruited calnexin or Sec22b to their phagosomal membrane. Similarly, phagosomes containing d ead parasites recruited lower level of ER molecules after infection. To further gain insight into h ER L PV interactions, the intracellular trafficking pathway of ricin was exploited. We established that Raw264.7 m acrophages are a suitable system to study ricin trafficking in eukaryotic cells. Ricin was targeted into LPV compartments via hER, and this process could be block ed by BrefeldinA. Ricin accumulation in LPVs and hER was not immediate, suggesting that molecules in the hER are continuously delivered to LPVs by vesicular transport. Finally, we analyzed the Leishmania infection in Raw246.7 cells transiently expressing either dominant negative or over expression constructs of Sec22b, Syntaxin 18, or D12. Our data suggest that hER membraneassociated SNAREs are essential for both Leishmania replication and PV development, but not for their entry in macrophages. Altogether, this study demonstrated that Leishmania PVs are hybrid compartments composed of both endocyt ic and host ER components; hER are essential for a successful Leishmania survival and replication, and PV development in macrophages.

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19 CHAPTER 1 GENERAL INTRODUCTION Introduction This g eneral introduction will contain an overview of the disease and highlight Leishmania parasite development in mammalian cells We will also discuss the process of phagocytosis, which is the way that these parasites enter their mammalian hosts. In addition, we discuss some aspect s of the structure of the endoplasmic reticul um as well as its interactions with secretory pathways, and molecule trafficking in eukaryotic cells At the end of this chapter, we provide the rationale and design of this research project. Leishmania sis Leishmania sis is a vector borne disease, which exhibits a spectrum of clinical symptoms ranging from a self -healing cutaneous ulcer to a severe mucocutaneous and potentially fatal visceral pathology. Leishmania sis is endemic in 88 countries mostly in tropical and subtropical regions (F igure1-1) where a pproximately 350 million people are at risk and 12 million infected. The incidence rate of this disease is estimated at 2 million new cases each year. The overall death rate is estimated at 3%, but th e rate is 100% deaths among untreated individuals with v isceral l eishmania sis (Desjeux, 2004) Leishmania sis is caused by protozoan parasites of the genus Leishmania which belongs to the family Trypanosomatidae and order Kinetoplastida (Grimaldi and Tesh, 1993) Leishmania parasites are transmitted to mammals by female bloodsucking sandflies of the phlebotomine sub-family. Dogs, rodents and other small mammals serve as natural reservoirs for these parasites, and humans are consi dered accidental hosts of the parasite s (Neuber, 2008)

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20 The incidence of l eishmania sis is increasing, with many endemic areas reporting a 500% increase over the past seven years (Roberts et al., 2000) The incidence rate of l eishmania sis is also increasing in Western world populations including Europeans and North Americans, especially among Afghanistan and Iraq war soldiers and veterans. For example, 35% of Dutch soldiers wer e infected with cutaneous l eishmaniasis within 3 months of establishing a new base in northern Afghanistan (Neuber, 2008) More than 1000 American soldiers were infected with cutaneous leishmaniasis within 2 years afte r their arrival in Iraq in 2003 The control of this disease is stalled by several factors such as the absence of an efficient vaccine, limitation with current drugs, and the increased transmission as a result of co-infections with HIV, inadequate vector ( sandfly) control, and insufficient access to or impetus for developing affordable new drugs (Croft et al., 2006; Murray et al., 2005) Leishmania Life C ycle Leishmania sp are haemogagellate par asites which have two distinct morphological forms during their life cycle ( Figure 1-2 ): promastigotes and amastigotes. Promastigotes (Figure 1-3A) are the motile, flagellated, and extra cellular forms that replicate in the sand-flys gut as procyclic promastigotes. They migrate in their vectors mouthparts to become non -dividing and infective metacyclic promastigotes, which are transmitted to mammals during sand-fly (of Phlebotomus and Lutzomyia genera) bites. In mammals tissue site, 20omastigotes exploit the phagocytosis process to silently enter and to evade triggering host responses (Engwerda et al., 2004; Sacks and Sher, 2002) These parasites preferentially infect profes sional phagocytes such as macrophages, dendritic cells, and neutrophils; other host cell types can be infected as well (Laskay et al., 2003; Bogdan et al., 2000) Once inside cells, t hese

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21 metacycl ic promastigotes are enclosed in a membrane-bound structure called a parasitophorous vacuole (PV), which interacts with other host compartments and displays lysosomal properties (Courret et al., 2002) After a perio d of 24 to 72h, promastigotes transform into amastigotes (Figure 1 3B) which are aflagellated and strict intracellular pathogens. Amastigotes have the capacity to survive the host cell defense mechanisms; they replicate in a low oxygen and low pH environm ent to cause the disease (Besteiro et al., 2007) Leishmania lifecycle is completed when the vector bites and ingests blood from infected mammals. In the vectors gut, infected macrophages are lys ed and the free amastigotes transform into procyclic promastigotes, which proceed to divide rapidly and attach to the sand -fly gut. After about 3 days, sand-fl ies will evacuate the remnants of the blood meal, including unattached parasites The parasites that were attached by their flagellae to the vector s gut (midgut microvillae, the hindgut cuticular surface, the hind gut tr iangle or pyloric valve) are released. They divide repeatedly and differentiate as metacyclic forms, which are generally unattached, fast -swimming with small body and long flagellum (Rogers and Bates, 2007) Leishmania Structure Promastigotes and amastigotes share many ultrastructural characteristics (Figure 1 -3) Leishmania belong to the order of Kinetoplas tida (Figure 1-5) and members of this order are characterized by the presence of a special organelle called, the kinetoplast. Kinetoplasts are bar -like structure s which represent a portion of the single mitochondrion of kinetoplastids (de Souza, 2002) The kinetoplast is located within the mitochondrial matrix perpendicular to the axis of the flagellum ; its DNA is made of several thousand of minicircles of about 0.5 to 2.5 kb in size and few dozen maxicircles,

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22 whi ch vary in size between 20 and 40 kb depending on the species (Liu and Englund, 2007; Liu et al., 2005; Shapiro and Englund, 1995) Leishmania possess general structural characteri stics of eukaryotic cells such as the presence a nucleus, rough and smooth endoplasmic reticulum, Golgi apparatus, lysosomes and a mitochondrion. They also have a contractile vacuole believed to be involved in osmotic regulation and storage vacuol es Kine toplastids including Leishmania have a peroxisome like organelle called glycosome, which hosts many oxidation of fatty acids, ether lipid biosynthesis and purine salvage (Par sons et al., 2005) Leishmania possess a cidocalcisomes which enable these parasites to mobilize and store many chemicals such as calcium, sodium, phosphorous and various othe r cations (Zn+ and Mg+) (Miranda et al., 2004; McConville et al., 2002) Leishmania Genetics The sequencing of the genome of three Leishmania species: L. major (Ivens et al., 2005) and L. infantum and L. braziliensis (Peacock et al., 2007) (Peacock, 2007) has provid ed a powerful tool to understand the biology of the parasites and to identify new targets for drugs and vaccine development. Leishmani a genomes seem significantly conserved with only about 200 out 8000 genes that show a differential distribution between the three species (Peacock et al., 2007) Approximately 34% of the predicted proteomes may p otentially have a role in pathogenicity. Leishmania are diploid organism s with a 3.55x 107bp genome, which is organized into 34 to 36 chromosome pairs ranging in size from ~250 kilobases (kb) to ~ 4 megabases (Mb) depending on the species. Leishmania ch romosomes are characterized by the presence of repetitive telomeric sequences (Wincker et al., 1996)

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23 varying degrees of aneuploidy and extrachromosomal DNA in the form of linear or circular minichromosomes. These minichromosomes are believed to be generated spontaneously, or by Leishmania genome sequence amplification or exposure to adverse conditions such as drug selection or nutritional stress within these parasites (Segovia, 1994; Ouellette and Borst, 1991; Beverley, 1991) H locus specific genes such as P glycoprotein-related gene ltpgpA, were shown to be d irectly involved in arsenite resistance (Papadopoulou et al., 1994a) and a short chain dehydrogenase reductase gene confers resistance to methotrexate, by reducing pterins (Papadopoulou et al., 1994b) This H locus seems to be quite conserved in different Leishmania species, but they do not necessarily favor extra chromosomal gene amplification in response to drug resistance induction, as shown by a recent experimental study comparing L. major and L. braziliensis (Dias et al., 2007) H loci were reported among natural antimony -resistant Indian isolates of L. donovani (Mukherjee et al., 2007) while no episomal amplification was encountered in antimony res istant Iranian isolates of L. tropica (Hadighi et al., 2007) Leishmania genome has a GC content of approximately 60% and the typical gene structure include s a 5 untranslated region (UTR) of 197 nucleotides on aver age, coding region and a 3 UTR, about 1021 nucleotides in length. Leishmania genes have no introns and they exist as single copy, paired or multi -copy genes that are arranged in tandem repeats or may alternate with other repeated genes (Stiles et al., 1999) Leishmania p arasites have an estimated 8311 genes (Dujardin, 2009) distributed in an unusual pattern on all chromosomes, with clusters of genes present as a contiguous unit on one DNA strand with other similar units on the opposite strand

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24 (Smith et al., 2007; Myler et al., 1999) These genes clusters are transcribed as a single unit (Martinez -Calvillo et al., 2003) prior to trans -splicing and polyadenylation, which is consistent with the polycistronic transcription model of kinetoplastids (Pays et al., 1994) Leishmania d irectional gene clusters (DGCs) rang e in size from a few to hundreds of genes stretching over 1 Mb of DNA (Inga et al., 1998) DGCs are separated by AT rich strand-switch regions considered to contain sites for transcri ption initiation and termination (Martinez -Calvillo et al., 2003) DGCs do not contain clusters of genes of related function like in prokaryotic operons (Smith et al ., 2007) but may contain tandem arrays of genes (like the rRNA, (Inga et al., 1998) ) or multigene families (like the gp63 glycoprotein, (Victoir et al., 2005) ). Individual monosistronic mRNA are produced from polycistroni c transcripts by two RNA processing reactions: 1 -trans -splicing and 2 polyadenyation (LeBowitz et al., 1993) The mechanism of gene regulation is not fully understood, however the regulation of transcript abundance appear s to be post transcriptional, dependent on 3UTR and intergenic sequences with the involvement of labile protein factors (reviewed in (Stiles et al., 1999) ). Differences between the genomes of the three L eishmania species include the identification of potentially active retrotransposons and genes implicated in the RNAi pathway in L. braziliensis (Peacock, 2007) not present in L. major (El -Sayed et al., 2005) It was hypothesized that L. braziliensis could have retained RNAi as an antiviral defense mechanism (Smith et al., 2007) : indeed RNA viruses were often reported in that specie s as well as in other species of the subgenus Viannia (Widmer and Dooley, 1995) The specific role of these mobile elements and the reason behind their absence in L. major are still unknown (Dujardin, 2009) The amastin gene array, largest family of

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25 surfaceexpressed protein genes in Leishmania, i s twice as large as in L. major than in L. braziliensis while the gp63 gene array is 4 times larger in L. braziliensis than in the two other species (Dujardin, 2009) Leishmania Para sites and Vectors Leishmania sis (Figure 1-4) are caused by 20 species of the Leishmania genus protozoan parasites transmitted by female sandflies, w hich suck mammalian blood in ord er to obtain protein necessary to develop their eggs. Species of these phlebotomines belong to two genera, P hlebotomus in the "Old World" (Eurasia, Africa) and Lu t zomyia in the "New World" (the Americas). These small flies, 2 3 mm long, can pass through the holes in even fine mosquito netting to bite their hosts. There are around 800 known Phlebotomines species, and only about 70 of them carry Leishmania (Ashford, 2000) Leishmania promastigotes promote its transmission to mammalian hosts by enhancing the feeding behavior of the sand fly. The metacyclic promastigotes produce a secretory gel containing a Leishmania specific filamentous proteophosphoglycan (fPPG). The fPPG impairs vectors mechanorec eptors and also promotes the vectors hunger state, persistence of the fly, or alternatively, increase in the threshold blood volume at which blood -seeking behavior is inhibited (Rogers and Bates, 2007) The Leishman ia transmission is the product of the physical blockage of the gut with a filamentous proteophosphoglycan (fPPG) that ensures regurgitation of infective forms; and a subsequent exacerbation of infection in the mammalian host through the action of fPPG and vector saliva (Rogers et al., 2004; Stierhof et al., 1999; Jefferies et al., 1991) Leishmania sis Clinical S ymptoms The clinical outcome of Leishmania infection is d etermined by the parasite species, vector virulence factors and host immune responses. Leishmania species are a

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26 very complex and diverse group of parasites in terms of their numbers and the outcomes of their interactions with mammalian or human host cells. There are five main type of clinical manifestations (Figure 14) : cutaneous, mucocutaneous, diffuse cutaneous, visceral and post -kalaazar leishmaniasis Visceral leishmaniasis Visceral leishmaniasis (VL) (Figure 1 -4D ) also known as kala azar is caused by L. donovani in the Indian sub-continent, Asia, and Africa, L. infantum in the Mediterranean basin, and L. chagasi in South America. Other species such as L tropica in the Middle East and L amazonensis in South America are occasionally viscerotropic (Desjeux, 2004; Desjeux, 2001; Guerin et al., 2002) Newly acquired infection varies from subclinical (no clinical symptoms), to oligosymptomatic, to fully established (kala azar ). The kalaazar is characterized by irregular bouts of fever, substantial weight loss, swelling of the spleen and liver, and anemia (occasionally serious) Active visceral leishmaniasis may also represent relapse (recurrence 6 12 months after apparently s uccessful treatment) or late reactivation (recrudescence) of subclinical or previously treated infection (Murray, 2005) The reactivation of the infection from the latent phase can be spontaneous, but is often prov oked by changes in T cell (CD4) number or functioncorticosteroid or cytotoxic therapy, anti -rejection treatment in transplant recipients, or advanced HIV disease (Murray et al., 2005; Fernandez Guerrero et al., 2004; Pintado et al., 2001) The estimated annual global burden of VL is 500, 000 new cases and more than 60, 000 deaths, of which 90% occur in just five countries India, Bangladesh, Nepal, Sudan, and Brazil (W.H.O., 2009) (Raguenaud et al., 2007; Maltezou et al., 2000)

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27 Post kalaazar dermal leishmaniasis (PKDL) PKDL is a cutaneous manifestation of visceral leishmaniasis (VL), ch aracterized by skin lesions, nodules or papules, frequently on the face (Figure 14E). It often appears 27 years after the apparently successful treatment of VL with pentavelent antimonial drugs (Croft, 2008) amphote ricin B and miltefosine (Kumar et al., 2009) The incidence of PKDL varies according to regions, for example 5 15% in India (Ramesh and Mukherjee, 1995) and about 60% in Sudan. So far, little is known about the factors that drive the parasite to cause a shift in the site of infection from viscera to dermis and thereby the clinical manifestation of the disease (Croft, 2008) Cutaneous leishman iasis (CL) CL accounts for more than 50% of new cases of leishmaniasis and 90% of the cases are reported from eight countries, six in the Old World (Afghanistan, Iran, Iraq, Algeria, Saudi Arabia and Syria) and two in the New World (Peru, Brazil). CL is c aused by L.major L. tropica and L. aethiopica in the Old World, and by the L. mexicana species complex and the Vianna subgenus in the New World. The disease starts as papules which progress into nodules and ends as ulcerat ive le sions with a central depre ssion and indurated border that results in atrophic scars over time (Figure 1 4A) (Ashford, 2000) CL usually heals spontaneously in the Old World, but less frequently in New World, because species of the L. viannia subgenus ( L viannia braziliensis L viannia guyanensis and L viannia panamensis ) tend to disseminate in mammalian host (Lawn et al., 2004; Choi and Lerner, 2001; Herwaldt, 1 999) In the United States, CL is endemic in south-central Texas, and recently some autochthonous cases of cutaneous leishmaniasis have been reported in north ern Texas (Wright et al., 2008) suggesting an

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28 increase in transmission and distribution of leishmaniasis in the United States (McHugh, 2010) Diffuse cutaneous leishmaniasis (DCL) DCL is caused by normal CL species, L. aethiopica and L. tropica (in the Old World), L. am azonensis and L. mexicana (New World), mostly in anergic hosts with poor immune responses. Infection is characterized by disseminated, flaking, and nonulcerated nodular lesions on the skin that resemble lepromatous leprosy (Figure 1 -4B) DCL is considered to an hypersensitivity condition in which most parasites are eliminated but the infection is not completely cured (Ashford, 2000) Mucocutaneous leishmaniasis (MCL) or espundia MCL is primary caused by parasites of the L. braziliensis complex in the N ew W orld, and occasionally by L. d. infantum in the O ld World, especially in Sudan. The disease begins as simple skin lesions or oriental sore that can metastasize via the blood stream or lymphatics. L esions can lead to partial or total destruction of the mucous membranes of the nose, mouth and throat cavities and surrounding tissues (Figure 14C) Generally, less than 5% of patients with simple cutaneous leishmaniasis will develop MCL, which can occur while the prim ary lesion is still active or several years after the primary lesion has healed (Ashford, 2000) MCL can be either ulcerative or non ulcerative; the ulcerative form is characterized by a rapid and extensive mutilat ion of soft tissue and cartilage. The nonulcerative is marked by local edema and hypertrophy, particularly of the upper lip; but if not treated, the disease will progress and lead to severe pathology and deformity (Ashford, 2000) The parasite tropism for macrophages of the oronasopharyngeal region is a poorly

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29 understood phenomenon and may involve several factors including sensitivity of the parasite to temperature, as well as permissiveness of the various macrophag e populations (Lodge and Descoteaux, 2005) Diagnosis of leishmaniasis In active endemic areas, all forms of leishmaniasis can be diagnosed with some reliability by clinical e xamination, with an additional blood count to show anemia and leucopoenia in the specific case of visceral leishmaniasis The confirmation of leishmaniasis diagnosis is generally done by demonstrating the presence of the amastigote parasites in stained microscopical preparations or cultures from skin biopsies (CL) or aspirates from bone marrow or spleen specimens (VL). VL displayed a very strong serological response, to the extent that the albumin /globulin ratio is reversed. The raised serum proteins are used diagnostically in a non -specific formol -gel test. Various DNA and monoclonal antibody probes have been developed, but none of these has reached routine practice. The formol gel test, which consists of the addition of a drop of formalin to 1 ml of the patients serum is generally used as/bec ause a positive reaction is indicated by the rapid coagulation of the serum Alt h ough this test is insufficiently specific to be recommended, it is still widely used in endemic areas. For MCL parasites are difficult to find in lesions, therefore the patie nts history of CL and serology (ELISA, IFA) are used as diagnostic tools (Ashford, 2000) Treatment Pentavalent antimony such as sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime) are the co re or first -line of treatment for visceral leishmaniasis CL, and m ucosal leishmaniasis These drugs are prescribed at a dose of

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30 20 mg/kg/d parenterally ( by piercing the skin or the mucous membrane) for a 3to 4 week period (Blum et al., 2004; Berman, 1996; Herwaldt and Berman, 1992) Limitations of these drugs, which include a high cost, difficulty of administration, toxicity, length of treatment, and drug resistance or rel apses in up to 25% of cases with some Leishmania str ains, have lead to the search for new anti -Leishmania sis compounds (Schwartz et al., 2006) Pentamidine isethionate is generally prescribed as an alternative for individuals, who are intolerant to antimonial treatment, or in cases of antimonial resistance. It is the first line of treatment of cutaneous leishmaniasis (3 mg/kg/d IM every other day for 4 injections) in French Guiana where more than 90% of infections a re caused by L guyanensis (Desjeux, 2004) Paromomycin sulfate is also a topical anti leishmaniasis agent to treat Old World CL ( L major L tropica, L aethiopica ). Injectable paromomycin has been experimentally used to treat visceral l eishmania sis (primarily caused by L donovani ) (Davis and Kedzierski, 2005) and only L mexicana infections in the New World CL (Lawn et al., 20 04; Blum et al., 2004) A less costly combination of 15% paromomycin/12% methylbenzethonium ointment to treat L mexicana infection is accepted (Mitropoulos et al., 2010; Soto et al., 1998) Amph otericin B preparations have typically been used in treating visceral and mucosal leishmaniasis unresponsive to antimonial therapy. Amphotericin B is associated with infusion-related toxicity and thus needs to be administered slowly and nephrotoxicity (Mitropoulos et al., 2010)

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31 Miltefosine, a phosphocholine analogue, was initially developed as an anti neoplastic agent. It was found to have anti -l eishmanial activity in vitro and in vivo, probably via effects on cel l -signaling pathways and membrane synthesis (Kuhlencord et al., 1992; Croft et al., 1987) Miltefosine is administered orally at a dosage of 2.5 mg/kg/d for 28 days, to cure visceral leishmaniasis in India and CL in Pakistan rates comparable with pentavalent antimony. The assessment of the miltefosine efficacy on New World CL is still an ongoing process (Mitropoulos et al., 2010) Imiquimod is an imidaz oquinoline amine, which induces interferontumor necrosis factor alfa, interleukin (IL) -6, and IL8 (Wagner et al., 1997) and has been shown to effectively stimulate leishmanicidal activity in macrophages (Buates and Matlashewski, 1999) Allopurinol (4 -hydroxypyrazolo[3,4d] pyrimidine) is a drug traditionally used for the treatment of gout. It has been shown to inhibit the growth of Leishmania in vitro at concentrati ons that are attainable in human tissues and body fluids. This compound is believed to act by prohibiting the de novo synthesis of pyrimidines, probably through the formation of allopurinol ribotide, which leads to the inhibition of protein synthesis in th e Leishmania parasite (Pfaller and Marr, 1974) Oral allopurinol (20 mg/kg/d for 15 days) is mainly recommended as pentavalent antimonial adjunct to treat New World CL (Martinez and Marr, 1992; Guderian et al., 1991) Other experimental drugs that have demonstrated leishmanicidal activity comprise some plant extracts, chalcones, alkaloids, terpenes, and phenolics (El -O n, 2009) Cryotherapy, heat application, curettage, electrodessication, and surgical excision have also been implemented in the treatment of early, small, cutaneous lesions in leishmaniasis These physical

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32 approaches constitute an alternative to costly an d toxic proposed first -line agents, or when systemic therapy is contraindicated, such as in cases of pregnancy, heart conditions, and impaired renal or hepatic function (Alkhawajah, 1998) Vaccines T o date, t here i s no FDA approved vaccine; however a live Leishmania prophylactic vaccine in Uzbekistan and a dead Leishmania vaccine, as an adjunct to antimony therapy, have been registered for clinical use against human disease in Brazil (Palatnik -de -Sousa, 2008) Three main approaches are generally pursued in the development of a potential vaccine against leishmaniasis The leishmanization consists of inducing the first infection by injecting live virulent parasites in an aesthetically acceptable site of the body in healthy individuals; this first natural infection with L. major is highly protective against subsequent infections (Belkaid et al., 2002) Leishmanization adverse side effects including the development of large persistent lesions, complications in immuno -compromised individuals, and the fact that this technique is n ot suitable for large-scale use have led to the discontinuation of this technique in many countries (Hepburn, 2003) The f irst -generation vaccine approach uses fractions of the parasite or whole killed Leishmania with or without adjuvants; and second-generation vaccines using live genetically modified parasites, or bac teria or viruses containing Leishmania genes, recombinant or native fractions (Khamesipour et al., 2006; Selvapandiyan et al., 2006) The monophosphoryl lipid A in a stable emulsion (MPL-SE), multi antigen vaccine, is the only second -generation vaccine against leishmaniasis that has reached human trials. MPL -SE is being developed for prophylaxis, a therapy based on studies in mice,

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33 and preliminary trials in human beings on a compassionate basis (Mitropoulos et al., 2010; Khamesipour et al., 2006) Synthetic anti Leishmania l vaccines, which involve e lements of the immune response such as CD4+ and CD8+ T cells, have been developed (Gurunathan et al., 2000) It is believed that CD8 cells also play a significant role in the maintenance of long -term vaccine induced immunity (Belkaid et al., 2002) The capacity to induce both CD4+ and CD8+ cell responses has, traditionally, been known to be a feature of DNA vaccines. However, some proteinbased vaccines have demonstrated the same capacity (Rhee et al., 2002; Reed, 2001) More than thirty nonapeptides, which specifically trigger IFN -gamma secretion by human CD8+ cells through the MHC class system hav e been identified, and have been of interest in many vaccine projects (Basu et al., 2007) Recombinant BCG expressing Leishmania antigens. R ecombinant BCG (Mycobacterium bovis bacillus Calmette Guerin) has been used in vaccination against leishmaniasis (Gicquel, 1995) It is being used in clinical trials (Basu et al., 2007) as an adjuvant in vaccine preparations with dead L. mexicana or with L. braziliensis promastigotes but its efficacy is still controversial (Khamesipour et al., 2006; Noazin et al., 2008; Convit et al., 2004; Handman, 2001) Vaccines based on sand fly salivary antigens. Although most vaccines strategies are genera lly directed against antigens of the infectious agent transmitted by the vector, other approaches are focused on the arthropod itself (Grimaldi and Tesh, 1993) It is known that saliva of sand flies enhances the infectivity of Leishmania parasites in their mammalian hosts (Lima and Titus, 1996) Vaccines have been designed against components of saliva or insect gut antigens that can protect from infection and decrease

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34 the v iability and reproducibility of the insect (Titus et al., 2006; Milleron et al., 2004) These vector components include the protein MAX or MAXADILAN ( Brodie et al., 2007) and the SP15 antigen obtained from Phlebotomus papatasi and have been shown to induce substantial resistance in mice to L. major infection (Valenzuela et al., 2001) Leishmania versus Mam malian Host D efense Important leishmanial virulence factors V irulence factors that enable Leishmania to e stablish and maint ain the infection in mammals differ according to parasite species and stages In the sandfly vector, the metacyclogenesis process th at transforms log-phase procyclic promastigotes to stationary phase metacyclic promastigotes is critical in equipping Leishmania parasites with infectiv e capacities for mammalian. Metacyclic promastigotes selectively increase the expression of virulence fa ctors, such as the surface lipophosphoglycan (LPG) and the metalloprotease gp63 (Yao et al., 2003) LPG has been identified as a virulence factor for L. major and L. donovani (Spath et al., 2000; McNeely and Turco, 1990; Turco, 1990) but not L. mexicana (Ilg, 2000) while gp63 has been recognized as a vital virulence factor for L. major L. mexicana/ L. amazonensis and L. donovani (Joshi et al., 2002; Joshi et al., 1998; Chen et al., 2000; Seay et al., 1996; Wilson et al., 1989) The glycoprotein of 63 KDa (GP63) GP63 also known as leishmanolysisn or major surface protease (MSP), is the most abundant surface protein of Leishmania promastigotes, and may account for approximately 1% of the total protein content (Bahr et al., 1993 ) It is not highly expressed in amastigotes (Yao et al., 2003; Hsiao et al., 2008) Gp63 on parasites surface membrane inhibits complement -mediated lysis by cleaving and converting C3b to C3bi, and this will favor parasite uptake via host

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35 macrophage CR3 (Brittingham et al., 1995) Extracellular release of gp63 (McGwire et al., 2003) may also facilitate the pr omastigote's migration and dissemination through tissue by degrading extracellular matrix components such as fibronectin. Other functions attributed to extracellular gp63, which may serve to subvert or circumvent immune responses, include cleaving major hi stocompatibility complex (MHC) class I molecules (Garcia et al., 1997) and limit CD4 T -cell responses (Hey et al., 1994) Gp63 may deactivate macrophages by down modulating MA RCKS-related (myristoylated alanine-rich C kinase substrate) protein (Corradin et al., 2002; Corradin et al., 1999) T he role of gp63 in promoting amastigote pathogenesis remains unclear, as its expression is significantly down regulated in the intracellular amastigote stage Lipophosphoglycan (LPG) LPG is the most predominant glycoconjugate of infective metacyclic promastigotes, at approximately 5 million copies per cell (Lodge and Descoteaux, 2005) and distributed along the entire cell surface and the flagellum. Its main structure consists of a polymer of Gal 1 surface via a glycophosphatidylinositol (GPI) anchor. LPG is present in all Leishmania species, but its structures and role vary among species (McCo nville, 1995; Turco, 1992) Gene knock out studies have demonstrated that LPG is a virulence factor in L. major and L. donovani (Spath et al., 2000) LPG forms a compact glycocalyx that act as a first line of the p arasite defense and also contributes to the evasion of complement mediated lysis (Puentes et al., 1988) LPG can bind to the C3b component of the complement cascade, and in conjunction with GP63 promotes parasite internalization via the macrophage C3 receptor (C3R) (Brittingham et

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36 al., 1995) The LPG has been demonstrated to be critical in a number of biological/virulence m echanisms (Turco et al., 2001; Ilg et al., 200 1; Moore et al., 1994; Descoteaux et al., 1991) important to Leishmania survival. LPG modulates nitric oxide (NO) production (Proudfoot et al., 1996) inhibition of apoptosis (Moore and Matlashewski, 1994) (Moore et al., 1994) delay in phagolysosome maturation (Holm et al., 2001) and inhibition of macrophage signal transduction (Descoteaux et al., 1991) LPG -deficient L. mexicana organisms are as virulent and infective to their murine hosts as wild type organisms, suggesting that LPG is not a critical virulence factor for L. mexicana, as it is for both L. major and L. donovani parasites (Turco et al., 2001; Ilg et al., 2001) T h erefore, L. mexicana may use alternate ligands or mechanisms to enter cells and evade host immune system The A2 gene lo cu s. It encodes a family of amastigote stage-specific 42 100 kDa proteins, localized to the parasite cytoplasm (Charest and Matlashewski, 1994) The A2 gene locus has been demonstrated to be important for the virulence of L. donovani (Zhang et al., 2003; Zhang and Matlashewski, 2001) ; L. donovani organisms made genetically deficient for A2 fail to be infective to macrophages and mice (Zhang and Matlashewski, 1997) Although the A2 genes appear to be required for the virulence of L. donovani they are not expressed in L. major Further, the transformation of L. major to express the A2 proteins (Zhang et al., 2003; Zhang and Matlashewski, 2001) results in increased visceralization and/or survival of L. major in the spleen but diminished parasite survival in cutaneous tissue. The diminished infection in th e skin appears to be due, in part, to increased migration of Leishmania -infected cells (dendritic cells and

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37 macrophages). Thus, the loss or gain of A2 expression in Leishmania appears to contribute to the species -specific tissue 'tropism'. However, the mec hanisms involved remain to be elucidated and may be complex and involve other species -specific parasite genes (Zhang et al., 2003) The Leishmania homolog of receptors for activated C kinase (LACK) LACK antigen pr omotes IL-4 production through the activa++CD4+ T cells (Malherbe et al., 2000; Julia et al., 1996) and is critical for the susceptibility of BALB/c mice to L. major infection. Further, studies indicate that LACK is required for parasite persistence within macrophages and the mammalian host (Kelly et al., 2003) Although LACK -re sponsive T cells develop during L. mexicana infection, susceptibility to L. mexicana is independent of LACK induction of IL-4 (Torrentera et al., 2001) In fact, LACK -tolerant BALB/c -LACK transgenic mice are resistant to L. major and yet these mice are fully susceptible to L. mexicana The development of 'healing' versus 'non healing' for L. major has been related to LACK -peptide T -cell receptor affinity. However, the LACK epitope involved is identical in both the L. major and the L. mexicana LACK proteins, suggesting that additional parasite molecules contribute to the developing pathogenesis in BALB/c mice during cutaneous infection caused by L. mexicana. C athepsin L-like cysteine protease B (CPB) enzymes CP B enzymes are expressed in high abundance and localized to the megasome organelles unique to strains of L. mexicana complex and are considered to be a critical for virulence (Denise et al., 2003; de Araujo Soares et al., 2003; Alexander et al., 1998a) CPBdeficient L. mexicana amastigotes have reduced ability to induce lesion growth in BALB/c mice (Denise et al.,

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38 2003; Alexander et al., 1998a) and the lesions of other susceptible strains of mice (C3H and C57BL/6) heal (Buxbaum et al., 2003) The virulence of L. mexicana for BALB/c mice has been associa ted with the ability of CPB (Denise et al., 2003; Alexander et al., 1998a) to induce IL4 production and a Th2 response. CPB -deficient parasites fail to induce IL4, resulting in a T -helper 1 cell (Th1) response and inhibition of lesion growth. Genetic restoration of CPB expression restores virulence together with the ability to induce BALB/c IL -4 production (Denise et al., 2003) Sand fly saliva It in duces vasodilatation, inhibition of coagulation and immunomodulatory effects (Sacks and Kamhawi, 2001) ; and attraction of PMNs as well as macrophages (Anjili et al., 2006; Zer et al., 2001) The parasite itself also produces a chemoattractant protein called Leishmania chemotactic factor, which can attract PMNs (van Zandbergen et al., 2002) Two hours after saliva injectio n, an intense and diffuse inflammatory infiltrate comprising PMNs, eosinophils and macrophages is induced only in mice preexposed to saliva (Silva et al., 2005) PMNs are the first cells to arrive at the site of Leishm ania infection (Muller et al., 2001) H uman PMNs infected with Leishmania secrete chemokines such as IL -8 (CXCL8 ) (Laufs et al., 2002) that are essential in attracting more PMNs to the site of infection Upon experimental infection with L. major MIP 2 and keratinocyte -derived cytokine (KC; also known as CXCL1 ), the functional murine homologs of human IL 8 (Modi and Yoshimura, 1999) are rapidly produced in the skin (Muller et al., 2001) In vitro studies have also shown that L. major promastigotes induce rapid and transient expression of KC by murine m acrophages (Raco osin and Beverley, 1997) and of IL -8 by human macrophages (Badolato et al., 1996) All of these chemokines are chemo attractants for PMNs (Baggiolini, 2001)

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39 PMNs can func tion as phagocytic cells, taking up and killing Leishmania (Lima et al., 1998) and they have been implicated in early parasite control. Chemokines have different roles in Leishmania infection including the recruitme nt of sentinel immune cells ( DCs macrophages T lymphocytes ) to the site of parasite delivery. They also play an important role in the adaptive immunity, macrophage activation and parasite killing (Teixeira et al., 2006) These cells possessed Toll li ke receptors (TLRs) (Muzio et al., 2000) and phagocytic receptors (Ross, 2000) which enable them to detect pathogen associated molecular patterns (Gordon, 2002) and uptake pathogens and opsonized particles. Sentinel cells also express various receptors for cytokines, which induce the production of chemokines to initiate the innate responses (Spellberg and Edwards, 2001; Spellberg, 2000) L. major infected mice induce overall upregulation of CCL5 also known as regulated on activation normal T cell expressed and secreted (RANTES), MIP CXCL10 and CCL2 in the footpads and LNs (Antoniazi et al., 2004) The role of PMNs in the context of the early response to Leishmania Leishmania extends the lifespan of PMNs (Aga et al., 2002) and can survive within these cells for hours or days after infection (van Zandbergen et al., 2004) Leishmania Infected PMNs induce the release o f MIP macrophages to the site of infection (van Zandbergen et al., 2004) Infected PMNs taken up by macrophages do not activate macrophage microbicidal function (van Zandbergen et al., 2004) In macrophages containing apoptotic PMNs, proinflammatory cytokine production is inhibited through mechanisms involving transforming growth factor 2 and platelet activating factor (Ribeiro -Gomes et al., 2004; Fadok et al., 1998a; Fadok et al., 1998b)

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40 These events contribute to a silent entry of Leishmania into macrophages its main host cell type (Laskay et al., 2003) Macrophages are the second wave of cells that enter the site of Leishmania infection, and are considered the ultimate host cells for Leishmania parasite development They perform antigenpresentation and also produce cytokines that will modulate the T cell -mediated immune response. Moreover, after appropriate activation by Th1 cells, they serve as effector cells for intracellular parasite killing (Teixeira et al ., 2006) This suggests that Leishmania parasites, especially the amastigotes form have evolved mechanisms to survive in macrophages. Monocytes are attracted in the early stages of infection by products of sand fly saliva (Zer et al., 2001; Anjili et al., 1995) and, two to three days later, by chemokines such as MIP (van Zandbergen et al., 2004) Leishmania can also induce other monocyte attractant che mokines. For example, L. major promastigotes induce rapid and transient expression of JE, a protein inducible by platelet -derived growth factor in murine macrophages (Racoosin and Beverley, 1997) and of its homol og CCL2 in human macrophages (Badolato et al., 1996) Besides attracting monocytes and macrophages CCL2 can attract other cells such as NK cells and DCs that are positive for the chemokine receptor CCR2 (Ritter and Moll, 2000) In human Leishmaniasis CCL2 and MIP macrophage activation in the skin lesions In L. mexicana localized CL, synergistic action of CCL2 and IFN kill parasites within the macrophage (Muzio et al., 2000) But if the parasite stimulates the production of IL 4 which can suppress CCL2 expression and increase the MIP progress into non -healing DCL lesions (Ritter et al., 1996)

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41 Leishmania affect host cell signaling. Host invasion by pathogens is frequently associated with the activation of nuclear factor kappaB (NF kappaB), which modulates the expression of genes involved in the immunological response of the host. However, pathogens may also subvert these mechanisms to secure their survival. L. amazonensis has developed an adaptive strategy to escape from host defense by activating the NF kappaB repressor complex p50/p50. The activation of this specific host transcriptional response negatively regulates the expression of iNOS, favoring the establishment and success of L. amazonensis infecti on. NF kappaB mediated repression of iNOS expression in Leishmania amazonensis macrophage infection {{798 Calegari -Silva,T.C. 2009}} L. donovani induces less CD2 on the surface of CD4+ T cells, which once activated orchestrate the protective IFN -gamma dom inant host defense mechanism via PKC mediated signal transduction and cell cycle (Calegari -Silva et al., 2009) (Bimal et al., 2008) Pathogenesis study of experimental cut aneous and visceral Leishmania sis using mannose receptor R deficient [MR -knockout (KO)] C57BL/6 mice, has revealed that the host mannose receptor MR is not essential for blocking IFN gamma/LPS -induced IL12 production and MAPK activation by Leishmania. Mannose receptors are not essential for host defense against Leishmania infection or regulation of IL -12 production (Akilov et al., 2007) Leishmania macrophage interactions Th ese interactions are complex because Leis hmania species modulate macrophage signaling pathways and metabolism to different extents. For example, activation of L. major -in fected macrophages with low

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42 levels of IFN incubation of L. amazonensis -infected macrophages with IFN IFN ot stimulate NOS2 activity or decrease arginase activity in the infected macrophages (Wanasen et al., 2007) Leishmania amazonensis amastigotes therefore appear to induce a unique activation state in the host cell in which cytosolic levels of arginine are elevated but not further catabolized. Modulation of host cell responses to L. major amastigote infection has also been observed in human THP -1 macrophages (Dogra et al., 2007) Interestingly, apoptotic cells can induce a similar activation state in macrophages in which NOS2 activity is not down regulated, but arginase II activity is elevated and the flux into the polyamine pathway increased). Leishmania amastigotes appear to mimic apoptotic cells and could direct subsequent host cell responses by engagement of specific receptors and associated signaling pathways that limit cell activation. Alternatively, there is increasing evidence that some amastigote proteins can be exported to the host cytoplasm and directly modulate host cell signaling pathways, although the mechanisms underlying this process are poorly defined (Kima, 2007) Leishmania versus the Host Adaptat ive Immune R esponses The develop ment of Leishmania infection depends in part on the parasite species, parasite and species specific factors, and importantly, the host immune response (Murray et al., 2006) In complex organisms such as Leishmania a susceptible phenotype can be associated with one or multiple genes/genetic loci (Havelkova et al., 2006) The innate and acquired immunity play a significant role in controlling Leishmania infection. IFN 12 driven T cell activation results in the polarization of a

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43 T Helper 1 (Th1) response a nd control of the disease (Mattner et al., 1996) ; w hereas a predominantly IL 4 driven polarization and IL10 dependent maintenance of a T Helper 2 (Th2) response (Alexander et al., 1998b; Nylen et al., 2007) will inevitably result in disease progression and a noncuring phenotype (Sacks and NobenTrauth, 2002; Naderer et al., 2008) Early stud ies particularly on L. major largely defined the Th1/Th2 paradigm of resistance/susceptibility to infection and the role of IL12 and IL4 respectively in driving Th1 and Th2 cell development (Reiner and Seder, 1995) While more recent studies using in particular gene deficient mice have largely substantiated the beneficial activity of IL -12 and the Th1 re sponse in controlling infection, the previously proposed paramount role for IL4 in disease exacerbation has been significantly questioned with other mediators being identified as playing influential roles (Sacks and Anderson, 2004; Scott and Farrell, 1998) As the major Leishmania species complexes diverge d some 40 80 million years ago (Stevens et al., 2001) it is unsurprising that different virulence factors have been identified for different species (McMahon -Pratt and Alexander, 2004) and consequently the growth of the different species is subject to different immunological controls (Alexander and Bryson, 2005) Thus while IL4 has been implicated in the nonhealing response o f BALB/c mice to infection with L. mexicana and L. amazonensis (Alexander et al., 2002) it has not been shown to promote chronic disease following visceral infection with L. donovani (Satoskar et al., 1995) and indeed may play a protective role (Stager et al., 2003a) Moreover, IL4 under certain circumstances can potentiate IL -12 production (Stager et

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44 al., 2003a; Stager et al., 2003b) and a type-1 response and Th-2 cell development can take place independently of IL-4 (Mohrs et al., 2000) Resolution of Infection and the Th1 response I t is now well established that a protective immune response against both cutaneous Leishmania sis caused by L. major L. mexicana or L. amazonensis as well as visceral Leishmania sis caused by L. donovani is dependent on the development of potent type 1 imm unity (Figure 1-6A ) (Alexander et al., 1999) The general consensus is that IL -12 from antigen presenting cells (APC ), macrophages and dendritic cells possibly augmented by cytokines such as IL 1 (Von Stebut et al., 2003) IL -18 (Dinarello and Abraham, 2002) IL 23 (Langrish et al., 2004) and IL -27 (Artis et al., 2004b) drives the differentiation and proliferation of T helper 1 cells. MHC Class II antigen presentation alone is not sufficient to stimulate T cell responses and ligation of co -stimulatory molecules, B7 -1 / B7 -2 and CD 40 on the APC with CD28 and CD40L on the T cell respectively is also a prerequisite (Bogdan, 1998; Bogdan et al., 1997) Ligation of CD40 / CD40L enhances APC IL -12 production (Heinzel et al., 1998) as can tumor necrosis factor [ TNF ] related activation induced cytokine (TRANCE)-receptor activator of NF (RANK) interactions (Padigel et al., 2003b) IFN from Th1 cells and probably to a lesser extent CD8+ T cells as part of the acquired immune response but also from IL12 activated NK cells as part of the innate response, mediates macrophage activation, nitric oxide production and parasite killing (Cunningham, 2002) Macrophage leishmanicidal activity induced by IFN has been shown to be enhanced by other cytokines such as TNF (Mannheimer et al., 1996) migration

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45 inhibition factor (MIF) (Juttner et al ., 1998; McSorley et al., 1998) and type 1 interferons (Diefenbach et al., 1998) as well as CD40/ CD40L interactions (Campbell et al., 1996; Kamanaka et al., 1996; Soong et al., 1996) Interestingly a recent study demonstrated that C57BL/6 mice can control L. mexicana independently of IL 12 (Buxbaum et al., 2003) Nevertheless immunity in this study was dependent on the transcription factor STAT4 which is required for IL -12 signaling a type -1 response and IFN and inducible nitric oxide synthase expression. Recently, the IL-12 related cytokine IL -27 signaling via WSX -1 has been shown to counter -regulate the early disease exacerbatory effects of IL -4 in this mouse strain during L. major infection (Artis et al., 2004a) by promoting a Th1 response. In the absence of WSX -1 signaling early lesion growth is greatly exacerbated although healing under the influence of IL12 occurs late in infection. Could the IL-12 independent control of L. mexicana described above occur via this alternative mechanism of Th1 induction? Significantly a further study on L. mexicana (Aguilar Torrentera et al., 2002) demonstrated early but not late resistance to this parasite in C57 BL/6 mice in the absence of IL-12 although the mechanisms involved remain uncharacterized Collectiv ely these studies would suggest elements of redundancy in the induction of type1 responses. Following resolution of patent infection concomitant immunity is dependent upon antigen -specific natural CD4+ CD25+ T regulatory cells producing IL -10 that moderat e the activity of T effectors to maintain latent infection (Belkaid et al., 2002) In the absence of persistent infection protective immunity is significantly reduced. Non -healing L. major infection: Th2 dependent or defective Th1 response The Th1/ Th2 paradigm of resistance/susceptibility to intracellular infection is largely based on investigations using L. major (Figure 1 6 A). Initial studies suggested

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46 that the resolution or progression of disease was dependent on distinct CD4+ T cell subsets Th1 and Th2 producing the counter regulatory and signatory cytokines IFN and IL -4 respectively (Heinzel et al., 1989) Furthermore, the exquisite sensitivity of BALB/c mice + T cells producing IL 4 which rendered T cells unresponsive to IL12 (Launois et al., 1995) by suppressing expression of IL(Himmelrich et al., 1998) Thus a strong case for the predominant role of IL 4 and a Th2 response in nonhealing disease was clearly established. Nevertheless, early IL -4 production was not necessarily be an indicator of susceptibility to L. major as resistant C57BL/6 mice also produced this cytokine early in infection yet proceeded to resolve infection as a Th1 response is developed (Scott et al., 1996) (Scott et al., 1996) In addition, the use of IL -4 and IL mice did not definitively establish the role of IL 4 in the disease process, as a series of apparently contradictory reports have been published (M ohrs et al., 2000; Noben-Trauth et al., 1996a; Kopf et al., 1996) with some but not others indicating a disease exacerbatory role for IL -4 While further studies have shown that these observed differences may be either due to the strain of parasite used (Noben -Trauth et al., 1996b) or even due to the age of the mice (Kropf et al., 2003) they also posed two significant questions: firstly could other regulatory cytokines be substituting or compensating or indeed responsible for the immunosuppressive activity previously attributed to IL 4 and/or secondly could the well documented defective Th1 responses of BALB/c mice be playing a major role in progressive disease (Sacks and Anderson, 2004) The inability to mount a Th1 response irrespective of a Th2 response has been attributed to an inability to produce or respond to IL12 (Belkaid et al., 1998; Kropf et al.,

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47 1997; Guler et al., 1996) While IL 4 is able to downregulate IL12 production and expression of ILIL 4 independent mechanisms (Jones et al., 1998) Consequently intrinsic defects in BALB/c antigen presenting cell function or in T cell development may underlie the susceptibility of BALB/c mice for L. major Defective antigen presenting cell (APC ) function attributable to an in ability to produce IL -12 is initially hard to reconcile with the ability of the parasite to suppress host cell IL 12 equally as well with cells from resistant as well as susceptible mice (Belkaid et al., 1998) Howev er, two recent studies have demonstrated BALB/c dendritic cell populations to be comparatively deficient in IL 1 production (Von Stebut et al., 2003; Filippi et al., 2003) compared with L. m ajor resistant strains. Furthermore, inoculation of IL -1 or IL into BALB/c mice at the onset of infection enhanced protective responses while IL 1 type 1 receptor deficient mice develop enhanced Th2 responses (Satoskar et al., 1998) Significantly not only is the abil ity of IFN to inhibit Th2 production dependent on IL 1 but IL 1 also stimulates dendritic cells to upregulate IL-12 production as well as MHC Class II co -stimulatory molecule expression (Eriksson et al., 2003) The defective Th1 cell development attributed to BALB/c mice was originally identified as an inability to respond to IL -12 (Guler et al., 1996) which was associated with a down regulation of CD4+ T cells IL xpression (Hondowicz et al., 1997) While it is well established that ILdown regulated by IL -4 (Himmelrich et al., 1998) this can also be an IL -4 independent mechanism as it also occurs in IL mice (). However, this is not necessarily a Th2 independent process as IL L. major infected mice continue to mount an unimpaired Th2

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48 response. However, as transgeni c IL BALB/c mice exhibit a nonhealing phenotype despite IL -12 signaling the importance of this defect remains questionable (Nishikomori et al., 2001) Nevertheless BALB/c CD4+ T cells have been demonstrat ed as having an intrinsic ILTh2 bias (Bix et al., 1998) This could be a consequence of the recently described defective co-polarization of the TCR and IFN receptor complex of nave CD4+ T cells at the immunological APC /T precursor synapse in BALB/c mice compared with C57Bl/6 mice (Maldonado et al., 2004) This would significantly favor a commitment to Th2 development. Nev ertheless, IL -4 signaling via STAT6 also serves to block this co localization indicating that both intrinsic T cell defects as well as immune regulatory mechanisms can serve to impair Th1 responses in BALB/c mice Studies using gene-deficient and transgen ic mice have clearly identified other cytokines in addition to IL 4 as playing major roles in the nonhealing response of BALB/c mice to L. major IL mice were found to be more resistant than IL 4 mice suggesting at least a compensatory role for IL 13 whose receptor also utilizes the IL (Mohrs et al., 1999; Noben-Trauth et al., 1999) In addition, a study using transgenic mice over expressing IL 13 on the resistant C57BL/6 background demonstrated that this cytokine was a key factor determining susceptibility (Matthews et al., 2000) IL 13 was found to act independently of IL 4 and studies using IL 13, IL 4 and IL -4 / IL 13 mice suggested the effects of IL 4 and IL 13 to be additive. However, as L. major infected ILBALB/c mice failed to upregulate ILexpression compared with wild-type mice and continued to be more susceptible than C57 BL/6 mice this su ggested other factors could be contributing to susceptibility.

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49 Recent studies using gene deficient mice would suggest that IL 10 is at least as influential as IL 4 / IL 13 at promoting L. major disease progression in BALB/c mice (Noben -Trauth et al., 2003) of antibody coated amastigotes (Kane and Mosser, 2001) or produced by Th2 cells or CD4+ CD25+ T regulatory cells (Sco tt and Farrell, 1998) T regulatory cells are also significant producers of TGF (Mills, 2002) which also is partly responsible for suppressing protective response s (Gumy et al., 2004; Jones, 2000; Reed, 1999; Letterio and Roberts, 1998) Paradoxically T regs producing IL 10 are responsible not only for persistence of parasites in C57BL/6 resistant mice but in maintaining concomitant immunity and in the absence of IL 10 sterile immunity accrues and concomitant immunity is lost (Figure 16) (Belkaid et al., 2002; Belkaid et al., 2001) Cutaneous (L. mexicana / L. amazonensis) infections and IL4 dependent The dichotomy in evolutionary terms between Old World and New World Cutaneous leishmaniasis as well the visceral disease is some 40 80 million years (Steve ns et al., 2001) which is comparable with the divergence of the mammalian orders. Therefore, it is hardly surprising that different Leishmania species have evolved specific adaptations to insect vectors and disparate vertebrate hosts. Consequently differ ent virulence factors have been identified for different Leishmania species and while the vast majority of mouse strains are resistant to L. major infection most develop non-healing lesions when infected with L. mexicana or L. amazonensis (McMahon-Pratt and Alexander, 2004) Furthermore, although BALB/c mice are susceptible to both Old and New World cutaneous leishmaniasis and the L. mexicana LACK antigen is recognized by CD4+ T cells producing IL -4 LACK tolerant BALB/c mice while resistant to L. major remain susceptible to L. mexicana (Aguilar Torrentera et al., 2002)

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50 Consequently the virulence of the L. mexicana complex for most mouse strains must be a resul t of additional factors and cysteine peptidases expressed specifically by amastigotes have been identified as likely candidates (Mottram et al., 2004) Studies examining IL 4 IL (Satoskar et al., 1997) and IL 10 (Padigel et al., 2003a) deficient BALB/c mice infected with L. mexicana have indicated IL 4 to be the major contributor to nonhe aling disease phenotype with lesser though significant complementary roles for IL -13 and IL -10 Experiments utilizing severe combined immunodeficiency disease (SCID ) BALB/c mice reconstituted with IL 4, IL or wild -type splenocytes demonstrated th at a nonlymphocyte source of IL -4 could initiate lesion growth but only in the presence of lymphocytes able to respond to IL4/ IL 13 However, in the absence of IL 4 producing lymphocytes (SCID mice reconstituted with IL -4 splenocytes) lesions healed. T he Cathepsin-L like cysteine peptidase, CPB has been identified as the likely L. mexicana virulence factor inducing IL 4 (Alexander et al., 1998b; Pollock et al., 2003) While studies on L. mexicana clearly identified a major disease exacerbatory role for IL 4 in susceptible mice other than the BALB/c strain similar studies on the closely related parasite L. amazonensis suggested an insignificant role for IL -4 and signaling via STAT6 in non-healing infection (Jones, 2000) At the footpad site in C57BL/6 and C3H mice studies, a comparison of cysteine peptidases B ( CPB) deficient mutants and wild -type parasites has identified the cathepsinL like cys teine peptidase to be responsible for the inhibition of Th1 responses (Buxbaum et al., 2003) This could be directly related to the recently identified ability of wildtype but not CPB deficient amastigotes to pro signaling proteins in infected

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51 bone marrow derived macrophages (Cameron et al., 2004) Thus L. mexicana amastigote cysteine peptidases have been associated with both promoting IL 4 production and a Th2 response and inhibiting a Th1 response independently of IL -4 production. The relative importance of these different mechanisms of inducing progressive disease is probably in part mouse strain dependent. As the parasite cysteine p eptidases also degrade MHC Class II molecules within the parasitophorous vacuole (De Souza Leao et al., 1995; Prina et al., 1993) this probably compounds the inability of most mouse strai ns to resolve infection with parasites of this complex Non -curing visceral Leishmania sis and Th2 independent A disease exacerbatory role for IL 4 and the Th2 response during the course of L. donovani infection has yet to be demonstrated (Figure 1 -6B) Ear ly studies in both mice (Kaye et al., 1991) and humans (Kemp et al., 1993) suggested that cure was independent of the differential production of Th1 and Th2 cytokines. Consequent ly studies in B6/129 (Satoskar et al., 1995) and BALB/c mice have shown that IL -4 and IL-type counterparts (Stager et al., 2003a) (Stager et al., 2003b) Furthermore while cure in susceptible BALB/c mice is IL 12 dependent this cytokine was also demonstrated to promote the expansion of the Th2 as well as the Th 1 response (Stager et al., 2003a) (Stager et al., 2003a) In addition, not only is IL -4 and ILsignaling essential for optimal clearance of L. donovani from the liver and limiting infection in the spleen (Stager et al., 2003a) following primary infection but also for effective T cell dependent chem otherapy (Alexander et al., 2002) and vaccine induced resistance mediated by CD8+ T cells (Stager et al., 2003a) In the absence of IL -4 type 1 responses and IFN production fail to be maintained following chemotherapy or fail to be induced by

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52 vaccination. Studies in mice (Murphy et al., 2001) and humans (Kar p et al., 1993) indicate that IL -10 is the major immunosuppressive cytokine in visceral leishmaniasis although TGF om a latent form by a parasite c athepsin -B like cysteine peptidase (Somanna et al., 2002 ) has also significant disease promoting activity (Wilson et al., 1998) Phagocytosis and Leishmania Inert particle phagocytosis Phagocytosis (Figure 1 -7) is a process by which phagocytes engulf and degrade large (0.4the host immune systems. It begins when ligands on the foreign particles surface interact with host cell membrane receptors (mannose, complement, Fc -, integrins, phosphatidyl serine or Toll -like receptors). Some phagocytosis processes occur in a Zipper mode, where phagocytes develop pseudopod-like projections to surround and engulf the particle (Cossart and Sansonetti, 2004) The particle engulfment is only completed when late and recycling endosomes fuse to the PM through a process called focal exocytosis (Bajno et al., 2000) or direct fusion of endoplasmic reticulum to the PM at the site of nascent phagosomes (Gagnon et al., 2002) Finally, the particle is enclosed in a membrane bound vacuole called a phagosome. Alternatively, phagocytosis can involve mannose receptors, and the particle will sink into phagocytes without initiating pseudopod protrusions (Le Cabec et al., 2002) Interactions with phagocytic receptors trigger different signaling cascades (involving Rho -family GTPases, PI3Kinases, phospholipase C, Tyr and Ser/Thr kinases). These signaling cascades have been shown to influence downstream host cellular functions such as cytoskeleton rearrangement (actin or tubulin polymerization), release of cytokines, oxidative stress, and calcium mobilization. Many

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53 mechanisms related to this initial particl e host cell interaction (ligands/receptors, signaling cascades, cytoskeleton rearrangement, membrane fusion or fission) are still controversial or not fully characterized (Haas, 2007; Touret et al., 20 05b) ER -mediated phagocytosis By convention, plasma membrane and endosomes seem to be the only source of membrane for nascent phagosome during phagocytosis. However, recent studies point at the endoplasmic reticulum as an alternative source of internal m embranes for forming early phagosomes. In 2001, gene knock out experiments involving two markers of endoplasmic reticulum, calnexin and calreticulin, resulted in a significant impairment of the process of phagocytosis in Dictyostellum sp. organisms (Muller -Taubenberger et al., 2001) More interesting, proteomic analyses, in combination with electron microscopy and glucose-6 phosphatase cytochemistry, have led to the suggest ion that during particle engulfmen t the ER fuses with the plasma membrane at the base of the phagocytic cup (Gagnon et al., 2002) The establishment of continuity between these two membranes may establish a pathway whereby the target particle could "sl ide" into the lumen of the ER, and scission of the ER and resealing of the plasmalemma was envisaged to complete the phagocytic event (Gagnon et al., 2002; Gagnon et al., 2005) (Gagnon et al., 2002) A contribution of the ER to phagosome formation and maturation is attractive in several respects. First, as the largest single intracellular compartment, the ER can potentially provide enormous amounts of membranes t o satisfy the need for entrapment of multiple large particles. Second, by delivering foreign particles to the ER, this mode of phagocytosis could favor antigen cross -presentation (the presentation on class I

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54 histocompatibility complexes of antigens internalized by endocytosis). However, because antigens require proteasomal degradation before class I presentation, direct exposure to the ER lumen would not simplify the loading procedure; the antigens would purportedly be retrotranslocated across the ER membrane to the cytosol, where proteasomal degradation would ostensibly occur, followed by re uptake of the resulting peptides into the ER by the TAP transporter. However attractive, the notion that the phagosome is composed largely of ER derived membranes is i nconsistent with a plethora of earlier biochemical and immunostaining data and is seemingly incompatible with at least some of the established physical attributes of phagosomes. In dendritic cells and macrophages, the lumen of the nascent phagosome becomes acidic shortly after sealing. This acidification has been attributed to the inward pumping of protons by V -ATPases, which are thought to be acquired through fusion with endosomes. The acidification becomes more accentuated as phagosomes age, and this corr elates with the graded acidification of the compartments of the endocytic pathway that fuse sequentially with maturing phagosomes. It is difficult to envisage how acidification would develop in a phagosome composed largely of ER, which is believed to be de void of V -ATPases and is inherently permeable to protons (Paroutis et al., 2004) Moreover, results from another independent study employing a variety of quantitative methods, including biochemical, immunological, fluorescence imaging, and electron microsco py techniques, did not support the fusion of the ER with plasma membrane during phagocytosis or a contribution of the ER to phagosome maturation (Touret et al., 2005b) Instea d, the limiting membrane of phagosomes was confirmed to

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55 derive largely from the plasma membrane, becoming subsequently modified through a series of fusion reactions with sub-compartments of the endocytic pathway. I ndependent evidence in support of this mod el was provided by targeting the SNARE proteins of the ER. In one report, trapping antibodies to ERS24/Sec22b inside macrophages depressed the efficiency of phagocytosis (Becker et al., 2005) A related study re eval uated the role of ERS24/Sec22b and additionally analyzed two other SNARE proteins of the ER: syntaxin 18 and D12. Interference with the function of syntaxin 18 and D12 produced a modest reduction in the phagocytic activity, whereas impairment of ERS24/Sec2 2b had no discernible effect (Hatsuzawa et al., 2006) Altogether, it appears that phagocytosis is a complex process which involves a wide range of mechanisms that lead to the internalization of multiple particle s or particles as large as the size of the phagocyte. Although, these mechanisms are still controversial or not fully understood, more evidence suggests that phagocytosis requires additional intracellular membranes, which are contributed by host organelles such as endosomes or ER. Phagosome maturation After phagocytosis, nascent phagosome s interact with the host endocytic pathway in an orderly and sequential manner involving respectively early endosomes, sorting endosomes, late endosomes and lysosomes eith er by direct fusion (Figure 17) (Braun et al., 2004; Hackam et al., 1998) or fission in kiss and run type interactions (Desjardins et al., 1997; Desjardins, 1995; Desjardins et al., 1994) Through these interactions or exchange, the phagosome will acquire a membraneembedded protonpump adenosine triphosphatase (ATPase) complex, (vacuolar ATPase) that med iates acidification or lowering of the pH (4-5) of the phagosome lumen.

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56 The phagosome also acquires microbiocidal factors such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex to produce superoxide radicals from molecular oxygen, and a nitric oxide (NO) synthetase to make NO radicals from arginine. In addition, acidic hydrolases, lysozymes, defensin peptides and other nonoxidative tools will make the phagosome a biocidal milieu. Antigens generated from degraded particles or pathogens are presented by major histo-compatibility class II (MHCII) molecules at the surface of infected cells to prime or activate an efficient immune response against the invading entities. There is evidence that the port of entry used by the particle greatly influences the microbicidal pathways that are activated; f -receptors will stimulate an increased production of superoxide radicals, as compared to the involvement of complement or other receptors. Although still very controversial (Touret et al. 2005b) the transient interact ion of ER organelles with phagosome can also enable foreign antigens cross -presentation by MHC class I molecules (Gagnon et al., 2002; Garin et al., 2001) In genera l, microbiocidal and immunological factors make the mature phagosome also called phagolysosome, a very harsh environment or killing machinery for foreign particles or pathogens (Haas, 2007) To date, information about ph agocytosis and phagosome biology has come mainly from studies based on dead particles such as Zymosan, latex and magnetic beads because vacuoles containing live parasites are delicate and very hard to isolate. The results with inert particles cannot easil y be applied to live organisms because the interactions of live organisms with host cells are more dynamic and complex.

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57 Leishmania phagocytosis Phagocytosis of parasites appears to follow the same path as that of dead particle, but with an unexpected outc ome; at the end of the process, internalized parasites are generally not destroyed. In addition, phagocytosis of Leishmania also yields PVs with distinct morphological features. Leishmania parasites of the L. donovani complex ( L. donovani L. infantum L. chagasi ) tend to live in tight individual primary PVs and, after binary replication, daughter parasites form their own vacuoles called secondary PVs. Whereas strains responsible for cutaneous and diffuse cutaneous leishmaniasis of the L. mexicana complex ( L. mexicana, L. amazonensis L. pifanoi ) generally replicate and dwell in a communal PV that distends as the number of parasites increases (Kima, 2007; Chang et al., 2003; Ch ang, 1978) The mechanism of how Leishmania parasites control different features of their PVs biology such as the source of membranes to form primary and secondary PVs in mammalian cells are still poorly understood. Given the complexity of Leishmania par asites, it is not surprising that these pathogens have adapted a wide range of mechanisms including virulence factors, selection of specific host receptors, and use of Trojan horse to successfully enter and develop in macrophages (review in (Lodge and Descoteaux, 2005; Bogdan, 2008) Leishmania parasites express many specific molecules that would help them to complete their life cycle. The selection and use of these molecules also called virulence factors or pathogen associated molecules vary with the strain of parasites involved. An example of such molecules is the lipophosphoglycan, which enables Leishmania major and L. donovani but not L. mexicana (Ilg et al ., 2001) to resist the lytic action of the host complement system (Puentes et al., 1988)

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58 Leishmania parasites can also select a specific mode of cell entry as a survival strategy. They can engage the host cell mannose receptor and enter cells without -receptors will lead to their rapid clearance by host cell defense system. It has been reported that there is low production of superoxide and cytok ine during Leishmania sp infection (Pham et al., 2005; Olivier et al., 2005) Therefore Leishmania parasites have evolved yet to be fully uncovered mechanisms to silently enter host cell (Laskay et al., 2003) and evade/subvert the host immune system (Dogra et al., 2007; Olivier et al., 2005) Membranes lining Leishmania primary and secondary PVs Plasma membrane (PM), late endosomes and even ER seem to be the source of membranes lining the nascent phagosome (Braun et al., 2004; Hackam et al., 1998) PM components generally recycle back v ery quickly after phagocytosis. This might imply that host endocytic compartments (Desjardins et al., 1997; Desjardins, 1995; Desjardins et al., 1994) that interact w ith nascent phagosome are most likely the source of additional membranes for phagolysosomes. Such intracellular interactions usually occur within an hour or less after phagocytosis of dead particles. Live organisms such as Leishmania parasites, survive man y days after phagocytosis, increase the size of their PVs and/or form new secondary PVs. It is likely that Leishmania pathogens might have developed a more selective form of interactions with the host cell endocytic pathway during their maturation. Endoplasmic Reticulum Endoplasmic Reticulum Structure The endoplasmic reticulum (ER ) is a continuous network of interconnected tubules, cisternae, and highly organized lamellar sheets (Figure 18) These elements

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59 form ER building blocks including rough ( RER), sm ooth (SER), transitional (tER or exit sites), sinusoidal, crystalloid, sarcoplasmic reticulum (SR), karmellae, myeloid bodies, and the nuclear envelope (NE) (Figure 1 -9 ) (Lavoie and Paiement, 2008; Mullins and NetLibrary, 2005) Sometime, ER is simply identified morphologically by two subdomains: rough ER (rER) with a granular texture due to the presence of bound ribosomes, and smooth ER (SER) is deprive of these organelles and is often more convoluted than RER (Prinz et al., 2000) The ER can also be grouped in three distinct membrane regions: the nuclear envelope, the peripheral reticular ER and the peripheral ER sheets (English et al., 2009; Shibata et al., 2006) The nuclear envelope (nER) is stabilized by the interaction of inner nuclear membrane proteins with chromatin and the nuclear lamina The peripheral reticular ER (pER) is shape d by to threeway junctions between ER tubules (Farhan and Hauri, 2009) The peripheral ER network is a complex and highly dynamic structure. It was originally thought to be generated and maintained motor proteins and the cytoskeleton (Terasaki et al., 1986) in conjunction with cytoskeleton-linking membrane proteins (CLIMPs), such as CLIMP 63 (Klopfenstein et al., 1998) R ecent stud ies suggest the peripheral ER development is complex and required reticulons and DP1 / Yop1 family members which deform membranes by providing the curvature needed to form tubules (Voeltz et al., 2006; Dreier and Rapoport, 2000) Atlastin proteins mediate fusion of these tubules to create a network (Shibata et al., 2009) which would be stabilized and modeled by the cytoskeleton, motor proteins (Terasaki et al., 1986) and CLIMPs (Klopfenstein et al., 1998)

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60 Endoplasmic Reticulum Functions The ER has multiple functions including the synthesis of membrane lipids, membrane and secretory proteins, drug metabolism and the regulation of intracellular calcium These functions are distributed in distinct ER sub -regions or domains (Borgese et al., 2006) Most membrane proteins are shared betw een RER and SER, but several proteins involved in translocation or processing of newly synthesized proteins are enriched in RER (Vogel et al., 1990; Amar -Costesec et al., 1989; Kreibich et al., 1978) Endoplasmic reticulum and ERAD Secretory and membrane proteins which constitute about one-third of all cellular proteins, are folded in the ER ER is equipped with a rigorous quality control system (Figure 1 9) which c an differentiate between the correctly folded proteins and the misfolded or unfolded proteins (Ellgaard and Helenius, 2003) P roteins that can not be folded properly in the ER are processed through ER associated de gradation (ERAD), transported from the ER into the cytosol, and subsequently ubiquitinated and degraded by the proteasome (Vembar and Brodsky, 2008) The accum ulation of misfolded proteins in the ER activates the u nfolded protein response (UPR), which induces the expression of molecular chaperones and ERAD components This results in an increase ER capacity to fold and clear of accumulating misfolded polypeptides (Malhotr a and Kaufman, 2007) The ER -Golgi intermediate compartment (ERGIC) In mammalian cells, the secretion pathway (Figure 1-11) is essential and this process starts with molecules traffick ing from the endoplasmic reticulum (ER) to the Golgi complex through th e tubulovesicular membrane clusters of the ER -Golgi

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61 intermediate compartment (ERGIC ) (Appenzeller -Herzog and Hauri, 2006) The exchange of proteins and membrane between ER and Golgi in eukaryotic cells is directed by COP I and COP II coat proteins. These coat proteins are essential in both selecting proper cargo proteins and deforming the lipid bilayer of appropriate donor membranes into buds and vesicles. COP II proteins are required for selective export o f newly synthesized proteins from the endoplasmic reticulum (ER). COP I proteins mediate a retrograde transport pathway that selectively recycles proteins from the cis Golgi complex to the ER. COP I coat proteins also have complex functions in intra -Golgi trafficking and in maintaining the normal structure of the mammalian interphase Golgi complex (Budnik and Stephens, 2009) Studies involving the transport of a temperature -sensitive mutant G protein from vesicular st omatitis virus (tsO45 -VSV-G) and the E1 glycoprotein of Semliki forest virus were to demonstrate that ERGIC 53 -positive compartment is a distinctive intermediates in ER -to -Golgi protein transport (Saraste and Svensson, 1991; Schweizer et al., 1990) The protein composition of ERGIC membranes differs from that of the neighboring ER and Golgi (Schweizer et al., 1991) ; serial sectioning and three-dimensio nal ultrastructure reconstruction and other analysis of pancreatic acinar cells confirmed the ERGIC as an independent structure, which is not continuous with the ER or the cis Golgi (Klumperman et al., 1998; Bannykh et al., 1996; Sesso et al., 1994) ER -Golgi transport and SNAREs Structure of SNAREs. SNAREs (soluble NSF attachment protein receptors) (Figure 1 11) are a superfamily of proteins that function in all membr ane fusion steps of the secretory pathway within eukaryotic cells (Weber et al., 1998; Sollner et al., 1993) Their

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62 core structure is characterized by an evolutionary conserved stretch of 60 70 amino acids containing eight heptad repeats, also known as SNARE motif (Brunger, 2005) The number of different SNAREs varies between different organisms, ranging from 25 in yeast, 36 in mammals, to over 50 in plants. Each fusion step requires a specific set of four different SNARE motifs that is contributed by three or four different SNAREs, and each of the membranes destined to fuse contains at least one SNARE with a membrane anchor (Lang and Jahn, 2008) Membrane traffic usually consists of a sequence of steps involving the generation of a transport vesicle by budding from a precursor compartment, the transport of the vesicle to its destination, and finally the docking and fu sion of the vesicle with the target compartment (Figure 1-10) SNAREs operate in the very last step of this sequence (Jahn and Scheller, 2006) Classification of SNARES SNAREs were originally classified functionally into t SNAREs (target membrane SNAREs) or v -SNAREs (vesicle membrane SNAREs ). While the v and t SNARE nomenclature appropriately describes SNAREs that function in the exocytic pathway, this distinction does not fit well with SNAREs involved in fusion betw e en internal membranes, and even homotypic fusion (Wickner, 2002; Fukuda et al., 2000) Therefore, another way of grouping SNAREs is suggested, and it is based on ionic layer found in the neuronal SNARE complex (Sutton et al., 1998) This region of the canonical coiled coil sequence contains polar or charged residues in place of hydrophobic residues, which are either a glutamine (Q) for Q -SNAREs or an arginine (R) residue for R SNARES. According to the position of the SNARE motif in the structurally conserved SNARE complex, t he Q -SNAREs are further subdivided into Qa

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63 (Syntaxin1a), Qb, and Qc (Kloepper et al., 2007; Bock et al., 2001; Fasshauer et al., 1998) However, t he Q SNARE and R -SNARE designation is not also universal because SNARE complexes such as the yeast ER -Golgi v -SNARE Bet1p has neither a Q nor an R, but an S in the relevant location. Vti1b contains an aspartic acid (D) residue in this location (Zwilling et al., 2007) SNAREs involved in ER -Golgi transport. Several SNARE co mplexes have been defined to function in various transport events in the secretory and/or endocytic pathways of mammalian cells (Hong, 2005) The complex consisting of Syn5 (Qa), GS27 (Qb), Bet1 (Qc), and Sec22b (R) ap pears to function in mediating homotypic fusion of ER -derived COPII vesicles into the ERGIC ( ER -Golgi intermediate c ompartment) also known as VTC (vesicular tubular cluster) (Zhang et al., 1997) Based on systematic analysis of yeast SNAREs (Parlati et al., 2000) a similar SNARE complex is found in yeast Since yeast Bet1p is the functional v -SNARE, Bet1 is likely the v SNARE, while Syn5, GS27 and Sec22b may form the t SNARE, although this remains to be experimentally investigated. EGTCs are dynamic structures that undergo maturation events (including recycling of proteins back to the ER ) as they move towards the Golgi apparatus (Horstm ann et al., 2002) The SNARE complex consisting of Syn5 (Qa), GS28 (Qb), Bet1 (Qc), and Ykt6 (R) is suggested to act in the late stage of transport from the ER to the Golgi and is likely to mediate the fusion of matured EGTCs with the cis -face of the Golg i apparatus (Horstmann et al., 2002) A likely possibility is that Bet1 stays on the EGTC and acts as the v -SNARE responsible for interaction with

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64 another t -SNARE assembled from the same heavy chain (Syn5) but two different light chains ( GS28 and Ykt6 ) at the cis -Golgi (Hong, 2005) COPI coat proteins mediated the retrograde transport between ER and Golgi, which help to maintain a dynamic balance of membrane traffic in the ear ly part of the secretory pathway The ER SNARE complex consisting of Syn18 (Qa), Sec20 (Qb equivalent ), Slt1 / Use1/p31 (Qc), and Sec22b (R) may serve as target or t -SNAREs during fusion of retrograde transport vesicles with ER (Hatsuzawa, 2004; Dilcher et al., 2003; Burri et al., 2003; Nakajima et al., 2004) The SNARE complex consisting of Syn5 (Qa), GS28 (Qb), GS15 (Qc), and Ykt6 (R) functions in intra-Golgi traffic and a similar complex is also found in yeast Based on analysis in yeast GS15 acts as the v -SNARE, interacting with t -SNARE assembled from Syn5, GS28, and Ykt6 (Parlati et al., 2002) In addition, a recent study suggests that this SNARE complex also mediates traffic from the endosomal compartments to the Golgi apparatus (Tai et al., 2004) Ricin Trafficking in Eukaryotic Cells and t he E R Ricin is a type II rib osome inactivating protein (RIP ) found in castor beans which are the seeds of the castor plant Ricinus communis Due to its high toxicity and ready availability, ricin has been listed as a Category B Select Agent by the National Institutes of Health and the Centers for Disease Control and Prevention (Audi et al., 2005) Ricin is a heterodimeric glycoprotein composed of a catalytically active 32 kDa A -chain (RTA) linked by a disulfide bond to a 34 kDa B -chain (RTB), a ga lactoseand N acetylgalactosamine -specific lectin The molecule enters the cells through endocytosis and undergoes retrograde translocation to the Golgi apparatus / endoplasmic reticulum Protein disulphide -isomerase reduces ricin to its A and B chains in t he endoplasmic

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65 reticulum (Figure 1-13) () After dissociation, a portion of the RTA reaches the cytosol where it inactivates ribosomes by depurinating a single adenine nucleotide (Spooner et al., 2006; Marsden et al., 2005) This depurination prevents binding of elongation factors, which leads to the inhibition of protein synthesis However, recent evidence in yeast suggests that ribosome depurination may not by itself cause cell death (Li et al., 2007) Ricin also activates stressactivated protein kinase (SAPK) signaling pathways that are induced by ribosome damage (ribotoxic stress) (Iord anov et al., 1997) and induces apoptosis (Rao et al., 2005; Wu et al., 2004; Higuchi et al., 2003) (Bhaskar et al., 2005) However, the role of SAPK pathways in ricin -induced apoptosis has not been well delineated. W hile the RTB subunit is thought to enhance the entry of ricin into cells, several studies have shown that the RTA subunit can enter the cell on its own and induce cytotoxicity (Vago et al., 2005; Svinth et al., 1998; Wales et al., 1993; Casellas et al., 1984)

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66 Figure 11. World map of leishmaniasis This map highlights areas where cutaneous, visceral, and mucocutaneous Lei shmaniasis are endemic (Adapted from (Handman, 2001) ).

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67 Figure 12. Life cycle of Leishmania parasites. Leishmaniasis is transmitted by the bite of infected female phlebotomine sandflies. The sandflies inject the infective stage (i.e., promastigotes) from their proboscis during blood meals Promastigotes that reach the puncture wound are phagocy tized by macrophages and other types of mononuclear phagocytic cells. Promastigotes transform in these cells into the tissue stage of the p arasite (i.e., amastigotes) which multiply by simple division and proceed to infect other mononuclear phagocytic cells Parasite, host, and other factors affect whether the infection becomes symptomatic and whether cutaneous or visceral leishmaniasis results. Sandflies becom e infected by ingesting infected cells during blood meals ( ). In sandflies, amastigotes transform into promastigotes, develop in the gut (in the hindgut for Leis hmania organisms in the Viannia subgenus; in the midgut for organisms in the Leishmania subgenus), and migrate to the proboscis

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68 Figure 13. Schematic representation of the main intracellular organelles from Leishmania promastigote (left, A) or amastigote (right, B) forms. The flagellar pocket marks the anterior end of the cell (Adapted from (Besteiro et al., 2007) ) Figure 14. Clinical symptoms of leishmaniasis A Cutaneous leishmaniasis B Diffuse cutaneous leishmaniasis C Mucocutaneous leishmaniasis D Visceral leishmaniasis and E Post kala azar (Murray et al., 2005; Chappuis et al., 2007; Perez et al., 2006) ; http://bearspace.baylor.edu/Charles_Kemp/www/ leishmaniasis .htm) A D E B C

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69 Figure 15 Leishmania taxonomy (adapted from (Ashford, 2000; Handman et al., 2000) )

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70 A Fig ure 16. Leishmania major : healing and non -healing immunological res ponses. During a blood meal an infected sandfly transmits metacyclic promastigotes to the vertebrate host, which convert to the amastigote form on entering macrophages and dendritic cells. IL12 production from infected cells induces NK cell activation and CD4+ T helper 1 differentiation and IFN IFN which mediates parasite killing and therefore a healing response. Failure to produce IL -12 or alternatively IL-4/IL 13 production r esults in unregulated parasite replication within the infected cells facilitated by host cell IL10 production. IL10 production by CD4+ CD25+ T regulatory cells can both facilitate nonhealing disease as well as maintaining latent infection and concomitan t immunity. Ingestion of infected cells by a sandfly during a blood meal initiates the life cycle of this protozoan parasite within the insect vector (Adapted from (Alexander and Bryson, 2005) ).

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71 B Figure 16 Continued Macro phage metabolism and intracellular growth of Leishmania amastigotes. Leishmania infection of healing or non-healing mice is associated with a transient or prolonged TH2 response, respectively, and the alternative activation of macrophages (green panel). This activation state can be induced by cytokines (IL4/IL-13 and IL-10) that predominate in nonhealing mice or the presence of apoptotic host cells (macrophages, neutrophils) and the uptake of Leishmania amastigotes expressing surface phosphatidylserine. A lternative activation results in the upregulation of endocytic activity, mitochondrial respiration and arginase-1, that increase levels of amino acids and polyamines required for amastigote growth. Conversely, parasite growth can be controlled by an IL12 driven TH1 response and classic activation of infected macrophages with IFN costimulatory molecules (red panel). Classic activation results in the upregulation of glycolysis, NO synthesis, and down-regulation of arginase 1 activity. Enzymes inv olved in depleting other amino acids may also be upregulated, restricting growth of amastigotes and increasing their sensitivity to oxidative stress. NOHA, NGhydroxy l arginine methyl ester; IDO, indoleamine 2,3dioxygenase (Adapted from (Naderer et al., 2008) ).

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72 Figure 17. Schematic simplified view of the endocytic (phagocytic) pathway. Maturation along the degradative pathway is indicated by red and the recycling pathway by blue arrows. Endosomes mature as they consecutively undergo fusion with membranes of the next stage of maturation, followed by fission of recycling vesicles whose identity is largely unknown (small, colored vesicles with black arrows in backward direction). In macr ophages, sametype endocytic organelles fuse particularly avidly with each other (doublesided black arrows). Traffic between early and late endosomes may be predominately accomplished by a vesicle (202). These are vesicles with multiple internal membranes that contain transmembrane proteins destined for degradation. Microorganism would not fit into such comparably small vesicles. It is more likely that early phagosomes fuse directly with late endosomes to form late phagosomes. Most of the killing and digestion is accomplished in a late phagosome and in phagolysosomes. Antigen presentation through MHC class II occurs predominately from a late phagosome compartment (not included here). Vesicles with biosynthetic cargo from the TGN can fuse with early and lat e endosomes. Times indicate the approximate periods of time required for a particle to appear in the respective compartment. For example phagolysosomes can normally be observed starting 15 min after ingestion by macrophages, while most phagosomes have matu red into phagolysosomes by 60 min of infection (times can vary between macrophage type and activation status). Other endocytic compartments may exist (20), but this simplified four compartment view has proven valuable in the discussion of most features seen. CGN, cis Golgi network; EEA1, early endosome antigen 1; LE, late endosomes; M6PR, mannose 6 phosphate receptor; MO, microorganism; MVB, multivesicular body; I(3)P, phosphatidylinositol 3 phosphate; RE, recycling endosomes; SE, sorting endosomes; TGN, t rans Golgi network. (Adapted from (Haas, 2007) ).

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73 Figure 18 Diff erent sub -compartments of the endoplasmic reticulum. The ER is composed of continuous but distinct subdomains. a The nuclear envelope (NE) is shown wi th nuclear pores and ribosomal particles attached to the outer membrane. b The rough ER (rER) is continuous with the NE and consists of stacked Xattened saccules, whose limiting membranes have numerous attached ribosomal particles. c Transitional ER (tER) is composed of a rER subdomain continuous with the rER and a smooth ER (sER) subdomain consisting of buds and tubules devoid of associated ribosomes (arrowhead points to a coated bud). d In some cells (e.g., steroid secreting cells and hepatocytes) the sER is composed of a large network of interconnecting tubules showing tripartite junctions ( arrows ) and fenestrations (Adapted from (Lavoie and Paiement, 2008) ).

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74 Figure 19 Schematic representation of the endoplasmic reticulum quality control system. Protein trafficking from the ER: upon translocation of polypeptides through the Sec61 proteinaceous channel, asparagine residues are frequently modified by covalent addition of a preassembled oligosaccharide core ( N acetylglucosamine2mannose9glucose3). This reaction is catalyzed by the oligosaccharyltransferase (OST). To facilitate unidirect ional transport through the translocon, nascent polypeptide chains in the ER lumen interact with BiP, a molecular chaperone that binds to exposed hydrophobic residues. Subsequently, rapid deglucosylation of the two outermost glucose residues on the oligosaccharide core structures, mediated by glucosidase I and II (GlcI and GlcII), prepares glycoproteins for association with the ER lectins calnexin and calreticulin. The calnexin/calreticulin associated oxidoreductase ERp57 facilitates protein folding by catalyzing formation of intra and inter molecular disulfide bonds, a ratelimiting step in the protein folding process. Release from calnexin/calreticulin (Cnx/Crt) followed by glucosidase II cleavage of the innermost glucose residue prevents further interact ion with calnexin and calreticulin. At this point, natively folded polypeptides transit the ER to the Golgi compartment, in a process possibly assisted by mannosebinding lectins, such as ERGIC 53, VIPL, ERGL. As an essential component of protein folding q uality control, non native polypeptides are tagged for reassociation with Cnx/Crt by the UDP glucose:glycoprotein glucosyltransferase (UGT1) to facilitate their ER retention and prevent anterograde transport. Polypeptides that are folding incompetent are t argeted for degradation by retrotranslocation, possibly mediated by EDEM and Derlins, into the cytosol and delivery to the 26S proteosome. Triangles represent glucose residues, squares represent N acetylglucosamine residues, and circles represent mannose r esidues (Adapted from (Malhotra and Kaufman, 2007) ).

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75 Figure 110 Distinct membrane trafficking steps that can be controlled by a Rab GTPase and its effectors (indicated in orange). a) -An active GTP bound Rab can activate a sorting adaptor to sort a receptor into a budding vesicle. b )Through recruitment of phosphoinositide (PI) kinases or phosphatases, the PI composition of a transport vesicle might be altered (the conversion of PI x into PI y) and thereby cause uncoating through the dissociation of PI -binding coat proteins. c) -Rab GTPases can mediate vesicle transport along actin filaments or microtubules (cytoskeletal tracts) by recruiting motor adaptors or by binding directly to m otors (not shown). d | Rab GTPases can mediate vesicle tethering by recruiting rod -shaped tethering factors that interact with molecules in the acceptor membrane. Such factors might interact with SNAREs and their regulators to activate SNARE complex format ion, which results in membrane fusion. e | Following membrane fusion and exocytosis, the Rab GTPase is converted to its inactive GDP -bound form through hydrolysis of GTP, which is stimulated by a GTPase activating protein (GAP). Targeting of the Rab GDP di ssociation inhibitor (GDI) complex back to the donor membrane is mediated by interaction with a membrane-bound GDI displacement factor (GDF). Conversion of the GDP bound Rab into the GTP bound form is catalyzed by a guanine nucleotide exchange factor (GEF) (Adapted from Stenmark, 2009)

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76 Figure 11 1 Structure of the (neuronal) SNAREs. Upper panel: domain structure of the three neuronal SNARE proteins involved in synaptic vesicle fusion. Syntaxin 1A and SNAP -25 (contains two SNAR E motifs) are associated with the presynaptic membrane, whereas synaptobrevin 2 is synaptic vesicle associated. The SNARE motifs form a stable complex (core complex) whose crystal structure has been analyzed (lower panel). In the complex, each of the SNARE motifs adopts an alpha-helical structure, and the four alpha helices are aligned in parallel forming a twisted bundle (modified from Sutton et al. 1998). Stability of the complex is mediated by layers of interaction ( 7 to +8) in which amino acids from each of the four alpha helices participate (Adapted from (Lang and Jahn, 2008) ).

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77 Figure 11 2 The SNARE conformational cycle during vesicle docking and fusion. As an example, we consider three Q -SNAREs (Q -soluble N ethylmaleimidesensitive factor attachment protein receptors) on an acceptor membrane and an R SNARE on a vesicle. Q -SNAREs, which are organized in clusters (top left), assemble into acceptor c omplexes, and this assembly process might require SM (Sec1/Munc18-related) proteins. Acceptor complexes interact with the vesicular R -SNAREs through the N terminal end of the SNARE motifs, and this nucleates the formation of a four helical trans -complex. T rans complexes proceed from a loose state (in which only the N terminal portion of the SNARE motifs are 'zipped up') to a tight state (in which the zippering process is mostly completed), and this is followed by the opening of the fusion pore. In regulated exocytosis, these transition states are controlled by late regulatory proteins that include complexins (small proteins that bind to the surface of SNARE complexes) and synaptotagmin (which is activated by an influx of calcium). During fusion, the strained trans -complex relaxes into a cis configuration. Cis -complexes are disassembled by the AAA+ (ATPases associated with various cellular activities) protein NSF ( N ethylmaleimide sensitive factor) together with SNAPs (soluble NSF attachment proteins) that function as cofactors. The R and Q -SNAREs are then separated by sorting (for example, by endocytosis).(Adapted from (Jahn and Scheller, 2006) ).

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78 Figure 113. P athway of ricin uptake by cells and the mechanism toxic activity of the A chain in the cytoplasm results in cell death. (Adapted from the Consortium of Glycobiology Editors, La Jolla,;California http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=glyco2&part=ch)

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79 Project Rational and Design Within the past decade an increasing number of studies have proposed that the endoplasmic reticulum (ER) is also a key participant in phago cytosis of large particles and in the uptake of microorganisms by phagocytes. In 2001 Muller -Taubenberger et al (Muller -Taubenberger et al., 2001) showed that the knockout of genes that encode two ER prot eins (calnexin and calreticulin) severely inhibits inert particles phagocytosis in Dictyostelium sp. During the same year, proteomic analyses revealed that purified latex bead phagosomes contained ER resident molecules such as calnexin, calreticulin and GR p78 (Garin et al., 2001) Proteomic analysis of phagosomes combined with confocal microscopy and electron microscopy (EM) studies later showed a direct association between ER and PM during early phagocytosis (Gagnon et al., 2002) (Garin et al., 2001) These authors then proposed that ER -mediated phagocytosis is an alternative mechanism employed by phagocytes to internalize large particles and micr oorganisms without significant depletion of their surface area (Gagnon et al., 2002) Further confirmation of the participation of ER in phagocytosis has been obtained in gene silencing and overexpression studies invol ving the soluble N ethylmaleimide -sensitive factor attachment protein receptors (SNAREs) syntaxin 18 (Hatsuzawa et al., 2006) and Sec22b (Becker et al., 2005) Both of these ER associated SNAREs were shown to regulate fusion between ER and PM during phagocytosis. However, a study on quantitative and dynamic assessment of ER contributions to latex bead phagosome biogenesis (Touret et al., 2005b) confirmed the presence of PM molecules at the phagosomal membrane, but could not confirm ER mediated

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80 phagocytosis of inert particles as well as live organisms such as Leishmania in macrophages. This notwithstanding, there have been several observations that have suggested that Leishmania parasitophorous vacuoles (PVs), which are formed after phagocytosis of Leishmania parasites, have interactions with the host ER. This evidence includes the fact that PVs that harbor Leishmania parasites display ER molecules (Garin et al., 2001; Gueirard et al., 2008; Kima and Dunn, 2005) ; in addition, it has been shown that Leishmania-derived molecules can access the MHC class I pathway o f presentation through a transporter associated with antigen processing (TAP) -independent mechanism (Bertholet et al., 2006) which implies that parasite -derived peptides in PVs have direct access to MHC class I mo lecules. Leishmania parasites reside in PVs with different morphologies: parasites of the Leishmania mexicana complex ( L. mexicana, Leishmania pifanoi and Leishmania amazonensis ) reside in communal PVs that continuously enlarge as the parasites replicate. In contrast, parasites of the Leishmania donovani complex ( L. donovani Leishmania infantum ) reside for the most part in individual compartments from which daughter parasites segregate into new compartments or secondary PVs after parasite replication. Alth ough the involvement of endocytic compartments including lysosomes in the biogenesis of Leishmania PVs is well established (Courret et al., 2002) the extent of the interactions of the host cell's ER with nascent and secondary PV has not been assessed. In this study the interactions between the ER and nascent and maturing PVs in macrophages was assessed. Murine Raw246.7 macrophages either transiently or

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81 stably transfected with representative green fluorescence protei n (GFP)or YFP tagged ER markers (calnexin and Sec22b, D12 and Syntaxin18) were infected; the resultant PVs were monitored for their recruitment of these molecules over the infection course both by fluorescence and Immuno -Electron microscopy. We also exploited the trafficking of ricin, a toxin that accesses the cytosol through a retrograde pathway that traverses the ER (Audi et al., 2005) to further characterize host ER and PVs association. Finally, we employed dominant negative constructs from ER -membrane associated SNAREs (Sec22b, D12, and Syntaxin18) that lack the transmembrane domain, to assess the consequences of host ER interaction on Leishmania replication and PVs development in macrophages.

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82 CHAPTER 2 MATERIAL AN D METHODS Material Chemicals and Reagents Most of the chemicals were purchased from Sigma Aldrich (St. Louis, MO) and Fisher Scientific (Pittsburg, PA). BrefeldinA, Prolong antifade mounting medium were purchased from Invitrogen Inc (Carlsbad, CA). DNA res triction endonuclease and 1kb DNA ladder were purchased from New England Biolabs, Inc (Ipswich, MA). Desalted oligonucleotides were from Integrated DNA Technologies (Coralville, IA). The fluorescein labeled Ricinus communis agglutinin II (RCA II) was purch ased from Vector Laboratories Inc (Burlingame, CA) Co mmonly use buffers and media : DNA Loading Dye (10X) 0.25% Bromophenol Blue, 0.25% Xylene Cyanol, 0.1M LB Broth10g Bacto Tryptone, 5g Yeast Extract, 10g NaCl. TAE40mM Tris -Acetate, 1mM EDTA (pH 8.0). TE 10mM Tris, 39mM Glycine, 15% Methanol. Biochemical Kits Plasmid extraction, DNA gel extraction and RNeasy kit s were purchased from Qiagen Inc (Valencia, CA). The Fast Link DNA Ligation Kit was purchased from Epicentre Biotechnologies ( Madison, WI). The n ucleofection kit was purchased from Amaxa Inc. (Gaithersburg, MD). Plasmids pShooter vector pCMV/ER/GFP/c -myc vector (Invitrogen Inc; Carlsbad, CA). The pmVenus, pmVenus/Sec22b, pmVenus/syntaxin 18, pmVenus/D12, -tm -Sec22b/RFP, -tm -Synatxin/RFP and -tm -D12/RFP were generously provided to us by Dr. Kiyotaka Hatsuzawa at the Fukushima Medical University, Japan)

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83 Antibodies Early endosome antigen 1 (EEA1), calnexin, GM130 (Santa Cruz Biotechnology, San Jose CA), BiP, (BD Biosciences, San Jos CA); the rat anti late endosome and lysosomal associated membrane proteins, 1D4B, was obtained from the Developmental Studies Hybridoma bank, Iowa City, IA. Cell Lines and Maintenance Parasites The Leishmania pifanoi promastigotes (MHOM/VE/57/LL1) line was obtained from the American Type Culture Collection (ATCC)( Manassas, VA ). It was grown in Schneiders medium supplemented with 10% fetal calf serum and 10 g/ml gentamicin at 23C. L. donovani strain 1S -CL2D from Sudan, World Health Organization (WHO) designation: ( MHOM/SD/62/1S -CL2D) was obtained from Dr. Debrabant (USDA, MD). Promastigotes of this parasite strain were grown in Medium 199 (with Hank's salts, Gibco Invitrogen Corp.) supplemented to a final concentration of 2 mM L-glutamine, 100 lic acid, 100 streptomycin, respectively, 1BME vitamin mix, 25 mM Hepes, and 10% (v/v) heat inactivated (45 min at 56 C) fetal bovine serum, adjusted with 1 N HCl to pH 6.8 at 26 C. Generation of amastigotes for ms was carried out as described (Debrabant et al., 2004) L. donovani axenic amastigotes parasites were maintained in RPMI 640/MES/pH 5.5 medium at 37 C in a humidified atmosphere containing 5.5% CO2 in air. Simila rly, L. pifanoi amastigotes were maintained in the amastigote medium above at 34 C. Macrophages The RAW 264.7 murine macrophage cell line (obtained from ATCC) was cultured in RPMI supplemented with 10 % fetal calf serum and 100 units Penicillin/Streptomyc in

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84 at 37C under a 5% CO2 atmosphere. G418 sulfate antibiotics at a final concentration of 1mg/ml were added to the complete growth medium of Raw 264.7 cells transfected with either pCMV/ER/GFP/c -myc or pCMV/ER/GFP -calnexin vector. Culture medium of Raw 26 4.7 or J774 cells expressing pmVenus/YFP or RFP based DNA constructs (Sec22b, syntaxin, and D12) was supplemented with puromycin antibiotics at a final concentration of 2ug/ml, as previously recommended by (Hatsuzawa et al., 2006; Hatsuzawa et al., 2009) These cells were routinely maintained every 2 to 3 days as follow, cells were dislodged from the bottom surface of a 75 cm2 culture flask or other culture vessels with a sterile cell scraper. An appropriate volume of cell suspension was transferred into a new culture vessel containing a fresh culture medium at a sub -cultivation ratio of 1:1 to 1:10. For long-term storage, these cells were frozen in complete growth medium supplemented with 5% (v/v) DMSO and stored at -80 C, as recommended by the provider. Bacteria routine cloning. Bacteria were grown on LB agar plates or LB broth. Media of transformed bacteria were supplemented with antibiotics at a final concentration of 50ug/ml (for kanamycin) and 100ug/ml (for ampicillin) depending of the resistant or selective gene on the plasmid of interest. Bacterial cultures were stored at 4C for up to a week in LB broth or a m onth on LB/agar plates. For a longbacterial culture was rapidly frozen in liquid nitrogen for 30 seconds, and stored at 80C.

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85 Molecular Cloning RNA Extraction and Purification Total RNA was extracted from murine Raw246.7 macrophages using the RNeasy Mini kit for animal cells (Qiagen). A volume of overnight fresh culture containing approximately 1 x 107 Raw246.7 cells was centrifuged at 300 g for 5 min. To lyse the -cooled (4C) Buffer RLN and incubate on ice for 5 min. Centrifuge the lysate at 4C for 2 min at 300 g Transfer the supernatant to a new centrifuge tube, and discard the pellet. Add the homogenized lysate, and mix well by pipetting. Transfer the sample to the RNeasy spin column placed in a 2 ml collection tube, and centrifuge at 1 3,000 rpm for 15 s. -Free DNase I solution to the column membrane and incubate at RT for 15 min. Wash the column by scard the flow -through. Buffer RPE (centrifuge for 2 min). Centrifuge again the RNeasy spin column in a new 2 ml collection tube for 1 min, to eliminate any possible carryover of Buffer RP E. To elute RNase-free water directly to the spin column membrane, and centrifuge for 1 min at 13000 rpm. The concentration of the total RNA was determined from A260nm. Quality of RNA was determined by 0.8 % (w/v) agarose gel electrophoresis in 1 TAE buffer after heating the RNA samples to 50 -70 C until further use.

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86 DNA Extraction and Purification One -step RT -PCR and DNA a mplification A DNA fragment containing the calnexin gene was directly amplified from purified total RNA using specific primer sequences (Forward: 5 AGC G GAT CC G GGA GGC TCG AGA TAG ATC ATG GA 3and Reverse: 5 AGC G GAT CC G GGA GGC TCG AGA TAG ATC ATG GA 3 ) and following a one-step RT -PCR protocol from Qiagen Inc. PCR and calnexin gene amplification The Calnexin gene was amplified from the DNA template obtained from the total RNA sample. Specific primer sequences containing a NotI restriction enzyme site (Forward: 5 AAG GAA AAA A GCG GCC GC C ATG ATG GAC ATG ATG ATG ACG 3 and Reverse: 5 AAG GAA AAA AA GCG GCC GC T CAC TCT CTT CGT GGC TTT CTG 3) Agarose gel electrophoresis Sizes of PCR products and plasmids were analyzed by electrophoresis using 0.7 1.2 % (w/v) agarose gels in TAE buffer (40 mM Tris acetate, 2 mM EDTA, pH 8.5) with 1kb DNA ladder (from New England Biolabs Inc) as the DNA molecular weight standard. The gels were stained with ethidium bromide at gels was captured with the Kodak Gel Logic 200 Imaging System, and the process with Carestream Molecular imaging software purchased from Carestream Health, Inc (Rochester, NY). Mini-prep plasmid DNA isolation Plasmids were iso lated by Qiagen Miniprep kit according to Manufacturers protocols (Qiagen Inc., Valencia, CA). When applicable, plasmids were purified from agarose slices by QIAquick gel extraction kit (Qiagen). The alkali lysis method of

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87 plasmid isolation was performed as previously described (Sambrook et al. 1989). Briefly, bacteria containing the desired plasmid were inoculated into 2ml of LB broth with the appropriate selectable drug and grown overnight with shaking at 37C. The following day, 1.5ml of the culture wa s pelleted by centrifugation at 10,000 rpm for 30 seconds. freshly prepared lysis buffer (0.2N NaOH, 1% SDS) and incubated at 24C for 5 mins. Following the incubation the lysed mixture, which was then mixed gently and incubated on wet ice for 5 mins. The mixture was then centrifuged at 4C for 15 mins. The supernatant was transferred to an empty microfuge tube to which 0.7 volumes of isopropanol was added, mixed and centrifuged for a further 15 mins at 4C. The pellet was air dried and resuspended in C for 30 mins. The plasmid preparation was then extracted s equentially with Tris -saturated Phenol (pH 8.0) and Chloroform. The aqueous phase was precipitated with equal volumes of 7.5M ammonium acetate and 2.5 volumes of 95% ethanol. The pellet was washed with 70% ethanol, air O. Endotoxinfree maxi -prep DNA isolation To generate a large-scale DNA production, 250 ml of fresh overnight culture of DH-5 cells transformed with a plasmid of interest was processed using the Endo-Free Plasmid Maxi Kit (Qiagen Inc. Chatsworth, CA) was used to generate a large amount of endotoxin -free and high purity DNA constructs for mammalian cell transfection. Kit ale plasmid isolations were performed using the Qiagen Maxi -Prep Kit Isolation of plasmid DNA from 250ml cultures was carried out accord ing to the manufacturers instructions.

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88 Plasmid DNA was purified using an anionexchange column. The purified DNA was stored at 20 C until further use. DNA Cloning Restriction enzyme digestion of DNA (vector and insert) The pCMV/ER/GFP/c -myc vector and th e calnexin gene were digested with NotI restriction enzyme purchased from New England Biolabs. digestion of plasmid DNA or C for 2 hours in the provided buffers from the manuf acturer. Dephosphorylation of digested DNA Dephosphorylation of digested plasmids was carried out to prevent subsequent religation of the plasmid and to facilitate sub -cloning of restriction digested fragments. Dephosphorylation of plasmid DNA performed b y digesting 1pico molar termini of DNA with 1 unit Shrimp Alkaline Phosphatase (Fermentas Inc; Glen Burnie, MD) at 37C for 30 min. To stop the reaction, the sample was incubated at 70C for 15 min, and chilled on ice for at 5min before usage or storage at 20C. DNA ligation Both insert and plasmid DNA digested with endonucleases was purified using a Gel extraction and purification Kit (Qiagen Inc.). Their concentration and purity were assessed using a NanoDrop spectrophotometer. The ligation reaction at t he ratio of 1:3 ( vector:insert ) was performed using the Fast Link DNA ligation kit purchased from Epicentre Biotechnologies Inc (Madison, WI). 1.5 ml 10X Fast -Link Ligation Buffer, 1.5 ml 10 mM ATP, x ml vector DNA (1); x ml insert DNA (2); x ml sterile wa ter to a volume of 14 ml 1 ml Fast -Link DNA Ligase. 2. Incubate the reaction at least 30 min. The reaction was transferred to 70oC for 15 minutes to inactivate the Fast -Link DNA ligase.

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89 After inactivation, the sample was chill on ice for at least 5min, an d centrifuge at 13000 for 15 seconds. DH 5 electro-competent cells were transformed with 1/10 of the ligation reaction, keeping the volume of the ligation to no more than 5% of the volume of competent cells,4 or follow the manufacturer's recommendations. If electroporating the ligated molecules, use no more than 2 ml of the ligation reaction with 50 ml of electrocompetent cells. To determine the extent of ligation, inactivate the ligase and run 5 ml of the ligation reaction on an agarose gel and visualize. DNA and RNA Analysis DNA and RNA quantity and quality measurement The quantity and quality of DNA and RNA were measured using a NanoDrop spectrometer equipped with the ND -1000 V3.3.0 software from NanoDrop Technologies Inc (Wilmington, DE). DNA sequencin g To confirm the nature and origin of DNA inserts amplified by PCR or RT -PCR, DNA constructs or clones were sent and sequenced at the University of Florida Interdisciplinary Center for Biotechnology Research (ICBR), DNA Sequencing Facilities. DNA agarose G els DNA was separated on 0.7% -.8% agarose gels depending on the size of the DNA fragments to be separated according to Sambrook et al. (1989). Gels were cast using pure Agarose (Invitrogen) in 1x TAE buffer by boiling. Ethidium bromide was added (0.5 -casting tray. DNA samples were mixed with DNA loading dye to a final concentration of 1x before separation in agarose gels. Electrophoresis was carried out at 100V constant in 1x TAE buffer.

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90 Preparation of E.coli DH5 Competent cells were created as previously described (Mandel and Higa 1970). and grown with shaking at 37C until th e OD600 = 1.0 (approx. 2 hours). Cells were pelleted at 6000rpm using a Sorval SS34 rotor for 10 mins at 4C. The pellet was resuspended in 10ml of sterile ice-cold CaCl2 solution (0.1M CaCl2, 15% glycerol) and incubated for 30 mins on wet ice. Cells were pelleted once again and the pellet was resuspended in 1ml of ice -cold CaCl2 solution and incubated on ice for one hour. The -frozen in liquid nitrogen and stored at 80C until required. For transformation of D H5 mins. The cells were then placed at 45C for 2 minutes, after which 1ml of LB media was added to the tubes, mixed and then placed at 3 7C for one hour. Cells were centrifuged at 10,000rpm for 30 seconds at 24C and the pellet was resuspended in warmed LB plates containing the appropriate selectable antibiotic. The plates were placed at 37C for 12 hours. Several bacterial colonies were then analyzed for the appropriate plasmid. Cells containing the appropriate plasmid were grown in LB media containing the selectable antibiotic for 12 hours with shaking at 37C. 850 glycerol and stored at 80C. Transformation of DH This process was performed following a modified protocol from Epicentre Biotechnologies Inc. Frozen bacteria sample was thawed on ice, and 20 ul of this sample was adde d into an ice pre-chilled 1.5 ml microcentrifuge test tube containing 1

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91 ul of inactivated ligation reaction. The bottom of the test tube was gently flicked to mix DNA and cells. This mixture was transferred into a 0.2 cm cuvette (Bio-Rad Inc); the electro poration was performed using the Gene Pulse electroporator (Bio -Rad, Inc) with 25uF capacitance, 200 immediately added to the cuvette; the electroporated cells in suspension were transferred into a sterile round bottom 15 ml culture tube, which was then incubated at 37 ter an hour, 100 ul of the transformation culture was spread on an LB/agar plate containing 100ug/ml ampicilin antibiotics. The culture plate was incubated at 37 was screened for colon y growth. Screening of bacterial colonies Colonies were picked and screened first using a PCR based approach. PCR positive either plasmid extraction/endonuclease or PCR based approach for potential positive clones, which were sequenced at University of Fl orida ICBR sequencing facilities. The true positive DNA constructs were purified at large scale using the endotoxin -free DNA Maxi prep kit (Qiagen Inc.) to transfect Raw264.7 macrophages. PCR based screening was only used when screening 10 or more distinct bacterial colonies. Each single colony was picked with a sterile micro pipet tip, transferred respectively on a master LB/agar/antibiotics plate and into a 0.2ml PCR tube containing the premix PCR reaction with specific primers to the gene or DNA segment of interest. The amplification was proceeded as described by the PCR kit provider. 0.8% agarose gel electrophoresis was performed and the image of the gel was analyzed to identify DNA fragment with the right expected size.

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92 Endonclease-based screening was performed when analyzing either less than 10 distinct bacterial colonies, or PCR positive colonies. Plasmid mini prep reaction was prepares for each single colony, and the plasmid was digested with one or multiples specific restriction enzymes. After dige stion, an agarose gel electrophoresis was performed and the gel image was screened for the DNA fragments with the appropriate or expected size. Vectors Construction and Expression To prepare the calnexin construct, total RNA was extracted from murine Raw2 64.7 macrophages using the RNeasy Kit from Qiagen Inc (Valencia, CA). The calnexin gene was directly amplified from total RNA employing specific primer sequences (Forward: 5 AAG GAA AAA A GCG GCC GC C ATG ATG GAC ATG ATG ATG ACG 3and Reverse: 5 AAG GAA AAA AA GCG GCC GC T CAC TCT CTT CGT GGC TTT CTG 3) in a one-step RT -PCR protocol (Qiagen Inc). The amplified gene was cloned in frame at the Not I site of the pShooter vector pCMV/ER/GFP/c -myc vector (Invitrogen Inc; Carlsbad, CA), such that GFP is express ed at the N terminus of the protein. The signal peptide sequence (from the vector) and retention signal (from gene) were selected to direct and localize the expressed protein in host ER compartments. The selected clone was sent to University of Florida Genetics and Cancer Institute, Sequencing Core facility (Gainesville, FL) for sequencing and to confirm the RT -PCR product. After sequencing, endotoxin free plasmids containing the calnexin/GFP tagged gene were obtained and used to transfect murine Raw 264.7 macrophages with a nucleofection kit from Amaxa Inc. (Gaithersburg, MD). Sec22b constructs in pmVenus plasmid as well as J774 cells expressing these Sec22b/YFP proteins were described previously (Hatsuzawa et al.,

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93 2006; Hatsuzawa et al., 2009) The pmVenus/Sec22b/YFP plasmid and the pmVenus/YFP plasmids were purified and used to transfect Raw 264.7 cells. Transfection (Nucleofection) of Raw264.7 Macrophages Approximately 1.7x10 7 cells at the exponential growth phase were put into 50 ml centrifuge tubes and centrifuged at 90xg (RCF) for 10 min. The supernatant was carefully discarded and 100 uL of the nucleofection solution was added to the cell pellet. Approximately 15 ug of DNA was transf erred into cells, and the mixture was gently transferred into a 0.4 cm cuvette (Amaxa). Specific conditions for nucleofection of Raw 264.7 supplied by the manufacturer (Amaxa Inc.) was selected to electroporate the cells. After electroporation, 500 uL of D MEM complete medium prewarmed at 37 was immediately added to the cuvette containing electroporated cells. They were transferred into a sterile cell culture dish holding 12 mm glass cover -slides, and incubated for 24h at 37 ore subsequent use. Infection of Raw 264.7 Macrophages with Leishmania Parasites Cell counting (macrophages and parasites) Macrophages The number of Raw264.7 macrophages was estimated using the Trypan blue assay. Under a safety hood, Raw 264.7 adherent cells in the culture flask or dish were gently detached with a sterile rubber policeman. The medium containing cells in suspension was diluted at a ratio of 1:1 with a Trypan-blue solution (0.4% Trypan bleu, 0.81% NaCl, 0.06% K2HPO4, 0.02%, NaN3) and incubat ed at RT for 5min. 10 ul the mixture was transferred on a hemocytometer, which was then placed under a 20x objective of a Leica Microsystems DM/LS microscope. The number of cells was counted using a cell counter device purchased from Fisher Scientific Inc.

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94 Parasites To estimate the number of Leishmania 5ul of the parasite culture was diluted in 95 ul of 2% PFA (ratio of 1:20) and incubated at RT for at least 20 min to fix the parasites. 10ul of the diluted culture was transferred in a hemocytometer, and the parasites were counted under a 40x objective of a Leica Microsystems DM/LS microscope. Plating Raw 264.7 macrophages on glass cover slips The number of Raw264.7 macrophages with the tissue culture flask was estimated using a hemocytometer. The appropr iate volume of cells in suspension was transferred on glass cover -slides inside a sterile cell culture dish. This dish was incubated at 37 night, to allow cells to adhere on cover -slides. Incubation of Raw264.7 macrophages with Leishmania parasites Infections were carried out following standard protocols previously described (Kima, 2007; Ruhland and Kima, 2009) Raw 264.7 macrophages were seeded on 12mm round glass cover slips inside cell culture Petri dishes and incubated at 37 under 5% CO2 atmosphere. The next day, Raw 264.7 macrophages on cover slips were co -incubated with Leishmania parasites in RMPI complete medium at a 1:10 cell to -parasites ratio. The infections were performed at either 37 L. donovani ) or 34 L. pifanoi and L. amazonensis ) under 5% CO2 atmosphere. In experiments in which the time course of ER recruitment to PVs was assessed, infections were initiated with 1:20 cell -to parasites ratio and then the cultures were washed at the first sam pling time to remove free parasites. To stop the infection, cover -slips of infected macrophages were washed three times in 1x phosphate buffered saline (PBS) and fixed in 2% paraformaldehyde (PFA)/1x PBS solution for at least 20min at room temperature.

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95 Im munofluorescence Labeling and Imaging Transfected or non-transfected cells infected on cover slips were fixed with 2% para -formaldehyde (PFA) for at least 20 minutes at room temperature and processed as previously described (Pham et al., 2005) These cover slips were washed twice in 1xPBS and incubated in 50mM NH4Cl/1xPBS solution for 5 min. After two washes in 1x PBS, the preparations were blocked in 2% fat -free milk/1x PBS binding buffer (BB) supplemented with 0.05% saponin, as a cell membrane permeabilizing agent. Some cover slips were then incubated with one of the following primary antibodies to early endosome antigen 1 (EEA1), calnexin, GM130 (Santa Cruz Biotechnology, San Jose CA), BiP, (BD Biosciences, San Jos CA) and/or 1D4B reactive with late endosome and lysosomal associated membrane proteins (LAMP 1), (obtained from the Developmental Studies Hybridoma bank, Iowa City, IA). Cover slips were then incubated with the appropriate Alexa Fluor secondary antibodies (Molecular Probes Carlsbad CA) into which the nucleic acid dye 4,6diamidino 2 phenylindole dihydrochloride (DAPI) had been added. Cover slips were mounted on glass slides with Prolong antifade (Molecular Probes). They were examined on a Zeiss Axiovert 2 00M microscope with a plan neofluar 100x/1.3 oil immersion objective. Images were captured with an AxioCam MRm camera controlled by AxioVision software. Images series over a defined z -focus range were acquired and processed with 3D deconvolution software s upplied with AxioVision. An extended focus function was used to merge optical sections to generate images presented in the figures. Immuno-Electron Microscopy Infected macrophage cultures were spun down and resuspended in the growth medium supplemented wit h 0.15 M sucrose. The macrophages were gently spun down

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9 6 and the cell pellet was rapidly frozen with a HPM 100 high pressure freezer (Leica, Bannockburn, IL). The whole process from cell harvesting to freezing was completed within several minutes. The frozen cell samples were freeze-substituted in 0.1% uranyl acetate and 0.25% glutaraldehyde in acetone at 80o C for 2 days. After freezesubstitution, the samples were warmed up to -50 oC over 30 hrs, washed 4 times with dry acetone at -50o C, then, embedded i n HM20 acrylic resin (Ted Pella, Inc., Redding, CA) at -50oC. The resin was polymerized under ultraviolet light at 50oC for 36 hrs. All the freeze-substitution, temperature transition, resin embedding, and UV -polymerization were carried out in the AFS2 automatic freeze substitution system (Leica, Bannockburn, IL). The HM20 embedded samples were sliced into 100 nm thin sections that were placed on nickel grids, which were then immunogold labeled with an anti -GFP antibody (1/50 dilution v/v) as described by Kang and Staehelin (Kang and Staehelin, 2008) The immunogold labeled sections were post -stained with an aqueous uranylacetate solution (2% w/v) and a lead citrate solution (26g L1 lead nitrate and 35g L1 sodium citr ate) and examined with a Hitachi TEM H 7000 (Pleasanton, CA) operated at 80 kV Ricin Experiment Ricin pulse -chase experiment Ice -cold fluorescein labeled Ricinus communis agglutinin II (RCA II) (called ricin here) purchased from Vector Laboratories Inc (B urlingame, CA) was added to cell cultures at a final concentration of 10ug/ml. The cultures were incubated on ice for 10 min to ensure the adherence of ricin molecules to the surface of cells. They were then incubated at 37C under 5% C02 atmosphere to ini tiate the internalization of ricininfected macrophages. After a five min pulse excess ricin was removed by rinsing the culture twice with RPMI complete medium. Ricin was chased into cells at 37C under

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97 5% C02 atmosphere; samples were collected at 30 min, 60 min, 3 h and 6 h respectively. In some experiments, cells were treated with 5ug/mL BFA (from Invitrogen) for 2 h prior to adding ricin. BFA was maintained in those cells for the duration of the experiment. In ricin experiments of infected cells, Raw 264.7 macrophages were incubated with either L. donovani or L. pifanoi parasites for at least 4 h at 34 or 37C with 5% C02, to generate mature PVs that would no longer interact with early endosomes containing ricin. The samples were washed three times in 1xP BS solution to remove noninternalized parasites. Growth medium was added to the infected cell culture and incubated for an additional 2hrs to ensure complete internalization of parasites attached to host cell membranes. The infected cell cultures were th en placed on ice for 10 min before incubation with ricin. Following the initiation of the ricin chase, infected macrophages on cover slips were collected at the same intervals listed above. Some samples of infected macrophages were incubated with BFA as de scribed above. Samples from different time points were processed in immunofluorescence assays as described above and cover slips were mounted on glass slides with Prolong antifade (Molecular Probes). They were examined on a Zeiss Axiovert 200M microscope w ith a plan neofluar 100x/1.3 oil immersion objective. Images were captured as described above. Targeting strategy of ricin into Leishmania parasitophorous vacuoles Green fluorescent Ricin molecules were targeted into LPVs following schematic design below.

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98 Statistical Analysis Data analysis and generation of graphs was performed on sigma plot software. Each data point is the mean standard deviation from at least three observations. T test was performed to assess differences; they were considered signifi cant at a P value of 0.05. Figure 21. Design of ricin targeting experiment Table 2 1. List of primers Names Sequences Usage NB02R DNA external fragment NB02F NB.Fi11: AAG GAA AAA A GCG GCC GC C ATG ATG GAC ATG ATG ATG ACG PCR c alnexin NB.Ri11: AAG GAA AAA AA GCG GCC GC T CAC TCT CTT CGT GGC TTT CTG Pcmv Forward3: CGC AAA TGG GCG GTA GGC GTG Sequencing BGH Reverse3: TAG AAG GTT CAC AGT AGG

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99 CHAPTER 3 HOST ENDOPLASMIC RETICULUM CONTRIBUTIONS TO THE LEISHMANIA PARASI TOPHOROUS VACUOLE MEMBRANE Introduction The endoplasmic reticulum (ER) is a single continuous membraneenclosed organelle made up of functionally and structurally distinct subdomains: the nuclear enve lope (NE) and the peripheral ER (pER) (Lavoie and Paiement, 2008) The pER is a network of tubules and sheets spread throughout the cytoplasm. ER functions include the translocation of secretory proteins and integration of proteins into its membrane (English et al., 2009) The ER is also the site of proteins folding and modificati on, synthesis of phospholipids and steroids and s torage and release of calcium ions (Ma and Hendershot, 2001; Meldolesi and Pozzan, 1998; Matlack et al., 1998) P roteins that transit the secretory pathway in eukaryotic cells first enter the endoplasmic reticulum (ER ) where they are fold ed, and in some cases assembled int o multi subunit complexes and glycosylated prior to transit to the Golgi compartment (Kaufman et al., 2002) The ER is equipped with a quality control system, which is a surveillance mechanism that scans and targets misfolded proteins for recycling, pe rmitting properly folded proteins to exit the ER en route to other intracellular organelles and the cell surface Misfolded proteins are either retained within the ER lumen in complex with molecular chaperones or are directed toward degradation through the 26S proteasome in a process called ER associated degradation (ERAD) or through autophagy Calnexin is a type I trans membrane protein that primarily resides in the ER (Leach et al., 2002; Schrag et al., 2001) To investigate the recruitment of host ER components to the membrane of PVs that harbor Leishmania parasites, we engineered a DNA construct in which the calnexin gene sequence was fused to the c terminus of a

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100 green fluorescence protein (GF P) gene. The ER signal sequence was supplied in the vector but the ER retention signals of calnexin were included in the construct. This construct as well as the empty plasmid, pCMV/myc/ER/GFP, was used to transiently transfect Raw 264.7 macrophages. The d istribution pattern of calnexin/GFP was assessed in transfected cells that were infected or not with Leishmania parasites. Membrane fusion in macrophages and other eukaryotic cells is thought to be mediated by soluble N ethylmaleimide -sensitive factor att achment protein receptor (SNARE) proteins (Coppolino et al., 2001) Although SNARE complexes are the critical machinery of membrane fusion, their role in determining the specific sites of fusion within the endom embrane system remains to be fully established (Okumura et al., 2006) SNAREs are a group of membrane proteins that localize and function in the diverse endomembrane system, where docking and fusion between membranes take place. All SNAREs are characterized by homologous stretches of 6070 amino acids referred to as SNARE motifs, which are adjacent to the membrane anchor domains. SNAREs can be classified into subgroups: the syntaxin and SNAP 25 families contain a c onserved glutamine at a central position called the 0 layer of the SNARE motif and are therefore called Q SNAREs, and the VAMP (also called synaptobrevin) family contains a conserved arginine at this position and are therefore called R -SNAREs (Jahn and Scheller, 2006) In this study Raw246.7 macrophages were transfected with DNA constructs co ntaining ER -SNAREs molecul es, Sec22b and D12 (R -SNAREs), or Syntaxin 18(Q SNARE), fused to yellow fluorescence protein. These transfected mac rophages expressing each SNARE molecule listed above were infected with either parasites or

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101 ZymosanA Samples were processed in immuno -fluorescence assays and analyzed by fluorescence microscopy to assess the recruitment of h ost ER molecules to Leishmania PVs during the course of infection. The recruitment of ER components to Leishmania PVs membranes was confirmed by immuno -EM analysis of J774 macrophages stably expressing the Sec22b/YFP fusion gene. Results Recruitment of Calnexin and LAMP -1 to the Leishmania PV Membrane Calnexin /GFP expression in Raw 264.7 Prior to analyzing the recruitment of calnexin to Leishmania PVs, a series of experiments were performed in order to determine whether the GFP -fusion to calnexin had achieved the natural distribution pattern of calnexin in cells. In cells transfected with the empty vector, pCMV/ER/GFP (appendix A, Figure A), the GFP signal was diffuse and non specific (Figure 3 -1A). However, in Raw264.7 macrophages expressing the calnexin/GFP chimeric protein, the GFP distribution was very specific; intense and high labeling of GFP around the nucleus (on the nuclear membrane), and a meshlike pattern in the cy tosol (Figure 31B). This GFP distribution in cells transfected with the calnexin/GFP vector, did not display any co localization with the fluorescent signal of LAMP -1, a marker of late endosome and lysosome compartments (Figure 3-1B, merged chanels). However, the staining of these calnexin/GFP expressing cells with antibodies against the endogenous calnexin, showed a complete co -localization between the chimeric and the endogenous calnexin (Figure 3 -1C). The calnexin/GFP distribution (Figure 31B) appeared identical to the calnexin sub-cellular distribut ion in wild type cells stained with anti -calnexin antibodies (Figure 31D).

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102 Recruitment of calnexin/GFP and LAMP -1 to L. pifanoi PV membranes After validating that calnexin/GFP localized in ER compartments within Raw264.7 macrophages, calnexin/GFP expressing cells were infected with L. pifanoi parasit es. Calnexin/GFP expressing Raw264.7 macrophages displayed a high level of GFP and LAMP -1 signals on the membrane of PV s containing L. pifanoi ; these two markers only co localized on Leishmania PV membranes within these transfected cells (Figure 32). PVs that harbor L. pifanoi parasites became progressively distended over the 24h time course (Figure 3-2) as the parasites within them replicated Similar observations were made in cells infected with L. donovani parasites. L. donovani infected cells had many PVs over time, and each PV had a tight lumen that generally contained only a single parasite (Figure 33). L. donovani PVs exhibited a high level of GFP and LAMP -1 signals on their membrane, where both markers only co -localized in host cells (Figure 33). Enumeration of calnexin/GFP and LAMP 1 recruitment to L. pifanoi PVs T he proportion of PVs that had their membrane positively stained with the c alnexin/GFP signal was e stimated. The data showed that at 15 min post infection, over 90 % of all LPV harbori ng live L. pifanoi parasites had their membrane positively stained with the calnexin/GFP marker (Figure 3-4). This calnexin/GFP rate was maintained through out the course of a 24h infection. In the same samples, the proportion of LPV s that were positively stained with LAMP -1, was approximately 60% at 15 min, and over 90% after about 2h and through out the rest of the 24h infection course. The pattern of calnexin recruitment to Leishmania PVs was compared to its recruitment to phagosomes that harbor Zymosan particles or dead parasites. When the transfected cells were incubated with heat killed parasites, the proportion of phagosome

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103 membranes positively stained with the calnexin/GFP signal starte d at about 65% at 15min, decreased to 30% at 1h, and returned to the initial level at 65% at 4h. After the 6h post infection, the heat -killed parasites were completely destroyed and their PVs cleared out within infected cells (Figure 3 4). Moreover, in calnexin/GFP expressing Raw264.7 macrophages that were fed with Zym osan particles, the proportion of phagosome membrane s positively stained with calnexin/GFP marker was approximately 23% at 15min, and 20% after an hour and through out the rest of the infection (Figure 34). In the same sample, the proportion of Z ymosan -co ntaining phagosome membranes intensely labeled with the LAMP -1 signal was about 55% at 15min, and more than 90% after an hour and during the rest of the infection (not shown on the graph) Recruitment of calnexin/GFP and LAMP -1 to L. donovani PV membranes L. donovani parasites cause the visceral leishmaniasis in their rather than the skin related form of the disease generated by members of the L. mexicana complex ( L. pifanoi L. amazonensis and L. mexicana). T o have a broad understanding of the scheme of Leishmania parasites interaction with the host ER, we also investigated t he recruitment of calnexin/GFP to PVs harboring L. donovani parasites. Our data showed that at 15 min post infection, over 90% of all LPV s harboring live L. donovani parasites had th eir membrane positively stained with the calnexin/GFP marker (Figure 35). This recruitment rate of the calnexin/GFP marker was relatively ret ained through out the course of 24h infection. The proportion of LPV that was positively stained with LAMP 1 was a pproximately 60% at 15 min, and over 90% after about 2h and through out the rest of the 24h infection course (Figure 3 5). When the transfected cells were incubated with heat killed L. donovani parasites, the proportion of phagosome membranes positively s tained with the calnexin/GFP

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104 signal started at about 65% at 15min, decreased to 40% at 1h, and returned to the initial level at 65% at 2h. This rate was maintained for 4h, but after 6h all the heat -killed parasites were completely destroyed and their PVs c leared out within infected cells (Figure 35). Recruitment of Host ER MembraneA ssociated SNAREs the L PV Membrane To obtain additional evidence on the interactions between PVs and the host cells ER we examined the recruitment of ER membrane associated S NAREs; Sec22b D12, and Syntaxin18 to Leishmania PVs. These SNARE proteins have been reported to me diate ER vesicles transport and fusion in eukaryotic cells (Aoki et al., 2008; Verrier et al., 20 08) Therefore, their recruitment to PVs would provide some insight into the mechanism by which ER molecules are recruited to PVs. These genes were inserted into the pmVenus vectors. The construction of the pmVenus vector s that encode YFP fused to the N -terminus of Sec22b D12, or Syntaxin18 was described by Hatsuzawa and collaborators (Hatsuzawa et al., 2006) Stable lines of J774 cells expressing these molecule s and transiently transfected Raw 264.7 were first assessed for appropriate expression before they were infected with Leishmania parasites. ER -SNARE ( Sec22b D12, and Syntaxin18) /YFP expression in Raw264.7 Even though the expression pattern of Sec22b/YFP, D12/YFP, and Syntaxin18/YFP was previously described (Hatsuzawa et al., 2006), in this study, a series of experiments were performed in order to determine what was the distribution of the YFP -SNARE fusion molecules in our system The data showed that in cells transfected with the empty vector, pmVenus, th e expres sion pattern of YFP was diffuse and non specific (Figure 3 -6A). Moreover, there was also no YFP signals on Leishmania PV membranes in these transf ected control cells (Figure 3-6B ).

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105 However, in Raw264.7 macrophages transfected with Sec/YFP, D12 or S yntaxin18, an intensely high level of YFP was observed around the cells nucleus or the nuclear membrane, and a meshlike YFP pat tern in the cytosol (Figure 36C ). T he distribution pattern of Sec22b/YFP, D12/YFP, or Syntaxin18/YFP observed in this study wa s consistent with their endogenous distribution pattern, which is representative of the ER distribution in mammalian cells. This was confirmed by the Sec22b/YFP distribution which overlapped with the pattern of endogenous BiP (Figure 36D) and calnexin (F igure 36E), but not with the endogenous late endosomes and lysosomes marker, LAMP -1 (Figure 3 6C). Raw 264.7 macrophages expressing Sec22b/YFP, D12/YFP, or Syntaxin18/YFP were then infected with Leishmania parasites and sampled over a 48h infection period. These infected cells were also stained with anti -LAMP-1 antibodies T he LAMP -1 distribution co-localized with the ER SNARE (Sec22b, D12, or Syntaxin18)/YFP signal only on the PV membrane containing at least a parasite (Figures 37 to 3 -10). Like with ce lls expressing calnexin/GFP, distinctive morphological features of the PVs form by parasites of both L.donovani and L. mexicana complexes were observed in cells expressing ER SNARE (Sec22b, D12, or Syntaxin18). Parasites of the L. mexicana complex ( L. pifa noi and L. amazonensis ) form a primary PV with a large lumen (Figure 3 -7A), which size significantly increased over time to accommodate multiple parasites (Figure 37B; Figure 39; Figure 3 -10) generated after replication. During our study, L. donovani pa rasites were only available when we had access to purified pmVenus and pmVenus/Sec22b/YFP vectors. For this reason, only the recruitment of Sec22b/YFP, but not D12/YFP or Syntaxin18/YFP, to PV membranes

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106 containing L. donovani parasites was investigated. In Sec22b/YFP expressing cells that were infected with L. donovani parasites, the majority of the primary PVs had a tight lumen with a single parasite (Figure 3 8A); over time these host cells were fill with many secondary individual PVs (Figure 3 8B). Enume ration of SNARE/YFP recruitment to L. pifanoi and L. amazonensis PV membranes The proportion of Leishmania PVs that had their membrane positively stained with ER SNARE (Sec22b, D12, or Syntaxin18)/YFP, as well as with the LAMP 1 marker was estimated through out a period of 48h infection. Our data showed that, in transfected cells infected with live L. pifanoi parasites, the proport ion of PVs positive for Sec22b/YFP signals was approximately 90% at 15 min and throughout the course of the 48h infection (Figure 3-11). Similar proportion was observed with all the transfected cell types (Sec22b, D12, and Syntaxin18)/YFP infected with L. amazonensis (Figure 312). Recruitment of Sec22b/YFP to L. donovani PV membranes The recruitment of Sec22b/YFP to L. donovani PVs was also determined. At 15 min post infection, over 90% of all LPVs harboring live L. donovani parasites had their membrane positively stained with the Sec22b/YFP marker (Figure 3 11). This high level of Sec22b/YFP signal was maintained on the membran e of approximately 90% of PVs, throughout the course of infection. The profile of LAMP -1 recruitment on Leishmania PVs in ER -SNARE transfected cells over time was identical of that in cell transfected with calnexin/GFP described above. Electron Microscopy (EM) Analysis To further confirm that ER membrane molecules are recruited to and displayed on the membrane of Leishmania PVs, we performed immuno -EM analyses on cells

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107 expressing Sec22b/YFP. The stable J774 cell line expressing Sec22b/YFP was used since all cells expressed the chimeric molecule, as oppose to the transiently expressed cell samples, in which only a portion of the cells contained expressed proteins. YFP distribution in these cells as well as in cells transfected with the pmVenus/YFP was evaluat ed with an antibody to GFP (see Materials and Methods section), which was followed with a secondary antibody conjugated to gold particles. Distribution of GFP/gold particles in control samples In Raw264.7 transiently transfected with the pmVenus plasmid, g old particles had a random distribution including in the cell nucleus, cytosol, and various organelles or vesicles (Figure 313A). This random distribution of gold particles was also observed in pm Venus transfected J774 cells (controls) that were infected with Leishmania parasites (Figure 313B). Distribution of Sec22b/GFP/gold particles in macrophages In transiently transfected Raw264.7 macrophages expressing Sec22b/YFP, the distribution of gold particles were restricted to the cell nuclear membrane, and to ER compartments within the cell cytosol (Figure 314A). This Sec22b/YFP distribution seems therefore to target the two subdomains of the ER in eukaryotic cells; the nuclear ER, and the peripheral ER. When Sec22b/YFP expressing cells were infected with Leishmania parasites ( L. donovani ), the specificity of the gold particle distribution was extended to membrane delimiting both primary (Figure 314B) and secondary PVs or multiples PVs (Figure 3-14C).

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108 These results complement the evidence obtained from flu orescence assays that had shown that the host cell ER membrane associated molecules are on the PV membrane. Discussion and Conclusion Phagocytosis enables professional phagocytes to internalize large particles, while their total cell surface remains relati vely constant. Numerous studies have considered the biogenesis and maturation of the new membrane bound structures that harbor large particles; nascent phagosomes sequentially interact with early endosomes, late endosomes and lysosomes to mature into phagolysosomes (Harrison et al., 2003; Vieira et al., 2003) T he evidence prevailing to date suggests that Leishmania parasites during the process of phagocytic internalization are directed into P Vs that fo llow a maturation scheme similar to that of phagosomes harboring inert particles. The focus of our studies was to assess the evolving composition of the PV membrane; specifically, their acquisition of ER components overtime. We generated a DNA c onstruct in which calnexin gene was fused to GFP, and demonstrated that the distribution of the chimeric proteins (calnexin/GFP, Sec22b/YFP, D12/YFP and Syntaxin18/YFP) was specific and identical to that of their wild type forms. First, thes e fusion protei ns were validated as appropriate markers to trace ER distribution in macrophages fluorescence and immuno-EM approaches. This result confirmed previous studies in which fusion of Sec22b, D12 and Syntaxin18 tagged with an YFP molecule, did not affect their d istribution (Hatsuzawa et al., 2006). A high proportion (more than 90%) of PVs containing Leishmania parasites had their membrane intensely labeled with ER markers at a very early time point, 15min. It

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109 strongly suggests that ER components such as calnexi n (Muller -Taubenberger et al., 2001) may be recruited to the site of phagocytosis; their fusion to the phagocytic -cup membrane to facilitate parasites entry in macrophage may be mediated by ER -SNAREs such as Sec22b, D12, and Syntaxin18. Our results corroborated previous studies that suggested ER SNAREs such as Sec22b (Becker et al., 2005), syntaxin18 and D12 (Hatsuzawa 2006, 2009) may play a significant role during phagocytosis. Our data showed a gradual recruitment of the LAMP -1 marker on Leishmania PV me mbranes before an hour, which confirmed the fact that PVs maturation is similar to phagosomes maturation that undergoes a sequential and orderly process. It respectively involved the fusion of nascent phagosomes with early endosomes at about 5min; late end osomes at about 15min and LAMP 1 after an hour. Although we did not provide EM data illustrating or confirming the co localization LAMP1 and Sec22b or other ER marker, our results strongly demonstrated recruitment of ER molecules. Both our fluorescence and immuno -EM data indicate that in addition to interactions with late endocytic pathway vesicles, Leishmania PVs interact co ntinuously with the host ER a nd acquire both membrane and membrane associated molecules Thus, t he biogenesis and maturation scheme of Leishmania PVs differs from the maturation of phagosomes that harbor inert particles; PVs are hybrid compartments. Both membranous and luminal contents of endocytic compartments are believed to be acquired by mechanisms including local or focal exocytosi s, which involve secretion and fusion of endosomes and lysosomes to the site of phagosome formation (Bajno et al., 2000; Tapper et al., 2002) The presence of endogenous ER resident proteins such as calnexin and calretic ulin on phagosome membrane s has been

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110 reported (Gagnon et al., 2002; Lee et al., 2010; Houde et al., 2003) However, the mechanisms by which these molecules a ccess phagosomal compartments are still being debated (Gagnon et al., 2002; Huynh and Grinstein, 2007) Suppression of genes responsible for both calnexin and calreticulin expression significantl y hindered the process of phagocytosis in Dictyostelium sp (Muller -Taubenberger et al., 2001) Other studies have also shown that Sec22b and syntaxin 18 (ER related SNAREs) form complexes with Sso1-Sec9C ( PM associated SNAREs) to regulate the fusion between ER and PM under the phagocytic cup (Becker et al., 2005; Hatsuzawa et al., 2006) Phagosome proteomic analysis revealed the abundance of E RS 24/Sec22b molecules at the early stage of phagosomes biogenesis (Gagnon et al., 2002) In the studies presented above we observed that at the earliest sampling time (15 min) the majority of nascent PVs displayed ER molecules, which suggested that these molecules were delivered / recruited at the earliest point of PV formation. As we noted, the situation with phagosomes containing dead parasites was intermediate between the observations of Zymosan particles and live Leishmania parasites. Differences between the Zymosan phagosomes that recruited ER molecules and those that were devoid of these molecules were not readily obvious. Since a higher proportion of PVs recruited ER marker molecules as compared to Zymosan phag osomes and phagosomes that harbor dead particles, this suggested that there is a unique interaction between Leishmania and host cells that promotes their PVs association with the ER. The observations of ER PV interactions are in part in agreement with a re cent report that found that a subpopulation of L. donovani promastigotes in neutrophils selectively develop in ER -like compartments (Glucose 6 -

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111 phosphatase and calnexin positive) that are non-lytic and devoid of lysosomal properties (Gueirard et al., 2008) Unlike the findings in that study that parasites in compartments with lysosomal properties were dead, our studies show that in macrophages, live Leishmania parasites reside in compartments that contain both ER like and lysosomal properties. The differences in PV characteristics observed in both of these studies might suggest that PV development is determined in part by the characteristics of the host cell.

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112 Figure 31. Distribution o f GFP and calnexin/GFP in Raw264.7 macrophages. Mur ine raw264.7 macrophages were transfected either with th e empty plasmid containing the GFP alone (pCMV/ER/GFP) (A) or the DNA vector in which calnexin was tag to a GFP marker (pCMV/calnexin/GFP) (B). These transfected cells were then processed in immuno-fluorescence assays (IFA) with 4',6 diamidino 2 -phenylindole (DAPI) to reveal nuclei (blue) and anti LAMP 1 antibody to label late endosomes and lysosomes (red). In addition, some cells transfected with pCM V/Calnexin/GFP were probed with DAPI and an antibody against an endogenous ER protein, BiP (C). Wild -type Raw264.7 cells were stained DAPI and an antibody against the endogenous calnexin (D), an important ER marker. Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. Optical sections from a Z -series were combined to create the images shown. Scale bar = 5 micrometers (5m).

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113 Figure 32. The r ecruitment of calnexin/GFP t o L. pifanoi PVs in Raw264.7 macrophages. Murine Raw264.7 macrophages t ransfected with the pCMV/calnexin/GFP plasmid were infected with L. pifanoi amastigotes for 2 h (A) or 12 h (B), then process ed in IFA with DAPI (blue) and anti LAMP 1 antibody (red). Thin white arrows point to parasites while thick white arrows show the PV membrane. Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. Optical sections from a Z series were combined to create the i mages shown. Scale bar = 5 micrometers (5m). Figure 33. The recruitment of calnexin/GFP to L. donovani PVs in Raw264.7 macrophages. Murine Raw264.7 macrophages were transfected with the pCMV/calnexin/GFP construct, and infec ted with L. donovani amastigotes for 2 h (A) or 24 h (B). The samples were processed in IFA with DAPI to reveal nuclei (blue) and anti LAMP 1 antibody to label late endosomes and lysosomes (red). Thin white arrows point to representative parasites while t hick white arrows show the PV membrane. Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. Optical sections from a Z series were combined to create the images shown. Scale bar = 5 micrometers (5m)

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114 Figure 34. Proportion of calnexin and LAMP 1 recruitment to L. pifanoi PVs. The proportion of PVs harboring live L. pifanoi parasites that are positively displaying calnexin/GFP (black circles) or LAMP 1 (open circles) during a 24 h course of infection were enumerated and plotted. Also included is the proportion of Zymosan phagosomes displaying calnexin/GFP (black triangles), the proportion of dead L. pifanoi parasites (Open triangles). Each data point is the mean value from at least three experiments in which at least 150 vacuoles were counted per experiment. Graphs were made using the Sigma plot software. The difference between the proportions of PVs that recruited calnexin as compared to Zymosan phagosomes was tested in a paired t test at all time points. The pvalue in each case was <0.001.

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115 Figure 35 Proportion of calnexin and LAMP 1 recruitment to L. donovani PVs. The proportion of PVs harboring live L.donovani parasites that are positively displaying calnexin/GFP ( black circles) or LAMP 1 (open circles) during a 24 h course of infection were enumerated and plotted. Also included is the proportion of Zymosan phagosomes displaying calnexin/GFP (black triangles), the proportion of dead L. donovani parasites (Open triangles). Each data point is the mean value from at least three experiments in which at least 150 vacuoles were counted per experiment. Graphs were made using the Sigma plot software. The difference between the proportions of PVs that recruited calnexin as compared to Zymosan phagosomes was tested in a paired t test at all time points. The pvalue in each case was <0.001.

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116 Figure 36. Distribution of YFP and Sec22b /Y FP i n Raw264.7 macrophages. Murine R aw264.7 macrophages were transfected either with the empty plasmid containing the Y FP alone ( pmVenus ) (A B ) or the DNA vector in which calnexin was tag ged to a Y FP marker ( pmVenus/Sec22b/Y FP) (C E ). Some cells transfected with the pmVenus plasmid were infected with Leishmani a parasites (B). These t ransfected cells were processed in IFA with DAPI to reveal nuclei (blue) and anti LAMP 1 antibody to label late endosomes and lysosomes (red) (A C) In addition, portion of cells transfected with the Sec22b/YFP vector probed with DA PI and antibodies against an endogenous ER protein, BiP (D ) or calnexin (E). Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. Optical sections from a Z series were combined to create the images shown. Scale bar = 5 micrometers (5m).

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117 Figure 37 The recruitment of Sec22b/YFP to L. pifanoi PVs in Raw264.7 macrophages. Murine Raw264.7 macrophages transfected with the Sec22b/YFP plasmid were infected with L. pifanoi am astigotes for 2 h (A) or 48h (B), then processed in IFA with DAPI (blue) and anti LAMP 1 antibody (red). Thin white arrows point to parasites while thick white arrows show the PV membrane. Images were captured with a Zeiss Axiovert 200M fluorescence micro scope controlled with Axiovision software. Optical sections from a Z series were combined to create the images shown. Scale bar = 5 micrometers (5m). Figure 38. The recruitment of Sec22b/YFP to L. donovani PVs in Raw264.7 m acrophages. Murine Raw264.7 macrophages transfected with the Sec22b/YFP plasmid were infected with L. donovani amastigotes for 2 h (A) or 48h (B), then processed in IFA with DAPI (blue) and anti LAMP 1 antibody (red). Thin white arrows point to parasites while thick white arrows show the PV membrane. Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. Optical sections from a Z series were combined to create the images shown. Scale bar = 5 micrometer s (5m).

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118 Figure 39 D12/YFP expressing Raw264.7 macrophages infected with L. amazonensis Murine Raw264.7 macrophages transfected with the D12/YFP plasmid were infected with L. amazonensis promastigotes for 4h (A), 24h (B) or 48h (C), then processed in IFA with DAPI (blue) and anti LAMP 1 antibody (red). The arrow head points to parasites while the full arrows show the PV membrane. Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiov ision software. Optical sections from a Z series were combined to create the images shown. Scale bar = 5 micrometers (5m). Figure 310 Syntaxin18/YFP expressing Raw264.7 macrophages infected with L. amazonensis Murine Raw 264.7 macrophages transfected with the Syntaxin18/YFP plasmid were infected with L. amazonensis promastigotes for 4h (A) or 48h (B), then processed in IFA with DAPI (blue) and anti LAMP 1 antibody (red). The arrow head point s to parasites while the full a rrows show the PV membrane. Images were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. Optical sections from a Z series were combined to create the images shown. Scale bar = 5 micrometers (5m).

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119 Figure 311. Recruitment of Sec22b to Leishmania PVs. Murine Raw264.7 macrophages transfected with Sec22b/YFP were co -incubated with L. pifanoi (close circle) and L. donovani (open circle) amastigotes at a ratio of 1:10 (one macrophage to five parasites). The cultures were washed twice with sterile 1xPBS 2h after the infection. Samples were removed from the culture and fixed in 2% PFA at several time points (2, 4, 12, 24, and 48h). Fixed samples were processed by IFA, and analyzed with a Zeiss fluorescence microscope. The proportion of L. pifanoi (close circle) and L. donovani (open circle) PVs that positively recruited Sec22b/YFP during a 48 h course of infection was estimated and plotted. More than 150 PVs containing live Leishmania parasites were screened. The experiments were repeated at least three times, graphs of the mean values were made using the Sigma plot software.

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120 Figure 312 Endoplasmic reticulum membrane associated SNAREs recruitment to L. amazonensis parasitoph orous vacuoles during infection. Murine Raw264.7 macrophages transfected with Sec22b/YFP, D12/YFP or Syntaxin18/YFP were co -incubated with L. amazonensis promastigotes at a ratio of 1:10 (one macrophage to five parasites). The cultures were washed twice wi th sterile 1xPBS 2h after the infection to remove free and unattached parasites in the culture. After washing, fresh medium was added to the cultures, which were returned in the incubator. Samples were removed from the culture and fixed in 2% PFA at several time -points (2, 4, 12, 24, 48, and 72h). Fixed samples were processed through immunofluorescence assays, and analyzed with a Zeiss fluorescence microscope. The proportion of Leishmania PVs that were positively stained with the YFP was estimated through out the course of 72h infection.

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121 Figure 313 A nti -GFP labeling of macrophages expressing pmVenus (YFP). A Raw264.7 expressing pmVenus (YFP) (A); J774 macrophages stably tr ansfected with pm Venus vector (B ) were infect ed with L. donovani parasites. These cells w ere processed for immuno-EM analysis. Sections on Nickel grids were incubated with a rabbit anti -GFP antibody followed by a secondary antibody conjugated to 18nM gold particles. The gr ids were post -stained with 0.5% uranyl acetate and lead citrate and were examined with a Zeiss EM 10CA transmission electron microscope. Arrows point to 18nM gold partic les r andomly distributed in both infected (B) and non-infected (A) cells. These images are representative of images obtained from at least 2 experiments. N= macrophage nucleus; P= Leishmania parasite A B Non infected macrophage I nfected macr ophage

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122 Figure 314 Immuno EM analysis of ER components recruitment to Leishmania PVs. J774 cells stably transfected with pmVenus/Sec22b/YFP (A) were infected with L. donovani parasites for 12h (B) and 48h (C), and then processed for immunoEM analysis. Sections on Nickel grids were incubated with a rabbit anti GFP antibody followed by a secondary antibody conjugated to 15nm gold particles. The grids were post stained with 0.5% uranyl acetate and lead citrate and were examined with a Hitachi TEM H 7000 (Pleasanton, CA) operated at 80 kV. A) A non inf ected J774 cell stably expressing Sec22b/YFP. B) Cell with a PV with single parasite; c) A cell with multiple PVs; and Zoomed out area. Bold white arrows point to perinuclear ER and PV membrane lined with gold particles. B A C

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123 CHAPTER 4 CONTRIBUTIONS OF HOS T ER TO LEISHMANIA PV LUMEN AND VESICULAR TRANSPORT Introduction In chapter 3, we made t he observations that an integral ER membrane protein and an ER membrane associated molecule s are recruited to PVs In this chapter, we assessed the accumulation of molec ules in the ER lumen in to the lumen of Leishmania PVs. Results Ricin trafficking in Raw264.7 macrophages To determine whether molecules in the ER lumen also gain access to the lumen of Leishmania PVs we elected to monitor the trafficking of ricin toxin in macrophages infected with either L. donovani or L. pifanoi strains. Ricin enters cells by endocytosis then traffics by a retrograde pathway through early endosomes, trans -Golgi network (TGN) and then the ER before reaching the cytosol (Audi et al., 2005; Skanland et al., 2007; Slominska -Wojewodzka et al., 2006) Some of the evidence in support of this pathway included the observation that ricin trafficking to the cytosol is independent of Rab7. We reasoned that if ricin is recruited to PVs, it would imply that the contents of the compartments in the pathway of ricin including the ER l umen could be delivered to PVs. Initial experiments were performed to confirm the trafficking scheme of ricin in uninfected RAW 264.7 macrophages ( Figure 41 ). After a brief pulse with ricin, the plasma membrane (PM) of cells became coated with fluorescent ricin molecules (green) (Figure 41 A); within 5 min, ricin was internalized and enclosed in endosomes. This was verified by demonstrating that ricin is found in vesicles that display the early endosome

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124 antigen (EEA1) (Figure 4-1 B [arrows point to endosomes labeling with both ricin and EEA1]). From early endosomes, ricin was transferr ed successively into the TGN (Figure 4 -1 C) and ER (Figure 41 D) before reaching the cytosol ( Figure 41 E ). At no point is ricin observed in LAMP -1 positiv e compartments. Further confirmation that ricin traverses the TGN and ER was obtained in experiments w ith brefeldin A (BFA), which blocks retrograde transport from the TGN to the ER (Plaut and Carbonetti, 2008; Mardones et al., 2006) Figure 41 F&G show that in the presence of BFA, ricin accumu lates in a compartment that is no longer reactive with anti -EEA1 antibodies (Fig. 4 -1 F). It however accumulates in a compartment that is partially reactive with anti GM130, which suggests that it is most likely retained in the TGN (Figure 4 1 G). GM130 is a matrix protein localized in the cis -Golgi, which explains its partial co localization with ricin (Nakamura et al., 1995) Targeting Ricin into Leishmania PVs Due to potential interactions of ricin in early endosomes (Sandvig et al., 2002) with nascent PVs, infe ctions were established for 4-6 h before pulsing cells with ricin for five minu tes. B y 4 h post -infection, PVs are LAMP 1 positive and therefore would not be expected to interact with early endosomes. After the ricin pulse, ricin was chased into infected cells, which were evaluated after an additional 5 min, 30 min, 1 h, 3 h and 6 h. Figure 42 and Figure 43 show representative images of ricin in infected cells at 5 min, 1 h and 3 h. After 5min, no ricin was found associated with PVs of both L. pifanoi (Figure 42A) and L. donovani (Figure 4 -3A). At 1h, ricin had reached the Leishmania PV membrane and lumen (Figure 4-2B). B y the 3h point ricin wa s within PVs that are clearl y delimited by a membrane that contains LAMP 1 ( Figure 42 C &D; Figure 43B ).

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125 Enumeration of ricin accumulation into Leishmania PVs The proportion of Leishmania PVs that had accumulated fluoresceinated ricin molecules was es timated over a 6h post -chase pe riod (Figure 44). At 30min, less than 5% of both L. pifanoi and L. donovani PVs contained ricin molecules on their membrane or lumen. At 1h post -chase, approximately 40% of both L. pifanoi and L. donovani PVs was positively labeled with ricin signals. Whe n the samples were chased for a longer period, the proportion of both L. pifanoi and L. donovani PVs that had accumulated ricin was about 75% at 3h, and 80% at 6h. This enumeration of PVs with ricin showed that there is a gradual increase in the number of PVs in both L. donovani and L. pifanoi infected cells that accumulated ricin ( Figure 4-5 ). When infected Raw264.7 cells were pretreated with BFA before the ricin pulse -chase experiment, the proportion of both L. p i fanoi and L. donovani PVs that had accumul ated the ricin marker was less than 5% throughout the 6h chase time. The gradual accumulation of ricin in PVs suggested that recruitment of ER contents into the PV might be the resul t of vesicular delivery. Discussion and Conclusion To further assess the extent of host ER association with Leishmania PVs, we exploited the pathway that ricin toxin traverses to access the cytosol of cells. Ricin has been shown to enter cells by endocytosis and to then migrate from endosomes through a retrograde pathway t hat t raverses the trans -Golgi network (TGN) and ER before reaching the cytoplasm (Sandvig et al., 2002) Access to this pathway is sensitive to BFA treatment (Audi et al., 2005; Skanland et al., 2007) Our data indicate d that ricin can access the Leishmania PV lumen via the TGN and ER. The transport of ricin from Golgi to ER, and subsequently from ER to Leishmania PVs was effectively blocke d by

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126 BFA treatment. A del ay was observed between the accumulation of ricin in the ER and its accumulation in Leishmania PVs. This suggests that these two compartments are not continuous. ER contents are most likely delivered to PVs through vesicular fusion; the presence of ER -SNAR Es which mediates ER vesicle fusion, on PVs supports this view. Our data are not consistent with an alternative delivery scenario in which ER and PV membranes fuse and become continuous, which would then permit the exchange of ER contents with PVs. Such a mechanism was suggested in a recent study on the interactions of the Toxoplasma containing vacuole and the host cells ER in which the existence of a pore between these compartments through which ER molecules could be delivered was propos ed (Goldszmid et al., 2009) The observation that Leishmania PVs recruit both endocytic pathway and endoplasmic reticulum components suggests that PVs are atypical compartments, which differ from other pathogen containing vacuol es that also interact extensively with the host cells ER. Legionella containing vacuoles (LCV) for example, avoid interactions with the endocytic pathway by elaborating molecules that are delivered to the cell cytosol via the type IV secretion apparatus (Paumet et al., 2009; Roy et al., 1998) LCVs recruit ER components through a mechanism that is sensitive to BFA w as shown to be ARF1 dependent (Shin and Roy, 2008) Experiments aimed at assessing the accumulation of ricin molecules in phagosomes containing dead particles or Zymosan was not feasible in this study. Dead particles within the phagosomes could not survive more 4h or 6h (see Chapter #3, Figure 34 and Figure 5), which was the required incubation time before the ricin pulse/chase experiment. There were additional technical difficulties with using

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127 Zymosan/Texas Red in these experiment s; there was the limitation of having to localize phagosome compartment s with a fourth marker, giving that ricin fluoresced green, and the host nucleus was stained in blue by the DAPI dye. The observations presented here documenting continuous interactions of PVs with the ER has implications for our understanding of how Leishm ania molecules might access the MHC class I pathway of antigen presentation. Although a few reports have addressed this issue, it is still not known where Leishmania molecules are processed and loaded onto MHC class I molecules for presentation to CD8+ T c ells by infected macrophages. The data presented here suggest that MHC class I molecules in the ER might be accessible to parasite molecules within PVs. Future studies on that topic and on how the presence of ER components in PVs benefits the Leishmania pa rasite should prove to be quite informative.

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128 Figure 41. Trafficking of ricin in non infected Raw 264.7 macrophages. Raw 264.7 macrophages on cover slips were pulsed with fluoresceinated ricin at a concentration of 10ug/ml in complete RPMI medium. The cultures were rinsed and incubated with complete medium at 37 under 5% CO2. Cover slips were removed after the pulse (0 min) (A) then after chase for 5 min (B); some cover slips were stained with anti EEA1 to visualize early endosomes. Bold arrow in the merged image shows vesicles that are both EEA1 and ricin positive. Representative images after 30 min (C), and 1 h (D) and 3 h (E) show labeling of cell nucleus with DAPI dye (blue) and LAMP 1 using an anti LAMP 1 antibody (red). Some samples were pre treated with BFA at a concentration of 5ug/ml for 2 h before ricin treatment. Cover slips obtained after 2 h chase were labeled with anti EEA1 (F) or anti GM130 (G) to localize compartment in which ricin was arrested after BFA treatment. Samples were captured with a Zeiss Axiovert 200M fluorescence microscope controlled with Axiovision software. These images are representative of images from 3 experiments. Scale bar = 5m.

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129 Figure 42. Ricin accu mulates in Leishmania PVs during infection in Raw 264.7 macrophages. Raw 264.7 macrophages were infected for at least 4h before the cultures were incubated with ricin at a concentration of 10ug/ml, and the reaction was chased at 37 atmosphere. At 0 min (A), 1 h (B) and 3 h (C) the cover slips were recovered and processed to reveal nuclei (blue) and LAMP 1 (red). Thin white arrows point to parasites in the infected cells. Thick arrow points to ricin within a PV. Scale bar = 5m. PVs harboring either L. pifanoi or L. donovani that were ricin positive were enumerated after 30 min, 1 h, 3 h and 6 h. The percentage of PVs that were ricin positive was plotted (E). Incubations were done in the presence of BFA and enumerated as well. The difference in the percentage of PVs that were positively displaying ricin was significantly different from the percentage of PV with ricin in cells incubated with BFA. denotes a P value < 0.01. Each data point was compiled from at least 3 experiments.

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130 Figure 43. Ricin accumulates in Leishmania donovani PVs during infection in Raw 264.7 macrophages. Raw 264.7 macrophages were infected for at least 4h before the cultures were incubated with ricin at a concentration of 10ug/ml, and the reaction was chased at 37 nd under 5% CO2 atmosphere. At 5min (A) and 3h (B) the cover slips were recovered and processed to reveal nuclei (blue) and LAMP -1 (red). Thin white arrows point to parasites in the infec ted cells. Thick arrow points to ricin within a PV. Scale bar = 5m. A B 5min n 1 2 1

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131 Figure 44 The proportion of ricin positive Leishmania PVs. Incubations were done in the presence of BFA and enumerated as well. The difference in the per centage of PVs that were positively displaying ricin was significantly different from the percentage of PV with ricin in cells incubated with BFA. denotes a P value < 0.01. Each data point was compiled from at least 3 experiments.

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132 Figure 45. The proportion of ricin positive Leishmania PVs. Incubations were done in the presence of BFA and enumerated as well. The difference in the percentage of PVs that were positively displaying ricin was significantly different from t he percentage of PV with ricin in cells incubated with BFA. denotes a P value < 0.01. Each data point was compiled from at least 3 experiments. The green line demonstrates a gradual accumulation of ricin in the PV; the red line represents a non-gradual accumulation of ricin in PVs.

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133 CHAPTER 5 EFFECT OF HOST ENDOPLASMIC RETICULUM ON LEISHMANIA DEVELOPME NT IN MAMMALIAN CELLS Introduction In the yeast Saccharomyces cerevisiae, the ER localized SNARE proteins Ufe1p, Sec22p, Sec20p, and Use1p/Slt1p form a SNA RE complex required for vesicular transport between the ER and the Golgi (Dilcher et al., 2003; Burri et al., 2003) In addition to the previously known mammalian orthologues syntaxin 18 and Sec 22b, and BNIP1 and D12 (also called p31) were recently identified as mammalian orthologues of Sec20p and Use1p/Slt1p, respectively (Nakajima et al., 2004; Okumura et al ., 2006; Hirose et al., 2004) BNIP1, unlike other ER -localized SNARE proteins, has been shown to be mainly required for apoptotic cell death and homotypic ER membrane fusion rather than for vesicle trafficking (Na kajima et al., 2004) The data presented in chapter 4 suggest that ER derived vesicles containing ricin molecules are transported in vesicles that fuse with both the nascent and maturing Leishmania parasitophorous vacuoles in Raw264.7 macrophages. In thi s chapter, the effect of ER membrane associated SNAREs (Sec22b, D12 and Syntaxin18) on L. amazonensis promastigotes entry, parasite replication and PV development in murine Raw246.7 macrophages is investigated. Results Effect of Host ER SNAREs on L. amazon ensis Entry in Raw264.7 Many pathogens such as Leishmania enter professional phagocytes through the process of phagocytosis. Recent studies have revealed the ER mediated phagocytosi s in which ER compartments fuse to the plasma membrane at the phagocytic cu p to

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134 facilitate the internalization of large inert or live particles by macrophages (Gagnon et al., 2002; Hatsuzawa et al., 2006) Knowing that membrane fusion is mediated by SNAREs; we assesse d the effect of ER SNAREs during L. amazonensis entry in macrophages. In this study, murine Raw264.7 m acrophages were employed and transiently transfected with vector s from which SNAREs (Sec22b, D12 or Syntaxin18) tagged with YFP were over expressed, or ve ctor s encoding dominant negative forms of these ER SNAREs ; the dominant negative SNAREs are devoid of their transmembrane domain (^tmSec22b/RFP; ^tm -D12/RFP or ^tm -Syntaxin -18/RFP) (Hatsuzawa et al., 2006) T ra nsfected cells were incubated with L. amazonensis promastigotes at 34C and 5% CO2 at a ratio of 1:10 (one macrophage for ten parasites) in RPMI complete medium. The samples were washed twice after 1h infection to remove all free pa rasites in the preparati ons. F resh culture medium was added to the preparations, which were returned to the incubator. One hour after washing, samples were removed and fixed in 2% PFA. They were processed through immunofluorescence assays, and analyzed with a Zeiss fluorescence m icroscope. After a 2h infection period, t he proportion of cells that had internalized at least one parasite was estimated. The number of parasites in each cell was also counted in order to assess the infection load. Non transfected cells (wild type) and c ells transfected with the empty vector, pmVenus/YFP, were used as control s in these experiments. We observed that approximately 20% of the cells in the control samples (non transfected Raw264.7 and pmVenus transfected cells) had internalized at least one L amazonensis promastigote during the course of the two hour infection (Figure 51). A mean infection

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135 rate of approximately 30% was scored for Raw264.7 macrophages transfected with the R -SNAREs, pmVenus/Sec22b/YFP or pmVenus/D12/YFP constructs; and 23% f or those transfected with the Q -SNARE, pmVenus/Syntaxin18 Cells transfected with dominant negative constructs were infected at 17% (^tm -D12), 19% (^tm -Sec22b) and 23% (^tm-Syntaxin18) (Figure 5 -1). A paired t -test statistical analysis of the data sugges ts the increase in the number of L. amazonensis -infected cells in the groups of macrophages transfected with either pmVenus/Sec22b/YFP or pmVenus/D12/YFP was statistically significant (P value 0.05) That analysis also revealed that there were no signifi cant differences between the control samples and the rest of the cells expressing Syntaxin18, and the dominant negatives ( -Sec22b, -D12, or -Syntaxin18)/RFP. We also observed that the number of parasites within each infected cell was the same acr oss all the cell types, except in those transfected with pmVenus/Sec22b/YFP construct, which have a significantly higher parasites load com pare to the controls (Figure 52 ). Taken together the over expression of ER membraneassociates R -SNAREs (Sec22b and D12), but not Q -SNARE (Syntaxin18), may significantly stimulate parasite entry into macrophages. D own -regulation of any single ER membrane associated SNARE molecules (Sec22b, D12, or Syntaxin18) does not affect significantly the efficiency of L. amazonens is promastigotes to enter Raw246.7 macrophages. Effect of Host ER SNAREs on L. amazonensis Replication During chronic infection, amastigotes of the Leishmania mexicana complex ( L. mexicana, Leishmania amazonensis and Leishmania pifanoi ) generally form a P V characterized by a large lumen; daughter parasites remain in a communal PV that

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136 increases in size after parasite replication (Kima, 2007) In this study, we assessed the effect of host ER SNAREs on the parasite load in L. amazonensis PVs throughout the course of 72h infection. Murine Raw264.7 macrophages transiently transfected with the SNAREs (Sec22b, D12 or Syntaxin18)/YFP, or their dominant negative forms (^tmSec22b/RFP; ^tm -D12/RFP or ^tm -Syntaxin 18/RFP) were i nfected with L. amazonensis promastigotes. The infection ratio was set at 1:5 ratio, which reduces the initial number of parasites entering the host Infections were s ampled and number of parasites inside the PV was scored at 2, 4, 12, 24, 48, and 72h time points. As illustrated in Figure s (5 5; 56; 5-7) we observed that the mean num ber of parasites within the PV wa s one, which remained relatively constant for all cell types investigated (both transfected and non transfected cells) up to 24h post infection After 24h post infection, the mean number of parasites per PV increases over time. At 72h post infection the mean number of parasites inside the PV was six parasites per PV; this was significantly higher in samples of macrophages over expressing Sec22 b and D12 molecules In comparison, there were four parasites per PV in the control samples (pmVenus, and non-transfected cells). A similar number of parasites per PV was obtained in samples over expressing Syntaxin18. However, after 72h infection, the mean number of parasites per PV was significant reduced by about a third in cells expressing dominant negative -Sec22b, and by half compare to controls expressing -D12. Dominant negative Syntaxin18 di d not affect the parasite per PV count. Taken together our data suggest that Sec22b and D12, but not Syntaxin 18, play a significant role in t he biogenesis of PVs, which ultimately affects the successful replication of Leishmania parasites in macrophages.

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137 Effect of Host ER SNAREs on L. amazonensis PV size For the studies in this section, our objective was to investigate whether host ER SNAREs co ntribute significantly to PV size during L. amazonensis chronic infection. To achieve this objective, we monitor ed the L. amazonensis PV size, in relation with the size of the host nucleus within infected Raw264.7 macrophages transiently transfected with t he SNAREs (Sec22b, D12 or Syntaxin18)/YFP, or their dominant negative forms (^tm -Sec22b/RFP; ^tm -D12/RFP or ^tm -Syntaxin 18/RFP) Representative images of some cell types infected with Leishmania parasites before and after 24h infections are illustrated in Figure 53 (controls cells) and Figure 5 -4 (cells transfected with D12/YFP (A-C) and ^tm D12/RFP (D -E)). T he ratio of the Leishmania PV s ize over the host nucleus size (pv/n) was estimated for all the infected groups at 4h and 48h time points. The contou r or perimeter of the Leishmania PV membrane and the host cell nucleus was measured using the ImageJ software from NIH. The pv/n ratio was estimated at about 0.6 for all groups a t 4h; which means that the size of the host cell nucleus at this early stage i s approximately twice the size of the Leishmania PVs. After the 24h infection time, the Leishmania PV size was at least one and an half times bigger that the host cell nucleus. T he pv /n ratio was significantly higher in more than 90% o f infected cells in t he control infected cells and in cells over expressing ER SNAREs (Sec22b, D12 and Syntaxin18). This increase in Leishmania PV size at the later time point during the infection is most likely to accommodate the increase in parasites after their replication However, in cells that were transfected with dominant negative constructs, the pv/n ratio was significant decreased (pv/n< 0.3) in more than

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138 80% of the cells investigated in this category. This indicated that the inhibition of ER vesicle fusion by target ing ER SNAREs hindered the size increase of Leishmania PVs (Figure 54 D & C). Discussion and Conclusion In this chapter, we investigated the effects of modulating the levels of the host ER membrane associated molecules, Sec22b, D12, and Syntaxin 18, with the goal of limiting the interaction of the ER and Leishmania PVs. The effect of this on Leishmania entry, Leishmania replication, and the development of their PV in Raw264.7 macrophages was determined Our data suggest that knocking down these SNAREs indi vidually does not significantly disrupt Leishmania promastigotes entry in macrophages. However, overexpression of the R -SNAREs (Sec22b and Syntaxin18) resulted in a modest but significant increase in the entry of Leishmania promastigotes The fact that dow n -regulating the ER SNAREs (sec22b, D12, and Syntaxin 18) did not significantly reduce L. amazonensis entry in Raw264.7 macrophages, can not ex clude the possibility that these SNAREs play an essential role during phagocytosis (Becker et al., 2005; Hatsuzawa et al., 2006) Based on the knowledge of how SNAREs act within fusion complex machinery, at least two scenarios can explain how the fusion machinery reacts so robustly to the down-regulation or loss of some of its main players. First, the function al redundancy of these molecules may enable SNAREs of the same subfamily to substitute for each other. For instance, some SNAREs such as syntaxin 1, synaptobrevin, Snc and Sso have several isoforms with identical functions, with their redundancy occasionally arising from recent gene duplication events (Aalto et al., 1993) ; Snc1/2 (Protopopov et al., 1993) Moreover, SNAREs of the same subfamily,

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139 even if not so clearly related, can funct ionally replace each other to varying degree s probably because SNAREs can and do associate in complexes rather promiscuously. For instance, in synaptobrevin knockout mice secretion of chromaffin cells remains normal because of full compensation by endogenous cellubrevin (Borisovska et al., 2005) in contrast to the severe synaptic dysfunction in these mice (with cellubrevin being absent in sy napses). Examples of incomplete compensation by related SNAREs include SNAP -25 that can be partially substituted by exogenously expressed SNAP -23 in chromaffin cells (Delgado Martinez et al., 2007) The yeast SNARE, Sec22 is partially rescue by Ykt6 (Liu et al., 2002) and Pep12p is also partially rescue by Vam3p (Darsow et al., 1997) Second, the SNAREs are constitutively expressed at high levels, which may drastically surpass the cellular needs. In a study in which there was 90% efficient knockdown of the early endosomal SNAREs syntaxin 13, 6 and vti1a, and the exocytic SNARE synaptobrevi in PC12 cell, the residual proteins were fo und to efficiently associate in SNARE complexes, at levels that were surprisingly close to the wildtype situation (Bethani et al., 2009) This led the authors to conclude that the fusion machinery is expressed in h igher levels than the cells demand because less than 11% of the total amount of SNAREs produced is still sufficient to enable fusion within the cell. F luor escence imaging revealed that the SNAREs are organized in multi -molecular clusters in the wildtype cells, which constitutes a possible mechanism to restrict the SNARE activity (Bethani et al., 2009) For example, neuronal exocytic SNAREs are expressed at hugely abundant levels (Holt et al., 2006; Walch-Solimena et al., 1995)

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140 with fusion persisting even when the free SNARE pool is substantially depleted (Kawasaki et al., 1998) The dominant -negative expre ssion of soluble mutants of syntaxin 18 and D12 lacking the transmembrane domain, but not the Sec22b mutant, was believed to significantly suppress the rate of phagocytosis in J774 macrophages by an amount similar to Arf6 T27N (Hatsuzawa et al., 2006) ; which causes inhibition of Fc receptor mediated phagocytosis (Uchida et al., 2001; Niedergang et al., 2003). Moreover, other independent studies confirm previous findings that functions of Sec22b (also called ERS24) functions selectively in phagocytosis triggered by IgG opsonized large particles (3.0 m in diameter) in J774 macrophages (Becker et al., 2005) Taken together, I have shown in this chapter that Leishmania entry i nto macrophages may be positively affected by host ER membraneassociated R SNAREs. Inside PV s, Lei s h mani a promastigotes required at least 24h to transform into amastigotes, which are the only Leishmania form that replicate in mammalian host cells. More im portantly, the functions of R -SNAREs (Sec22b and D12), but not Q -SNARE (Syntaxin18) seem to be required by Leishmania parasites to successful replicate and develop with in their hosts, ma crophages

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141 Figure 51. Effect of ER -membrane associated SNAREs on Leishmania parasites entry into macrophages. Murine Raw264.7 macrophages transfected either with over expressing constructs (Sec22b/YFP; D12/YFP or Syntaxin18/YFP) or their dominant negative constructs (^tm -Sec22b/RFP; ^tm -D1 2/RFP or ^tm Syntaxin -18/RFP) were co -incubated at 34C and 5% CO2 with L. amazonensis promastigotes at a ratio of 1:10. The samples were washed twice with the culture medium after 1h infection to remove all free parasites in the preparations. After adding a fresh culture medium, they were returned to the incubator. One hour later, samples were removed and fixed in 2% PFA. They were processed in immunofluorescence assays, and analyzed with a Zeiss fluorescence microscope. The proportion of cells that had internalized at least one parasite was estimated. The experiment was repeated at least three times. The graphs and t test analysis were done using the Microsoft Excel software. *Data are significantly different from the control (T -test value P

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142 Figure 52. Effect of ER -membrane associated SNAREs on Leishmania parasites load in newly infected macrophages. Murine Raw264.7 macrophages transfected either with over expressing constructs (Sec22b/YFP; D12/YFP or Syntaxin18/ YFP) or their dominant negative constructs (^tm -Sec22b/RFP; ^tm-D12/RFP or ^tm -Syntaxin -18/RFP) were co-incubated at 34C and 5% CO2 with L. amazonensis promastigotes at a ratio of 1:10. The samples were washed twice with the culture medium after 1h infect ion to remove all free parasites in the preparations. After adding a fresh culture medium, they were returned to the incubator. One hour later, samples were removed and fixed in 2% PFA. They were processed in immunofluorescence assays, and analyzed with a Zeiss fluorescence microscope. The number of parasites internalized by each infected cell was estimated. The experiment was repeated at least three times. The graphs and t test analysis were done using the Microsoft Excel software. *Data are significantly different from the control (T -test value P

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143 Figure 53 Representative images of the controls used. Nontransfected Raw264.7 macrophages and Raw264.7 in the study to the effect of host ER membrane associated SNAREs on L. amazonensis replication and PV development. Murine Raw264.7 macrophages nontransfected (C and D ) or transfected with pmVenus/YFP (A and B) Fixed samples were processed through immunofluorescence assays, and images taken and analyzed with a Zeiss fluorescence microscope. B D 12h 12h 48 h 7 2h C A

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144 Figure 54 Representative images of the effect of host ER SNAREs on L. amazonensis replication and PV development. Murine Raw264.7 macrophages transfect ed either with D12/YFP (A-C ) or ^tm -D12/RFP (dominant negative)(were co incubated with L. amazonensis promastigotes at a ratio of 1:5 (one macrophage to five parasites) (D E). Fixed samples were processed in immunofluorescence assays, and images taken and a nalyzed with a Zeiss fluorescence microscope. A D 12h 72h B E C

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145 Figure 5 5 Effect of Sec22b on parasite load in L. amazonensis PV with in Raw264.7 macrophages Murine Raw264.7 macroph ages transfected with Sec22b/YFP or dominant negative Sec2 2b (^tm-Sec22b), were co incubated at 34C and 5% CO2 with L. amazonensis promastigotes at a ratio of 1:5 (one macrophage to five parasites) in RPMI complete medium. The cultures were washed twice with sterile 1xPBS after 2h infection to remove all free pa rasites in the preparations. Samples were removed from the culture and fixed in 2% PFA at several time-points (2, 4, 12, 24, 48, and 72h). Fixed samples were processed in immunofluorescence assays, and analyzed with a Zeiss fluorescence microscope. The num ber of parasites within the PVs was estimated using a cell counter device purchased from Fisher Scientific Inc. The experiment was repeated at least three times. *Data are significantly different from the control (T -test value P

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146 Figure 5 6 Effect of D12 on parasite load in L. amazonensis PV with Raw264.7 macrophages Murine Raw264.7 macroph ages transfected with D12/YFP or dominant negative D12 (^tm-D12 ), were coincubated at 34C and 5% CO2 with L. amazonensis proma stigotes at a ratio of 1:5 (one macrophage to five parasites) in RPMI complete medium. The cultures were washed twice with sterile 1xPBS after 2h infection to remove all free parasites in the preparations. Samples were removed from the culture and fixed in 2% PFA at several time-points (2, 4, 12, 24, 48, and 72h). Fixed samples were processed in immunofluorescence assays, and analyzed with a Zeiss fluorescence microscope. The number of parasites within the PVs was estimated using a cell counter device purch ased from Fisher Scientific Inc. The experiment was repeated at least three times. *Data are significantly different from the control (T -test value P

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147 Figure 57. Effect of Syntaxin18 on parasite load in L. amazonensis PV with in Raw264.7 macrophages. Murine Raw264.7 macrophages transfected with Syntaxin18/YFP or dominant negative Syntaxin18 (^tm Syntaxin18), were co incubated at 34C and 5% CO2 with L. amazonensis promastigotes at a ratio of 1:5 (one macrophage to five parasites) in RPMI complete medium. The cultures were washed twice with sterile 1xPBS after 2h infection to remove all free parasites in the prepar ations. Samples were removed from the culture and fixed in 2% PFA at several timepoints (2, 4, 12, 24, 48, and 72h). Fixed samples were processed in immunofluorescence assays, and analyzed with a Zeiss fluorescence microscope. The number of parasites with in the PVs was estimated using a cell counter device purchased from Fisher Scientific Inc. The experiment was repeated at least three times. *Data are significantly different from the control (T -test value P

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148 CHAPTER 6 OVERALL CONCLUSION AND PERSP ECTIVES Overall Conclusion The goals of this study were to advance our understanding of the contributions of host ER on Leishmania parasitophorous vacuoles biogenesis and maturation, as well as its effect on parasites replication in macrophages. Recent stu dies have provided new insight on phagocytosis, which is the process deployed by phagocytes to capture and internalize large inert particles and pathogens such as Leishmania parasites. Proteomic analyses demonstrated enrichment of many ER markers such as c alnexin and calreticulin to latex bead phagosomes (Garin et al., 2001) The deletion of these ER markers in Dictyostelium sp significantly impaired the capacities of these cells to internalize latex bead particles by p hagocytosis (Muller -Taubenberger et al., 2001) A combination of several approaches including proteomics, confocal microscopy and electron microscopy (EM) to investigate latex bead phagosomes properties demonstrated a direct association between ER and PM during phagocytosis (Gagnon et al., 2002) Thus, the ER -mediated phagocytosis was suggested as an alternative mechanism employed by phagocytes to internalize large p articles and microorganisms without significant depletion of their surface area (Gagnon et al., 2002) Other studies involving both down regulation and up regulation of fusogenic molecules associated with the ER (Synta xin18, D12, and Sec22b), indicated that these ER SNAREs regulate fusion between the ER and PM during phagocytosis (Hatsuzawa et al., 2006) It has been also demonstrated that molecules in phagosomes can access MHC class I via crosspresentation in phagosomes (Houde et al., 2003; Ackerman et al., 2003; Guermonprez

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149 et al., 2003) as well as the export of antigens out of the phagosom e require ER components (Ackerman et al., 2006) However, the involvement of the ER in the process of latex bead phagocytosis in J774 cells, RAW264.7 cells, and dendritic c ells was not confirmed by some independent studies employing biochemical and immunogold EM techniques (Groothuis and Neefjes, 2005; Touret et al., 2005a) Therefore the role of the ER in the process of phagocytosis and phagosome fo rmation still remains controversial. Leishmania parasites enter macrophages by the process of phagocytosis and reside in PVs with different morphologies (see description above). Several studies have shown that like inert particles phagosomes, nascent Leis hmania PVs interact with endocytic compartments; early endosomes, late endosomes and lysosomes, to acquire phagolysosomal properties (Courret et al., 2002) However, the extent of the interactions of the host cell' s ER with nascent and secondary PV has not been assessed. The present Dissertation project assessed the contributions of the host ER to nascent and maturing Leishmania PVs in macrophages. M acrophages either transiently or stably expressing ER marker tagged fluorescent protein were employed to monitor the recruitment of host ER components to Leishmania PVs by fluorescence and Immuno Electron microscopy. We also exploited the retrograde pathway of ricin in macrophages to demonstrate the accumulation of host E R contents into Leishmania PVs lumen. Finally, dominant negative constructs generated from ER -SNAREs (Sec22b, D12, and Syntaxin18) were used to determine how the interaction of the ER and PVs affects Leishmania parasites entry and replication in macrophag es, as well as the size and development of their PVs in macrophages.

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150 Our data demonstrated that Leishmania parasites within macrophages, reside inside a membranes bound structur e called PV s, which is a hybrid compartment composed of components of both the host endocytic pathways (late endosomes and lysoso mes) and the ER. This study also provided evidence that host ER contents are continuously delivered to Leishmania PVs which may occur through the process of vesicular transport and membrane fusion involvi ng ER membraneassociated R SNAREs (Sec22b and D12), but not the Q -SNARE (syntaxin18). The R -SNAREs had a significant effect on PVs development at the later time point during infection, and on Leishmania parasite replication Therefore, Leishmania amastigo tes positively select host ER components to survive, replicate and established a chronic infection in their mammalian hosts. These findings are significant because they can lead to the development of new therapeutic strategies to combat Leishmania sis, whi ch affect more that 12 millions people around the globe. The evidence presented in this study and other previous ones had led us to propose a model summarizing (Figure 6) of our understanding of the interactions of the host ER with Leishmania PVs during their biogenesis and maturation within macrophages. In this model, Leishmania parasite ligands (reviewed in (Lodge and Descoteaux, 2005) ) make contact with the host cell surface receptors (reviewed in (Haas, 2007) ); this interaction should initiate a sequence of signal transductions within the macrophages (Haas, 2007) that will lead to the formation of a phagocytic cup by the plasma membrane (PM). ER derived vesicles will be transported along the microtubules to the phagocytic cup (PC), where there will fuse with the PM underneath the PC through a process mediated by ER SNAREs such as Sec22b (Becker et al.,

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151 2005) and D12, and even Syntaxin18 (Hatsuzawa et al., 2006), and other fusion molecules (Jahn and Scheller, 2006; Hong, 2005) The fusion of host ER -derived vesicles to the PC provide molecules and membranes (Gagnon et al., 2002; Muller Taubenberger et al., 2001) that will facilitate the phagocytosis process while keeping the cell surface area relative constant. The end of the phagocytosis process or parasite entry inside the host cell is marked by the internalization of the Leishmania parasite, which is enclosed in a newly formed PV. In order to mature, the na scent PV will continuously fuse with host ER derived vesicles through out t he course of the infection. The nascent PV will also in a sequential and orderly manner fused with early and recycling endosomes, late endosomes, and lysosomes, which are also transported by microtubules and associated motor proteins. A mature PV, which generally contains the amastigote form of the parasites, has a hybrid membrane that is mainly composed of host late endosomes, lysosomes and ER. Perspectives Future investigation as a continuation of this work will focus on four main points. First, the concept of vesicular transport of ER -derived vesicles proposed for the first time in this study, rather than that there is a continuity between Leishmania PVs and the host ER compartment. This can be further characterized by employing the a 3dimensional immuno-EM tomography approach. This approach has been previously used to successful define the 3-dimensional ultrastructural organization of the complex cellular sub-compartments and functions such as ERGIC and ER -to Golgi transport in plant cells (Kang and Staeh elin, 2008) Moreover, the possibility of the involvement of cell cytoskeleton components in ER derived vesicles transport remains experimentally untested. From the present study, the functions or role of ER -SNAREs in Leishmania

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152 parasites entry in macroph ages or phagocytosis is not fully characterized. Therefore, other approaches adapting tools such as siRNA, drug assays, gene knock down, to simultaneously inhibit the function of multiple ER -SNAREs could be applied. Although the present study clearly demon strated that Leishmania PVs are hybrid compartment s containing components of lysosomes and the host ER, the effect of host ER components on the properties of lysosomal molecules within the PVs, or on the PVs composition in general remains unknown. Equally the effect of the endocytic components on ER functions within the PV still need to be defined. Answers to these questions can significantly help uncover the mechanisms by which Leishmania parasites survive the microbiocidal factors generated by normal ph agosomes.

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153 Figure 61 Model of the macrophage endoplasmic reticulum contributions to Leishmania PV biogenesis and maturation. 1 con tact Leishmania parasite and host cell surface. 2 Formation of the phagocytic cup (PC). 3Transport and fusion of ER derived vesicles to the PC, mediated by ER SNAREs (Sec22b, D12, and Syntaxin18) and other fusion molecules. Fusion of host ER derived ves icles to the PC provides molecules and membranes and facilitates the phagocytosis. 4 The newly formed PV interacts with host ER derived vesicles, and early and recycling endosomes. 5 Fusion of the PV with late endosomes and ER vesicles, and later lysosom es. Microtubules and associated motor proteins enable the vesicular transport. 6 A mature PV has a hybrid membrane made of host late endosomes, lysosomes and ER. The formation and maturation of the inert particle phagosome (Left) do not require fusion or interaction with host ER components. 1 2 5 6 3 4

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154 APPENDIX A ANALYSIS OF THE CALNEXIN GENE AND THE PCMV/ER/GFP VECTOR Figure A 1 pCMV/myc/ER/GFP Map. Adapted from Invitrogen Inc. (www.invitrogen.com)

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155 Fi gure A2 Schematic design of the pCMV/GFP/calnexin vector construction

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156 Figure A 3 0.8% agarose gel electrophoresis pCMV/GFP and pCMV/GFP/Calnexin plasmids were digested with NotI res triction enzyme for 4h at 37

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157 Figure A 4 Experimental design to assess host ER recruitment to Leishmania PVs. EM: Electron microscope.

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192 BIOGRAPHICAL SKETCH Blaise Ndjamen was born in February of 1973 in Ndoungue, Littoral Province, Cameroon. He received a Bachelor of Science in biology from Un iversity of Yaound I in August of 1996 in Yaound, Cameroon. He also received a masters degree in parasitology in August 1998, and an Advanced Professional Degree in parasitology in 2000, both at University of Yaound. He won the prize of Best Student i n the Department of Animal Biology in 1999, and an Award of Academic Excellence from the Cameroon Ministry of High Education in 2000. He worked as Research Assistant at the Centre for Schistosomiasis, and a Research Associate at the Medicinal Research Cent er of the Ministry of Scientific Research and Innovation in 2002; where he did both laboratory and field work in project aiming at controlling the schistosomiasis disease, vectors, and parasites. His research work in public health had lead to the identific ation and characterization of a new focus of Schistosomiasis in the village of Yoro, in the Central Province of Cameroon. He received a Fulbright Fellowship from the United States of Americas Government in 2003, to travel to the United States of America t o complete a masters degree in public health at Tulane University. He joined the graduate program at the Department of Microbiology and Cell Science at the University of Florida in August of 2005. He began working with Dr. Peter Kima on host endoplasmic r eticulum interaction with Leishmania parasitophorous vacuoles. He married Ann Lee Grimstad on December 23, 2009 in Gainesville, Florida. He has attended and presented papers at several national and international scientific meetings. Blaise Ndjamen plans on pursuing a career in cancer biology and drug discovery.