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1 EXPORT OF PROTEINS FROM PARASITOPHOROUS VACUOLES THAT HARBOR LEISHMANIA PARASITES; FOCUS ON AN IN VIVO INDUCED VARIANT OF TRYPAREDOXIN PEROXIDASE By VIKARMA WAYNE BROOKS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNI VERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 Vikarma Wayne Brooks
3 To my family, and all others who have helped me reach this milestone
4 ACKNOWLEDGMENTS I thank my parents for supporting me throughout this endeavor. I thank my principal investigator, Dr. Kima, for guiding me through this project. I thank my fellow lab member, Johnathan Canton, for helping me within the laboratory.
5 TABLE OF CONTENTS pa ge ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF FIGURES ................................ ................................ ................................ .......... 7 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 Leishmania ................................ ................................ ................................ ............. 11 Leishmania Parasites and Vectors ................................ ................................ ... 11 Leishmaniasis ................................ ................................ ................................ ... 12 Forms of disease ................................ ................................ ....................... 12 Treatment ................................ ................................ ................................ ... 13 Leishmania Life Cycle ................................ ................................ ...................... 14 Leishmania and the Host Immune Response ................................ ................... 15 Promastigote cellular invasion ................................ ................................ ... 15 Amastigote cellular invasion ................................ ................................ ....... 17 Persisting inside the parasitophorous vacuole (PV) ................................ ... 18 Intracellular Pathogen Secretion Systems ................................ .............................. 21 Pathogenic Bacterial Secretion from Intracellular Vacuoles ............................. 21 Type III secretion system ................................ ................................ ........... 21 Type IV secretion system ................................ ................................ ........... 22 Type VI secretion system ................................ ................................ ........... 22 Apicomplexan Secretion from Intracellular Vacuoles ................................ ....... 23 Eukaryotic Endomembrane System ................................ ................................ ........ 26 Endomembrane System Overview ................................ ................................ ... 26 Endoplasmic Reticulum Recruitment to the PV ................................ ................ 29 Retro 2 ................................ ................................ ................................ ....... 30 CI 976 ................................ ................................ ................................ ........ 30 Tryparedoxin Peroxidase ................................ ................................ ........................ 31 Hypothesis ................................ ................................ ................................ .............. 31 2 MATERIALS AND METHODS ................................ ................................ ................ 40 Parasites and Cell Lines ................................ ................................ ......................... 40 Infections ................................ ................................ ................................ ................ 40 Retro 2 an d CI 976 Drug Treatments ................................ ................................ ..... 41 Transfection of RAW 264.7 Macrophages ................................ .............................. 41 Immunofluorescence Assays and Imaging ................................ ............................. 42 Fluorescence Intensity Quantification ................................ ................................ ..... 43 Western Blot Analysis ................................ ................................ ............................. 44 Streptolysin O P ermeabilization of RAW 264.7 Cells ................................ ............. 45
6 Statistics ................................ ................................ ................................ ................. 46 Enzyme Linked Immunosorbent Assay ................................ ................................ ... 46 3 RESULTS ................................ ................................ ................................ ............... 49 Export of IVI 16/TXNPx outside the PV ................................ ................................ .. 49 IVI 16/TXNPx Exports from the PV in COP II Vesicl es ................................ ........... 51 Blocking Endoplasmic Reticulum Recruitment Affects IVI 16/TXNPx Export .......... 56 Leishmania Utilizes COP II Vesicles to Modulate Host Cell Signaling .................... 58 4 DISCUSSION ................................ ................................ ................................ ......... 79 LIST OF REFERENCES ................................ ................................ ............................... 87 BIO GRAPHICAL SKETCH ................................ ................................ ............................ 94
7 LIST OF FIGURES Figure page 1 1 World distribution of Leishmania ................................ ................................ ......... 33 1 2 The life cycle of Leishmania ................................ ................................ ............... 34 1 3 Morphological differences between parasitophorous vacuoles (PV) of different Leishmania species ................................ ................................ .............. 35 1 4 Cysteine peptidase may be exported from the PV ................................ .............. 36 1 5 The Type Three Secretion System ................................ ................................ ..... 37 1 6 The Type Four Se cretion System ................................ ................................ ....... 38 1 7 COP II vesicles export cargo from the endoplasmic reticulum (ER) to the Golgi apparatus ................................ ................................ ................................ .. 39 2 1 Formula for calculating fluorescence intensity outs ide the PV ............................ 48 3 1 Export of IVI 16/TXNPx outside the PV ................................ .............................. 61 3 2 cTXNPx is not exported ou tside the PV ................................ .............................. 62 3 3 Quantification of IVI 16/TXNPx labeling outside the PV ................................ ..... 63 3 4 Expression levels of IVI 16/TXNPx and cTXNPx ................................ ................ 64 3 5 IVI 16/TXNPx resides in vesicles after PV export ................................ ............... 65 3 6 IVI 16/TXNPx co localizes with Sec23 ................................ ............................... 66 3 7 Parasites label for Sec23 ................................ ................................ .................... 67 3 8 IVI 16/TXNPx an d Sec23 co localize on vesicles ................................ ............... 68 3 9 CI 976 inhibits the labeling of 5C6 at the host cell surface ................................ 69 3 10 Quantification of 5C6 labeling at the host cell surface ................................ ........ 70 3 11 CI 976 treatment inhibits export of IVI 16/TXNPx to the host cell ....................... 71 3 12 Quantification of the effect of CI 976 on IVI 16/TXNPx export ........................... 72 3 13 Retro 2 mislocalizes Syntaxin 5 ................................ ................................ .......... 73 3 14 Quantification of the localization of Syntaxin 5 ................................ ................... 74
8 3 15 Retro 2 b locks ER recruitment to the PV ................................ ............................ 75 3 16 Retro 2 inhibits the export of IVI 16/TXNPx to the host cell ................................ 76 3 17 Quantification of th e effect of Retro 2 on IVI 16/TXNPx export to the h ost cell .. 77 3 18 CI 976 blocks Leishmania from suppressing macrophage tumor necrosis factor secretion ................................ ................................ ................................ 78 4 1 The IVI L ................................ ........ 85 4 2 L 16/TXNPx model structure ....................... 86
9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EXPORT OF PROTEINS FROM PA RASITOPHOROUS VACUOLES THAT HARBOR LEISHMANIA PARASITES; FOCUS ON AN IN VIVO INDUCED VARIANT OF TRYPAREDOXIN PEROXIDASE By Vikarma Wayne Brooks May 2012 Chair: Peter E. Kima Major: Microbiology and Cell Science Leishmania must manipulate host cell funct ions in order to persist within the parasitophorous vacuole (PV). Many intracellular pathogens have been shown to affect host cell function by secreting effector proteins, each of which has a unique role. It is not known whether Leishmania adopts this mo del to affect host cell function, although it has been assumed. The purpose of this study was to investigate the potential export of a putative effector protein, the tryparedoxin peroxidase termed IVI 16/TXNPx, from the Leishmania PV. The mechanism by wh ich the effector protein would exit the PV was also investiga ted. Immunofluorescence assays and corresponding fluorescence intensity quantification of labeled IVI 16/TXNPx suggested export of the protein into the host cell. Selective permeabilization of infected host cells with Streptolysin O uncovered that IVI 16/TXNPx exists within vesicles prior to entering the host cytosol, if at all. Co localization of IVI 16/TXNPx with Sec23, a COP II coat protein, suggested that the vesicles which IVI 16/TXNPx tra fficked out in were host COP II vesicles. Inhibition of IVI 16/TXNPx export into the host cytosol by treatment with CI 976, a COP II inhibitor, strengthened evidence that IVI 16/TXNPx export occurred via COP II
10 vesicles. Moreover, CI 976 inhibition rever sed the unresponsiveness of infected macrophages to activation by lipopolysaccharide and interferon pound Retro 2, which inhibits endoplasmic reticulum recruitment to the PV membrane, also inhibited the export of IVI 16/TXNPx to the host cytosol. This was presumably because COP II coat proteins could not be recruited to the PV.
11 CHAPTER 1 IN TRODUCTION Leishmania Leishmania are protozoan parasites which belong to the family Trypanosomatidae. The parasite is the causative agent of l eishmania sis, which is primarily a zoonotic disease. From the perspective of human infection, there are a numbe r of reservoir hosts including rodents, marsupials, edentates, monkeys, wild canids, and domestic dogs (Ready, 2008). Leishmania is vector borne, and is transmitted to humans via the bite of Phlebotomus sand flies (Kamhawi et al., 2000). Thus, the parasi tes geographical location is determined by where its sand fly host can flourish. Leishmania Parasites and Vectors There are about 30 species of Leishmania that infect mammals. Human infection may be caused by about 21 of these, including species such as L eishmania brazilensis, Leishmania guyanensis, Leishmania naiffi, Leishmania shawi, Leishmania lainsoni, Leishmania amazonensis, Leishmania mexicana, Leishmania panamensis, Leishmania major and Leishmania pifanoi (Santos et al., 2008). The different speci es are morphologically indistinguishable, but may be differentiated by methods such as isoenzyme electrophoresis or monoclonal antibodies (Mimori et al., 1989). The combination of these methods along with classical clinical and epidemiological criteria le d to a splitting of the genus into two subgenera. Subgenus Viannia comprises a number of species located exclusively in the New World. Subgenus Leishmania includes pathogenic species from the Old World and the New World (Britto et al., 1998). Overall, L eishmania species can be found in regions such as South America, Africa, and the Middle East (Figure 1 1).
12 The only vector of Leishmania species are diptera of the subfamily Phlebotominae. The genus Phlebotomus host Old World species while the Lutzomyia genus serves as host for the New World species (Almeida et al., 2003). It has been noted that some vectors support many different Leishmania while others may be more selective. For example, Phlebotomus papatasi transmit only L. major despite their wide spread presence in regions endemic for multiple Leishmania species. Such specific interaction typically depends on modified versions of an abundant parasite surface protein known as adhesin lipohosphoglycan (Dobson et al., 2010). Leishmania sis Overall, l eishmania sis can be found in certain areas of about 88 countries. The number of new cases of this disease is estimated to be more than 2 million per year. Despite this astonishing number there are currently no vaccines available and treatments suffer fro m severe limitations (Cruz et al., 2009). An alternative method of combatting the disease is vector control. The rapid growth of mega cities in developing countries where facilities for housing, water, and sanitation are inadequate creates increased op portunity for the transmission of this disease (Desjeux, 2001), highlighting the need for a cure. Forms of disease Leishmania sis can pr esent itself in 4 major forms: C utaneous, diffuse cutaneous, mucocutaneous, and visceral. Cutaneous l eishmania sis consis ts of a lesion that is frequently self healing in the Old World. However, if multiple lesions occur leading to disfiguring scars, the aesthetic damage can create a lifelong stigma (Desjeux, 2004). This form of the disease can be caused by multiple specie s of Leishmania such as Leishmania tropica (Handman et al., 1979). Diffuse cutaneous l eishmania sis typically
13 occurs in individuals with a defective cell mediated immune response. The resulting disseminated lesions resemble those of lepromatous leprosy an d will never heal spontaneously. Current treatment options are limited and may lead to a relapse. Mucocutaneous l eishmania of oral nasal and pharyngeal cavities via disfiguring lesions. Subseq uent mutilation of the face leads to great suffering for life. The disease is mostly related to species from the New World including Leishmania brazilensis and Leishmania guyanensis However, it has also been reported in the Old World due to the Leishman ia donovani Leishmania major and Leishmania infantum Visceral l eishmania the most dangerous form of the disease. If left untreated, it is typically fatal. Common symptoms include undulating fever, loss of weight, spl enomegaly, hepatomegaly, lymphadenopathies, and anaemia. If patients recover from the initial disease, they may develop a chronic form of cutaneous l eishmania l eishmania (Desjeux, 2004). Visceral l eishmania sis is caused by the Leishmania donovani species (Sundar, 2001). Treatment The classic treatments used for l eishmania sis are far from ideal due to their high toxicity and adverse effects. Furthermore, the available c ompounds are expensive, making widespread treatment a non viable option. Ordinary treatment against l eishmania sis includes pentavalent antimonial, which has been in use since the 1940s. In resistant cases, drugs such as pentamidine, amphotericin B, and p aromycin are used as a second option, even though they are highly toxic. In order to reduce drug toxicity as well as concentration in the tissues, associating certain drugs with liposomes has been found to be an effective treatment. Some researchers have turned to nature in
14 search of natural compounds which may lead to better treatments (Santos et al., 2008). A new synthetic drug, hexadecylphocoline, has come into the spotlight as a l eishmania sis treatment. Termed miltefosine, it is orally administered and was originally developed as an anticancer drug (Unger et al., 1989). In a study performed in India, oral treatment with miltefosine effectively treated 114 of 120 visceral l eishmania sis cases that had not responded to previously administered antimonia ls (Escobar et al., 2001). Leishmania Life Cycle The Leishmania parasite exists as one of two major developmental forms, the promastigote or amastigote. The promastigote form is an extracellular, flagellated version which multiplies in the midgut of the s andfly vector. The amastigote form is non motile and lives within the macrophages of the vertebrate host (Alves et al., 2005). A host acquires the Leishmania parasites upon being bitten by an infected phlebotomine sand fly (Tibayrenc, 2007). It is has b een noted that the sandfly saliva may actually promote a mammalian Leishmania infection. For example, mice that were inoculated with a mixture of Leishmania promastigotes and phlebotomine saliva grew larger lesions more quickly than those of mice which we re inoculated with only promastigotes (Almeida et al., 2003). The sand fly becomes infected when feeding on the blood of a previously infected host. A number of mammalian species, including humans and dogs, can serve as such hosts. During a feeding ses sion, the sandfly vector will ingest host macrophages containing amastigotes. These amastigotes are then released into the posterior abdominal midgut of the insect where they will transform into promastigotes. The extracellular promastigotes then migrate to the anterior section of the alimentary tract of the sandfly where they will under multiplication via binary
15 fission. After 7 days post feeding, the promastigotes become infectious by transforming into metacylic promastigotes via metacyclogenesis. Lat er, when the sandfly lacerates a the host. Host macrophages will then take up the metacylic promastigotes which will subsequently transform into the amastigote form The parasites then increase in number via binary fission until the host cell bursts, causing the release of more infective parasites which can continue the cycle (Tibayrenc, 2007). A simplified visual representation of the life cycle is presented at th e end of this chapter (Figure 1 2). Leishmania and the Host Immune Response Promastigote cellular i nvasion Promastigote entry into a host macrophage has been shown to occur by phagocytosis. The phagocytic process, primarily performed by macrophages, invol ves engulfing microbes and targeting them to a cellular compartment where they will be degraded. Phagocytosis, under normal circumstances, is an essential weapon utilized by the host to combat microbial threats. However, Leishmania has evolved to utilize the phagocytic process to gain entry to its macrophage host. Phagocytosis is comprised of two sequential events, the first being attachment via low affinity interactions. The second event is the internalization of the target after a high affinity intera ction has been achieved. There are multiple mechanisms by which a macrophage can internalize a microorganism. It has been noted that successful parasite invasion occurs via the zipper mechanism of phagocytosis. The mechanism begins with the attachment o f the parasite to receptors on the phagocytic host. There are two main families of parasite ligands which are thought to facilitate attachment to macrophages. The first is gp63, a zinc metalloprotease that is displayed abundantly on the surface of promas tigotes. The
16 second is the phosphoglycan family, which is comprised of glycolipids such as lipophosphoglycan (Handman and Bullen, 2002). These same molecules also act to protect the parasite from the microbicidal action of the macrophage. Gp63 has the c apability of inactivating proteolytic host enzymes, protecting Leishmania proteins from degradation within the phagosome. Lipophosphoglycan is able to suppress an oxidative burst, scavenge oxygen radicals, and inhib it the action of lysosomal galactosidases (Basu and Ray, 2005). After this initial attachment, the recruitment of additional receptors from the surrounding membrane is triggered, resulting in a simultaneous rearrangement of the cytoskeleton. This event allows the extension of a p seudopod that advances along the organism like a zipper, permitting entry into the macrophage via a phagosome. Complement receptors (CR) play a large role in the process, specifically CR1 and CR3, by facilitating parasite binding and subsequent uptake. E ngagement of the complement receptors by Leishmania does not trigger the ordinary respiratory burst, but rather improves parasite survival (Handman and Bullen, 2002). The phagosome by which the parasite enters lacks the ability to kill any ingested microb es. Such microbicidal property is acquired during the course of phagosomal maturation. Shortly after the phagosome is formed, the vacuole undergoes many compositional changes via vesicular fusion and fission events. The process ultimately yields a hybri d organelle, known as the phagolysosome. Phagolysosomal compartments possess a variety of degradative properties, such as low pH, hydrolytic enzymes, and toxic oxidative compounds (Vieira et al., 2002). The parasite will reside within a specialized compa rtment known as the parasitophorous vacuole (PV), which is derived from the
17 phagolysosomal compartment. It has been suggested that Leishmania parasites have evolved to survive in the acidic conditions provided by the phagolysosome (Antoine et al., 1990). Amastigote cellular invasion After an infection has been initiated with the promastigote form of Leishmania it takes about 24 to 72 hours for the parasite to transform into the amastigote form within the PV (Kima, 2007). The traditional view is that afte r sufficient replication, the host cell will burst to release infectious parasites. This view is based on the coincident observation of damaged host cells and released amastigotes. However, it has also been suggested that amastigotes might recruit the ex ocytic machinery of their host cells in order to release themselves (Rittig and Bogdan, 2000). These newly released amastigotes will need to infect new host cells in order to persist, and in doing so will utilize strategies distinct from their promastigot e form. Like promastigotes, amastigotes gain entry into their host cells via phagocytosis. Multiple studies have revealed that internalization of the parasites is mediated by Fc and complement receptors. Utilization of the Fc receptor for engulfment of p arasites results in the release of anti inflammatory cytokines such as IL 10 (Kima, 2007). The cytokine IL 10 has been shown to inhibit the mi crobicidal activity of interferon ( IFN ) activated macrophages against intracellular parasites (Gazzinelli et al., 1992). An important parasite surface molecule implemented in the phagocytosis of amastigote parasites is phosphatidylserine (PS). PS is a ligand that is ordinarily display ed by apoptotic host cells to indicate to phagocytes that they need to be removed within inducing an inflammatory response. More specifically, PS ligation causes the release of transforming growth factor and IL 10, which suppress macrophage
18 mediated inf lammation. It has been shown that Leishmania amastigotes display PS on their surface, allowing their internalization by phagocytes without setting off a microbicial inflammatory cascade (de Freitas Balanco et al., 2001). Phagocytic uptake of particles b y macrophages can result in superoxide production, which occurs in the case of promastigote internalization. However, this response is not normally seen with amastigote uptake of several Leishmania species. Superoxide, a powerful microbicidal molecule, i s the product of a multi subunit NADPH oxidase enzyme complex that is assembled on a target membrane. It has been noted that the assembly of a functional NADPH oxidase enzyme complex did not occur on PVs harboring L. pifanoi amastigotes. This suppressive activity is believed to be carried out by induction of haeme oxygenease by infecting parasites. It has also been suggested that parasites may affect the phosphorylation state of one of the subunits of the NADPH oxidase enzyme complex, causing a malfuncti on during membrane assembly. Additional reports have shown that nitric oxide production can also be blocked by Leishmania infection of macrophages (Kima, 2007). Persisting inside the parasitophorous vacuole (PV) The PV of Leishmania is late endosomal in n ature, has a pH of 4.5 5.2, and contains components such as lysosom al hydrolases, lysosome associated membrane proteins proton ATPases, and MHC class II molecules (Alexander, 1999). Although there are many similarities between the PVs of the different Leishmania species, there are also morphological and functional differences. For example, L. amazonensis or L. mexicana species exist in large, communal PVs while L. donovani or L. major propagate via small, individual PVs (Figure 1 3) (Antoine, 1998). N onetheless, while inside the
19 PV, all parasites must circumvent the microbicidal mechanisms of the macrophage host in order to persist and replicate. Once inside the PV, the Leishmania parasite has evolved to sustain some of the microbicidal processes of the macrophage, rather than suppress them. For example, a vacuolar acidic pH is maintained and hydrolases are still targeted to the PV (Handman and Bullen, 2002). To survive the acidic pH, amastigotes have evolved to become acidophilic organisms whose me tabolism functions optimally at the pH of the PV lumen (Antoine et al., 1998). Despite this acidic environment, the cytoplasm of the amastigote is maintained at a near neutral pH via proton extrusion (Burchmore and Barrett, 2001). In order to combat prot eases, amastigotes express low numbers of plasma membrane proteins while possessing large amounts of glycoinositolphospholipids which play a protective against hydrolases (Antoine et al., 1998). There are other detrimental host responses that the parasite has likely evolved to actively suppress. One such host response is the presentation of parasite antigens via MHC class II molecule complexes. In order to combat such antigen presentation, L. amazonensis parasites were found to internalize and degrade MHC class II molecules (De Souza Leao, 1995). It has also been suggested that certain Leishmania species may sequester antigenic complexes within the PV where amastigotes bind to the membrane, preventing their export (Antoine et al., 1998). Another host resp onse Leishmania parasites are believed to suppress is upregulation of cytokines which confer host protective immunity. The induction of protective immunity has been linked to the production of IL 12. More specifically, IL 12 production b y macrophages lea ds to IFN production by natural killer cells. In turn,
20 IFN induces nitric oxide production, which is harmful to intracellular parasites (Aliberti et al., 1996). It has been shown that infection with certain Leishmania species, such as L. mexicana re sults in sustained suppression of IL 12 production by macrophages (Kima, 2007). The exact mechanism of how Leishmania parasites can suppress such a vital immune function from within a vacuolar membrane remains a mystery, but it is suggestive of parasite p roteins which may manipulate host processes outside the PV. To date, there is limited evidence of Leishmania secreting parasite proteins outside of the PV in order to modulate host functions. However, it has been suggested that Leishmania secretes cystein e peptidases (CP) into the host cell in order to increase its survival. The process begins with the trafficking of a precursor form of the CP to the flagellar pocket of the parasite (Mottram et al., 2004). The flagellar pocket is a unique compartment for med by an invagination of the plasma membrane at the base of the parasite flagellum and is a major site for exocytosis of macromolecules (Stierhof et al., 1994). The CP becomes activated within the flagellar pocket before being released into the PV Due to the late endosomal nature of the PV, the CP then hitches a ride outside the PV into the host cytosol via the host endocytic network (Figure 1 4). Once in the cytoplasm, the CP degrades the transcriptional regulators NF kB and IkB (Mottram et al., 2004) These transcriptional regulators promote the expression of a multitude of genes. The majority of these genes participate in the host immune response, including proteins such as cytokines and chemokines (Hiscott et al., 2001). Although the study of par asite secreted CP is intriguing, the exact mechanism of secretion is still unknown. A discussion of intracellular pathogen secretion systems used for export of effectors
21 outside PVs similar to that of Leishmania species follows. Perhaps these systems can provide some insight into the PV export mechanism of Leishmania Intracellular Pathogen Secretion Systems Pathogenic Bacterial Secretion from Intracellular Vacuoles There are a number of bacterial species which can infect mammalian host cells. Like Leish mania some of these species exploit their own personal niche within their host in the form of a vacuole, similar to the PV. These intracellular pathogenic bacteria have also evolved to manipulate host cell functions from within their vacuole. They exert such control by secreting effector proteins which have specific roles within the host cell. The mechanisms by which they secrete these effector proteins into the host cell may provide some insight into how Leishmania controls its host functions. Type III secretion system Type III Secretion Systems (T3SS) are vir ulence devices employed by Gram negative bacteria to promote infections. The system is ancestrally related to the multiprotein complexes that assemble flagella. The complex structure consists of a basal body complex that spans the outer and inner membrane of the bacterial cell. From this basal structure a polymerized needle filament extends to a translocator complex that sits in the vacuolar membrane for final delivery of the effector proteins in to the host cell. An energy generating ATPase also aids in translocation of effectors. A diagram of the T3SS complex can be found in the Figures section (Figure 1 5). A representative bacterium that utilizes the T3SS is Salmonella typimurium which actu ally encodes for two separate pathogenic T3SS. One of these T3SS, encoded by Salmonella pathogenicity island SP 1, is utilized to transport effector proteins associated with internalization and early trafficking events. The second T3SS, encoded
22 by Salmon ella pathogencity island SP 2, delivers effectors that are required for development of an intracellular vacuole that can support replication (Galn, 2001; Cambronne and Roy, 2006 ). Thus, T3SS SP 2 is utilized in a situation that is similar to that of Leis hmania Furthermore, it has been shown that the T3SS SP 2 can prevent co localization of the intracellular Salmonella vacuole with the NADPH dependent oxidase which produces microbicidal reactive oxygen species within macrophages (Ohl and Miller, 2001). This ability mirrors the capability of Leishmania to prevent assembly of a functional NADPH oxidase enzyme complex on the PV (Kima, 2007). Type IV secretion system The Type IV secretion system (T4SS) is utilized by Gram negative bacterium to directly trans port virulence proteins into host cells. The T4SS likely emerged from the macromolecular complexes utilized during bacterial conjugation. This secretion system consists of structural components and a filament which span the bacterial inner and outer memb rane (Figure 1 6). An ATPase located at the bacterial inner membrane provides energy (Cambronne and Roy, 2006). This system has the capability of exporting protein substrates into host cells which can induce vast changes. An example of an intracellular pathogen that utilizes this system is Legionella pneumophila After entry into a host macrophage, L. pnemophila exists within a phagosomal vacuole. The T4SS wielded by L. pnemophila allows the bacteria to control phagosome trafficking, preventing fusion with the endosome and lysosomes of the host, thus creating an intracellular niche for survival (Christie, 2001). Type VI secretion system The Type VI Secretion System (T6SS), which is present in many pathogenic proteobacteria, is an apparatus used to secre te effectors into target host cells. Due to
23 structural similarities, it is believed that the T6SS mimics bacteriophage machinery to puncture target cell membranes and translocate proteins. Current knowledge on the structure of the T6SS is limited, but it is believed to span the outer and inner bacterial membranes, and yield a pilus like structure that punctures the lipid bilayer of target cells (Bnemann et al., 2010). Genomic data on the secretion systems suggests that the T6SS exists in most bacteria t hat come into close contact with eukaryotic cells, including animal pathogens (Cascales, 2008). An example of a bacterium which utilizes this secretion system is Burkholderia pseudomallei Like Leishmania this bacterium has the capability of invading ma crophages, and has been shown to exist wi thin an intracellular vacuole (Jones et al., 1996; Shalom et al., 2007 ). Furthermore, it was shown that components of a T6SS were upregulated after invading a macrophage, and suggests that signals within the endocy tic compartment trigger the expression and functions of the T6SS (Ma et al., 2009). Apicomplexan Secretion from Intracellular Vacuoles Leishmania is a member of the kingdom Protozoa, and thus is related to other members of the protozoan classification. Of the various organisms within the kingdom Protozoa, those of the subphylum Apicomplexa bear a strikingly resemblance to Leishmania in terms of mammalian host cell infection. According to a recent classification, Leishmania was grouped under the subphylum Saccostoma. Both Apicomplexa and Saccostoma are grouped under the Subkingdom Biciliata and thus share a number of similar features (Thomas, 2003). One trait shared by the two microorganisms is that they both contain single, well developed mitochondria (d e Souza et al., 2009). Considering this close relationship, the secretion methods that
24 Apicomplexans utilize within a PV may provide insight into the mechanism that Leishmania utilizes. The majority of Apicomplexans are obligate, intracellular parasites. They have the usual secretory pathways found in eukaryotes, with proteins trafficking through the endoplasmic reticulum (ER) and the Golgi. However, Apicomplexans also have other secretory pathways used to deliver specific proteins to secretory organelle s. Proteins from these secretory organelles are secreted into host cells which have been invaded. Two genera which feature such secretion are Toxoplasma and Plasmodium (Ravindran and Boothroyd, 2008). The parasite Toxoplasma gondii exhibits a wide host r ange, able to infect almost any nucleated mammalian cell type. An active penetration process involving the secretion of three discrete secretory organelles, termed the micronemes, rhoptries, and dense granules, permit the parasite entry into host cells (R avindran and Boothroyd, 2008). After internalization, Toxoplasma resides in a PV, which serves as a platform for manipulation of host cell functions (Lalibert and Carruthers, 2008). To date, almost nothing is known about the mechanism by which Toxoplasm a secretes protein into the host cell after invasion and PV establishment. A suggested model proposes a two step method, in which the rhoptries and dense granules first secrete into the nascent PV followed by the creation of a pore or transporter system i n the PV membrane that permits entry of proteins into the host cytosol (Ravindran and Boothroyd, 2008). Of the proteins identified that associate with the Toxoplasma PV membrane, one protein known as ROP14 was found that contains several predicted transme mbrane domains usually found in proteins involved in transport activity (Lalibert and Carruthers, 2008).
25 Plasmodium parasites are the causative agents of Malaria, a prominent disease of the human species. Like Leishmania Plasmodium is transmitted to hum ans via an insect vector, known as the mosquito. While inside the human host, most devastating effects take place while it infects erythrocytes. After formation of its PV within an invaded erythrocyte, the parasite introduces a number of pro teins that cause major changes to the overall architecture of the erythrocyte and the molecules expressed on its surface (Ravindran and Boothroyd, 2008). which are suggested to be organelles established by the parasite to route its proteins may play a role in cell signaling and biochemical pathways (Lanzer et al., 2006). However, before exported from the PV. Although the exact mechanism of PV export is not known at this time, a protein export machine has recently been identified in Plasmodium falciparum Known as PT EX, the multi protein complex is ATP powered (de Koning Ward et al., 2009). A host targeting motif, named the Plasmodium export element or PEXEL, is believed to be one of the signals t hat mark a protein for export (Ravindran and Boothroyd, 2008; de Koning Ward et al., 2009 ). Once a protein has been synthesized and properly modified, it is then deposited into the vacuolar space. After recognition by the PTEX complex, the protein will be unfolded so it can be fed through the central channel of the complex, and subsequently reach the host where it can be refolded (de Koning Ward et al., 2009). It has also been suggested that vesicular fusion alone could secrete parasite proteins to the host cytoplasm (Ravindran and Boothroyd, 2008).
26 Eukaryotic Endomembrane System Endomembrane System Overview The endomembrane system of eukaryotic cells is a complex of cellular machinery made up of dynamic membranous organelles, each of which has a specific protein and lipid composition that conveys a discrete function. One o rganell e is the ER which is the site of polypeptide entry to the secretory system and also functions in lipid biosynthesis. Another organelle is the Golgi apparatus, which performs post translational modifications on newly synthesized proteins and sorts them for transport to additional endomembrane organelles. The Golgi apparatus plays a pivotal role in both the endocytic and exocytic pathways and is important for extracellular matrix formation. The plasma membrane forms the interface between the cell a nd its surrounding environment and performs functions such as internalizing material and cellular signaling (Dacks et al., 2009). An additional organelle is the endosome, which is an intracellular, acidic, membrane bound, subcellular compartment which re ceives endocytosed ligands, receptors and plasma membrane proteins for sorting and delivery to destinations within the cell (Mullock et al., 1987). Early endosomes define those which have just internalized cargo. This cargo may be recycled by returning t o the cell surface via recycling endosomes. Cargo that is destined for degradation is transferred to late endosomes, which eventually transfer the cargo to terminal lysosomes (Rink, 2005). Proteins and lipids are transported between these compartments by small membrane bound vesicles and tubules that bud from one compartment and fuse to another, effectively delivering their contents and membrane. These transport intermediates are generated, targeted, and fused by four major classes of proteins: Rab GTPas es,
27 tethers, SNAREs [soluble NSF(N ethylmaleimide sensitive fusion protein) attachment protein receptor], and coat proteins (Fasshauer et al., 1998; Dacks et al., 2009 ). The primary function of a Rab GTPase is to control vesicle formation. They exist in t wo major states, GTP or GDP bound. Cycling between these two states provides a molecular switch of activity (Dacks et al., 2009). Rab GTPases were originally thought to define the identity of the various compartments of the endomembrane system. For inst ance, Rab1 is present on the ER, Rab6 is on the Golgi, Rab5 is on early endosomes, and Rab7 is on late endosomes. However, this was found to be only partially true. Currently, Rab GTPases are known to function by recruiting membrane tethering and docking factors that facilitate membrane traffic. They also aid in the formation of transport vesicles. Furthermore, they can recruit motor proteins onto vesicles for transport (Suzanne R, 2001). Tethers are complexes formed from a heterogeneous assembly of mu lti subunit proteins which mediate initial recognition and attachment of an incoming vesicle to a target membrane. These complexes vary in composition and structure, with each type of complex mediating transport to specific organelles with the proper orie ntation. For example, DSL1 is involved in anterograde ER to Golgi body transport, while TRAPP1 mediates the transport between the same organelles but in opposite directions (Dacks et al., 2009). In general, tethering of vesicles refers to an initial, loos e interaction of vesicles with their targets that precedes tighter, more stable docking interactions. Vesicle tethering and docking precede a more intimate interaction known as SNARE pairing (Pfeffer, 1999).
28 SNAREs are components which are required for ve sicle fusion. The SNARE hypothesis, which provides a model for SNARE function, proposes that a transport vesicle SNARE interacts with a second SNARE on a target membrane to form a stable complex. This interaction potentially brings the two membranes clos e enough to drive lipid bilayer fusion. Also proposed is that each transport step requires a specific set of SNAREs. Therefore, the selective interactions of these molecules could provide specificity to the transport of vesicles (Dacks et al., 2009). In deed, SNAREs are now considered to be the major players in the final stage of the docking and fusion of many vesicle transport events (Hong Wanjin, 2005). Coat proteins appear to conform to a general model of action, but are specific and characteristic f or the locations or pathways where they function. The COP II coat functions to form vesicles for transport from the ER to the Golgi apparatus (Dacks et al., 2009). The COP I coat functions in retrograde transport, from the endosomes to the Golgi network, as well as intra Golgi transport (Bonifacino and Rojas, 2006; Dacks et al., 2009 ). Clathrin/adaptin coats play a role in the transport of cargo between the trans Golgi network, endocytic organelles, and the cell surface (Dacks et al., 2009). The coat p rotein complex II, or COP II, functions to form vesicles which bud from the ER. The COP II coat is able to segregate biosynthetic cargo from ER resident proteins. COP II vesicle formation begins with the activation of Sar1 GTPase at export sites by the g uanine nucleotide exchange factor Sec12. Sar1 will then form transient complexes with cargo which will be stabilized by the binding of the Sec23/24 complex. Next, the Sec13/31 complex is recruited to the cargo complexes, facilitating membrane
29 curvature ( Figure 1 7). A vesicle will emerge and travel towards the Golgi complex (Barlowe, 2002). The coatomer, or COP I, complex transports material from the Golgi apparatus back to the ER. The process begins with the activation of ARF GTPase by Sec7 GEF. ARF is also recruited to the Golgi apparatus membranes by interaction with members of the p24 protein family. These steps create a priming complex where the coatomer complex can bind. The COP I coat is composed of a total of seven proteins. The F Cop subcompl ex contains the subunits CopB, CopG, CopD, and CopZ. An additional three CopG, and CopD subunits interact with a conserved motif in the cytoplasmic tails of cargo, thus selecting it for transport (Dacks et al., 2009). Endoplasmic Reticulum Recruitment to the P V The Leishmania PV is a compartment that interacts continuously with the host cell. Recently, it was shown that the PV can actively recruit an integral ER membrane protein known as calnexin (Ndjamen et al., 2010). Calnexin is a type 1 transmembrane protein that is resident in the ER, and funct ions as a molecular chaperone (Bergeron et al., 1994; Ndjamen et al., 2010 ). It was also shown that the ER membrane associa ted SNARE Sec22b, which mediates ER vesicle transport, is recruited to the PV. Due to the fact that SNAREs function to fuse vesicles, the recruitment of Sec22b might provide some evidence as to how ER molecules are recruited to the PV. This evidence stro ngly suggests that the PV interacts continuously with the ER of the host cell (Ndjamen et al., 2010). Considering this, it might be possible that the Leishmania PV recruits various components from the ER which could mediate PV export.
30 Retro 2 As previousl y mentioned, the Leishmania PV can actively recruit host ER to its membrane (Ndjamen et al., 2010). Therefore, if this process could be blocked, the PV would be deprived of some of its necessary components, and the intracellular parasites would likely suf fer. Recently, a compound named Retro 2 was discovered which can block the retrograde pathway of molecules traveling from the cell surface to the ER. Retro 2 is believed to cause this effect by altering the location of Syntaxin 5. Syntaxin 5 is a SNARE t hat is normally localized at multiple points between the Golgi and the ER. After Retro 2 treatment, Syntaxin 5 relocates to small vesicles in the cytoplasm, perturbing traffic between the Golgi and the ER (Seaman and Peden, 2010). Within our lab, this sa me compound has also been shown to cause the size of Leishmania PVs to shrink during an infection, as well as reduce the number of parasites per PV. Presumably, this is because Retro 2 blocks the recruitment of ER to the PV. CI 976 COP II vesicles bud fro m the ER to deliver cargo to the Golgi apparatus within cells (Dacks et al., 2009). Therefore, when the PV recruits ER components to its membrane, it could acquire COP II coat proteins which had not yet budded from the ER. These COP II components could t hen select cargo from inside the PV, and export it when they bud from the PV. COP II vesicle formation occurs via two main steps, formation of a spherical bud, followed by constriction at the bud neck with subsequent membrane fission into a vesicle (Brown et al., 2008). In addition to the COP II coat proteins that facilitate COP II budding, phospholipid modifications may also play a role in the final steps of vesicle budding. Specifically, lysophospholipid acyltransferases (LPAT), which catalyze the tran sfer of fatty acids from acyl CoA donors to
31 lysophospholipid acceptors, have been implicated in the final step of COP II budding. A compound known as CI 976 functions as a potent LPAT inhibitor. This compound has been shown to cause the accumulation of c argo at ER exit sites containing all the necessary COP II components, indicating that it inhibits the fission of assembled COP II budding elements (Brown et al., 2008). Tryparedoxin Peroxidase Leishmania contains a unique system of enzymes for eliminating toxic peroxides generated by oxidative stress. This enzyme cascade includes trypanothione reductase, tryparedoxin, and tryparedoxin peroxidase. Higher eukaryotes utilize catalase and selenium dependent glutathione peroxidase to eliminate peroxides, howev er these are absent in Leishmania and therefore the previously mentioned enzyme cascade is the primary means of detoxification of peroxides. Likewise, this enzyme cascade is absent in the mammalian host, making them possible molecular targets for anti p arasite drug research. Combatting oxidative stress is crucial for Leishmania survival because a large amount of reactive oxygen and nitrogen intermediates are generated by the host cell to create unfavorable conditions for the invading parasite. Many Lei shmania species are known to have multiple variant copies of the tryparedoxin peroxidase enzyme, including cytosolic and mitochondrial localized forms (Iyer et al., 2008). Hypothesis In addition to the cytosolic and mitochondrial form of tryparedoxin perox idase, additional variants have been identified. One tryparedoxin peroxidase variant termed IVI 16/TXNPx has been shown to be preferentially expressed during macrophage infection. The IVI 16/TXNPx molecule was identified using change mediated antigen tec hnology. In this method, serum reactive to Leishmania infected cells was raised by
32 immunization of mice with lysates from cells infected with parasites. This serum was then adsorbed against lysates of parasites cultured under normal laboratory conditions to deplete the serum of antibodies that were reactive to parasite structural and housekeeping molecules. The remaining serum antibodies were then used to screen inducible Leishmania genomic expression libraries for genes that were preferentially expresse d during infection. Immunofluorescence assays localized IVI 16/TXNPx to vesicles outside the PV (Kima et al., 2010). In this thesis, we propose that IVI 16/TXNPx after synthesis within the PV trafficks out. Furthermore, we provide evidence which suggests that Leishmania exploits the host cell machinery to aid in the export of IVI 16/TXNPx from the PV. Specifically, we suggest that the host COP II protein coat is involved in cargo export from the Leishmania PV.
33 Figure 1 1. World distribution of Leish mania Species of Leishmania can be found in regions such as South America, Africa, and the Midd le East. [Figure reprinted from Davies, C.R., Kaye, P., Croft, S.L., Sun dar, S. (2003). Leishmaniasis: N ew approaches to disease control. Brit. Med. J. 326 (7 385) 377 382. (Page 378, Figure 1) ]
34 Figure 1 2. The life cycle of Leishmania The parasite cycles through mammalian and insect hosts. [Figure reprinted from Handman, E., and Bullen, D.V (2002). Interaction of Leishmania with the host mac rophage. Trends Parasitol. 18 332 334 (Page 333, Figure 1).]
35 Figure 1 3. Morphological differences between parasitophorous vacuoles (PV) of different Leishmania species. A) Some species of Leishmania exi st within large communal PVs. B) Some species reside in small, individual PVs [ Figure adapted from Antoine, J.C., Prina, E., Lang, T., and Courret, N. (1998). The biogenesis and properties of the parasitophorous vacuoles that harbour Leishmania in murine macrophages. Trends Microbiol. 6 39 2 401. (Page 394, Figure 1).]
36 Figure 1 4. Cysteine peptid ase may be exported from the PV This diagram illustrates the mechanism by which a putative Leishmania effector may exit the PV. [ Figured reprinted from Mottram, J.C., Coombs, G.H., and Alexander, J. (2004). Cysteine peptidases as virulence factors of Leishmania Curr. Opin. Microbiol. 7 375 381. (Page 377, Figure 1).]
37 Figure 1 5. The Type Three Secretion System. The Type Three Secretion System is utilized by some Gram negati ve bacterium to export effector proteins into the host cytosol. [ Figure reprinted from Cambronne, E.D., and Roy, C.R. (2006). Recognition and Delivery of Effector Proteins into Eukaryotic Cells by Bacterial Secretion Systems. Traffic 7 929 939. (Page 93 0, Figure 1).]
38 Figure 1 6. The Type Four Secretion System. The Type Four Secretion System is utilized by some Gram negative bacterium to export effector proteins into the host cytosol. [Figured reprinted from Christie, P .J. (2001). Type IV secr etion: I ntercellular transfer of macromolecules by systems ancestrally related to conjugation machines. Mol. Microbiol. 40 294 305. (Page 297, Figure 3).]
39 Figure 1 7. COP II vesicles export cargo from the endoplasmic reticulum (ER) to the Golg i apparatus. The COP II coat proteins, SAR1 (not shown), Sec23/24 and Sec13/31, assemble at the ER membrane to promote vesicular budding. After budding from the ER, Sec23/24 remain on the COP II vesicle during transit. [ Figure reprinted from Zanetti, G. Pahuja, K.B., Studer, S., Shim, S., and Schekman, R. (2012). COPII and the regulation of protein sorting in mammals. Nat. Cell Biol. 14 20 28. (Page 22, Figure 2).]
40 CHAPTER 2 MATERIALS AND METHOD S Parasites and Cell Lines The L. pifanoi (MH OM/VE/57/LL1) promastigote line was obtained from the American Type Culture Collection (ATCC). The L. amazonensis (MHOM/BR/77/LTB0016) promastigote line was obtained from ATCC and Dr. Diane McMahon Pratt, Yale University. Both parasite lines were culture Drosophila Medium (Gibco) supplemented with 20% heat inactivated FB S (Atlanta Biologicals) and 10 g/mL Gentamicin (Gibco) and grown at 23 C. The RAW 264.7 murine macrophage like cell line (obtained from ATCC) was cultured in RPMI complet e medium at 37C under a 5.5% CO2 atmosphere. RPMI complete medium was composed of RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 units Penicillin/Streptomycin. Infections Infections of RAW 264.7 macrophages for both L. a mazonensis and L. p ifanoi parasites were carried out using the following protocol which has been described (Ndjamen et al. 2010). RAW 264.7 macrophages, previously counted using a hem ocytometer, were plated onto 12 mm round glass coverslips inside cell culture dishes and incubated at 37C under a 5.5% CO2 atmosphere overnight. The following day, the plated macrophages were co incubated with Leishmania parasites in RPMI complete medium at a 10:1 parasite to cell ratio. Parasite number was also obtained using a hemocy tometer. Infections were performed at 34C under 5% CO2 atmosphere. For all infections, parasites were incubated with RAW 264.7 host cells for up to 4 hours, before extracellular parasites were washed away with two rinses using
41 RPMI complete medium. Inf ections were then returned to 34C under 5% CO2 atmosphere for any remaining time points to be collected. Retro 2 and CI 976 Drug Treatments For Retro 2 treated infections, 100 M Retro 2 was prepared in RPMI complete medium. After 4 hours post infection, infected coverslips were rinsed twice with RPMI complete medium, and then placed in drug treated medium for the duration of the infection. For Retro 2 treated uninfected RAW 264.7 samples, cells were first plated overnight in RPMI complete medium. The f ollowing day, t he medium was aspirated and 100 M Retro 2 in RPMI complete medium was added to the cells. Coverslips were collected 24 hours later. The same procedure was applied for both 1 0 M and 50 M CI 976 drug treatments for infected and uninfected RAW 264.7 cells. Transfection of RAW 264.7 Macrophages The Calnexin GFP construct was previously generated in the lab and has been described (Ndjamen et al., 2010). DNA was introduced into cells utilizing the following nucleofection method, adapted from (Ndjamen et al., 2010). To begin, cells were placed into a 50 mL tube and centrifuged at 100g for 10 minutes. The sup ernatant was removed, and 100 L of nucleofection solution was added to the cell pellet. Then 15 ug of DNA construct was transferred ont o the cells, and the resulting mixture was transferred into an Amaxa 0.4 cm cuvette. The cells were then electroporated using the Amaxa Nucleofector syst em. After electroporation, 500 L of 37 C DMEM supplemented with 10% FBS and 100 units Penicillin/St reptomycin was immediately added to the cuvette. These cells were then plated onto 12 mm glass coverslips contained in a cell culture dish and incubated for 24 hours at 37 C under 5.5% CO2 atmosphere before subsequent use.
42 Immunofluorescence Assays and Imaging IVI 16/TXNPx antibody was previously generated in the lab and has been described (Kima et al. 2010). The cTXNPx antibody was gifted from another lab and has also been described (Kima et al., 2010). Lysosome associated membrane protein ( LAMP ) an tibody was obtained from the Developmental Studies Hybridoma Bank, Iowa City, IA. Sec23, Syntaxin 5, and HRP conjugated antibody were obtained from Santa Cruz Biotechnology, San Jose, CA. Hybridoma clone 5C6 was obtained from ATCC and hybridoma supernata nts were prepared in the laboratory. GM130 antibody was obtained from BD Biosciences, San Jose, CA. Anti Rat Green, Anti Rat Red, Anti Mouse Green, Anti Mouse Red and Anti Rabbit Green antibodies were obtained from Invitrogen Molecul ar Probes, Eugene, OR The fluorescent molecule termed diamidino 2 phenylindole dihydrochloride (DAPI) was obtained from Sigma, St. Louis, MO. To begin the immunofluorescence assay (IFA), coverslips were fixed in 2% paraformaldehyde (PFA) in phosphate buffered saline (PB S). They were then rinsed three times with PBS. Next, they were incubated in 50 mM NH4Cl in PBS for seven minutes. After three washes in PBS, they were incubated with 0.05% saponin in PBS to permeablize cell membranes. Following this, they were blocked in 2% fat free milk in PBS supplemented with 0.05% saponin. Coverslips were then incubated on the appropriate primary antibody for one hour. They were then rinsed three times with 0.05% saponin in PBS, and blocked in 2% fat free milk in PBS supplemented with 0.05% saponin. Next, the coverslips were incubated for one hour with the appropriate fluorescent secondary antibody to which DAPI had been added. They were then rinsed two times with 0.05% saponin in PBS, twice with PBS, then once more with triple
43 deionized water. To finalize the procedure, coverslips were mounted on glass slides with mounting media. Labeled coverslips were examined and used for capturing representative images with Axiovision software controlling a Zeiss Axiovert 200m fluorescent microscope. Representative images were captured at 1000X magnification using the Z stack feature of Axiovision, which allows the user to capture multiple planes within a cell. The Z stack images were then subject ed to a medium strength inverse filter dec onvolution algorithm available within the program. Selected planes were then compressed into a single image, capable of being split into individual color channels, using the extended focus feature. Parasite IFA was set up as follows. A slide was etched t o indicate the site of parasite deposition. A drop of parasites, pulled straight from culture, was placed in the etched area and air dried overnight. Parasites were fixed in 2% PFA in PBS, and then the slide was processed for IFA using the above describe d procedure. Fluorescence Intensity Quantification The following standardized process was used to acquire Z stack images with Axiovision software for measuring fluorescence intensity. All images were captured at 1000x magnification. First, an exposure time was selected that would not saturate the microscope camera when viewing the brightest sample of the molecule of interest. This exposure time was then utilized to capture all images of that channel for each time point to complete a data set. The halogen lamp was never turned off until a data set was captured in its entirety to control for bulb intensity fluctuation. For IVI 16/TXNPx quantification, DAPI and LAMP channels were adjusted accordingly to reveal nuclei and PVs. For Tryparedoxin Peroxidase qua ntification, a DIC channel was used for visualization of the cell outline and PV. After capture, all Z stacks were merged using
44 the Extended Focus function within Axiovision software to generate the final pictures for measurement. For 5C6 quantification, a medium strength inversed filter deconvolution algorithm, available within Axiovision software, was applied to all Z stacks before compression to ensure clarity of the cellular membrane. ImageJ was utilized to perform fluorescence intensity measurements of images. For IVI 16/TXNPx quantification, the Freehand Selection feature was used to measure both the mean fluorescence and area of an entire infected cell and PV. Background fluorescence wa s determined by measuring RAW 264.7 cells labeled with IVI 16/ TXNPx and corresponding secondary antibody. A formula was applied to acquire the fluorescence intensity of IVI 16/TXNPx outside the PV of an infected cell (Figure 2 1). The same method was applied for quantifying the localization of Syntaxin 5, however t he Golgi apparatus was used in place of the PV. For 5C6 quantification, the FreeHand line tool was used to measure mean fluorescence of cell membranes. Background were locate d. Microsoft Excel software was used for recording measurements and performing calculations. Western Blot Analysis The following protocol has been previously described (Kima et al. 2010). The RAW 264.7 macrophages were plated in 100 mm cell cultu re dish es and infected with L. p ifanoi parasites at the appropriate time, such that all infection time points ended on the same day. Parasites were then removed, cell monolayers washed once with PBS, and then cells were l ysed within the plate using 250 L of RIP A lysis buffer. The lysates were then processed with a dounce homogenizer. Protein concentrat ions were read using Pierce 660 nM Protein Assay Reagent (Thermo Scientific, Rockford, IL). SDS
4 5 PAGE (SDS Polyacrylamide Gel Electrophoresis) was run on each sa mple on 12% polyacrylamide gels, and proteins were transferred to Immobilon P membrane (Millipore, Billerica, MA). Membranes were blocked with milk and probed with primary antibodies. The IVI 16/TXNPx and cTXNPx antibodies used were the same mentioned in the IFA section above. After rinsing the primary antibodies, membranes were incubated with the corresponding secondary antibody conjugated to horse radish peroxidase. After washing, membranes were incubated with chemiluminescence (ECL, Amersham) reagent s. Antibody reactivity was subsequently visualized by exposure of the membrane to x ray film. The membranes were stri pped by incubating them in 62.5 mM Tris HCl pH 6.8 supplemented with 20 mM 2 mercaptoethanol and 2% SDS for 30 minutes at 50C. The memb ranes were then re probed using the above procedure. Actin was labeled using JLA20 anti actin antibody, obtained from Developmental Studies Hybridoma Bank, Iowa City, IA. Streptolysin O Permeabilization of RAW 264.7 Cells Selective permeablization was per formed using the Streptolysin O reagent obtained from Sigma. The following protocol has been previously described (Beuzon et al. 2000), but has modified details. RAW 264.7 cells were plated on coverslips and grown overnight. Cells were then infected fo r 4 hours before extracellular parasites were washed away. Coverslips were then collected 24 hours post infection, and rapidly washed with ice cold ICT buffer (50 mM HEPES KOH pH 7.9, 4 mM MgCl2, 10 mM EGTA, 8.4 mM CaC l2 78 mM KCl, 1 mM DTT, 1 mg/mL BSA; pH adjusted to 7.1). They were then incubated with 2.5 g/mL Streptolysin O for 5 minutes at 4 C Next, they were washed 3 times with room temperature ICT buffer. This was followed by incubation for 5 minutes at 37C in ICT buffer, and immediate fixati on in 2% PFA in
46 PBS. Coverslips were subjected to an IFA protocol as described above, however no cell membrane permeabilizing agent was used. Statistics Data analysis and generation of graphs was performed using Microsoft Excel. Each data point is repres ented as a mean generated from at least two data sets with standard error indicated using y error bars. A t Test was performed to determine significance (denoted by a *) at a p value of < 0.05. Enzyme Linked Immunosorbent Assay To set up the Enzyme Linke d Immunosorbent Assay (ELISA), RAW 264.7 cells were plated in 6 well plates overnight. The appropriate wells were infected for 4 hours before extracellular parasites were washed away. CI 976 was added to the appropriate sample s at these concentrations: 7 .5 M, 12.5 M, 25 M, and 50 M. After 2 hours post CI 976 treatment, lipopolysaccharide and interferon which needed activation. Supernatants were collected 24 hours after activation. The eBioscience tumor necrosis factor well ELISA plate was coated with 100 L per well of capture antibody diluted in co ating buffer. Plate was sealed and incubated overnight at 4 C. Wells were washed with wash buffer for five times using 200 L per well, this volume for used for all washes. W ells were then blocked with 200 L per well of assay diluent for 1 hour. Foll owing this, wells were washed with wash buffer for five times. Samples were diluted to 1/100 using assay diluent, then placed into the appropriate wells using 100 L per well. Triplicate wells were used for each sample. Samples were incubated for 2 hour s. Assay diluent alone served as background. Wells were washed 5 times with wash buffer. Wells were incubated for 1 hour with detection antibody diluted in assay diluent for 1 hour, used at
47 100 L per well. Wells were washed 5 times with wash buffer. Next, 100 L of avidin HRP (avidin horse radish peroxidase) was added to each well and incubated for 30 minutes. Wells were washed 7 times with wash buffer. Substra te solution was added using 100 L per well and left for 15 minutes. Stop solution was ad ded using 50 L per well to complete th e ELISA. Plate was read at 450 nM.
48 Figure 2 1. Formula for calculating fluor escence intensity outsi de the PV This formula was utilized for the quantification of IVI 16 /TXNPx outside the PV durin g infections, including those treated with Retro 2 and CI 976. It was also utilized for the quantification of Syntaxin 5 localization.
49 CHAPTER 3 RESULTS Export of IVI 16 /TXNPx outside the PV To assess whether IVI 16/TXNPx is exported into the host cell during Leishmania infection, an immunofluorescence assay (IFA) was performed (Figure 3 1). To set up the IFA, an infection of macrophage like RAW 264.7 host cells was performed on coverslips. Cells on coverslips were fixed at 4 hours, 12 hours, 24 hours and 48 hours post infection. The se coverslips were labeled for lysosome associated membrane p rot ein (LAMP), IVI 16/TXNPx, and diamidino 2 phenylindole dihydrochloride (DAPI). LAMP was labeled with a red fluorescent antibody, IVI 16/TXNPx with a gr een fluorescent antibody, and DAPI fluoresced as blue. As mentioned in Chapter 1, the Leishmania parasitophorous vacuole (PV) membrane contains the lysosomal marker LAMP (Alexander et al., 1999), and thus labeling for this protein enables the viewer to vi sualize PVs. At 4 hours post infection, there was no IVI 16/TXNPx labeling outside the PV in the host cell. At 12 hours, some IVI 16/TXNPx labeling was visible in the host cell as discrete dots, which indicated the protein was contained in vesicles. At 24 hours, a relatively large amount of IVI 16/TXNPx was seen in the host cell. This suggested 24 hours post infection was the time of highest export. At 48 hours, IVI 16/TXNPx can still be found in the host cell, albeit at lower levels when compared to 2 4 hours. As mentioned in Chapter 1, IVI 16/TXNPx is only one of the variants of tryparedoxin peroxidase that Leishmania contains. Another tryparedoxin peroxidase, known as cTXNPx, is localized solely in the cytoplasm of the parasite (Kima et al., 2010). An IFA was performed, in the manner described above, in which cTXNPx was labeled green, and nuclei as blue using DAPI (Figure 3 2). Since the LAMP and
50 cTXNPx antibodies were both made in rats, the PV and cellular outline were estimated using differential interference contrast microscopy. The pattern of labeling of cTXNPx was restricted to that of the parasite, with no labeling anywhere else in the host cell. This labeling revealed that antibodies against tryparedoxin peroxidase molecules are not reactive against any host molecules, strengthening evidence that the IVI 16/TXNPx labeling in the host cell was from secreted IVI 16/TXNPx molecules. The labeling of cTXNPx also verified that IVI 16/TXNPx labeling in the host cell was not from dead parasites. T he secretion of IVI 16/TXNPx and cTXNPx was quantified using images from IFAs performed as described above (Figure 3 3). The labeling of cTXNPx outside the PV remained at a relatively low level throughout the course of the infection. However, the labelin g of IVI 16/TXNPx was notably increased over that of cTXNPx at the 10, 24, and 48 hour time points, which indicated secretion of the molecule. The measured level of IVI 16/TXNPx outside the PV was highest at 24 hours, and was statistically significant fro m the measured level of cTXNPx outside the PV at this time point. The fluor escence intensity of IVI 16/TXNPx within the PV increased until 24 hours post infection. A western blot was performed to assess the levels of IVI 16/TXNPx and cTXNPx protein throug hout a 48 hour infection (Figure 3 4). Both proteins migrate to about 23 kDa. The level of the IVI 16/TXNPx protein appeared to increase as time progressed. This indicated that it was preferentially up regulated during the infection. The same membrane was stripped and re probed using an antibody against cTXNPx. The amount of the cTXNPx protein appeared to remain constant throughout the infection, suggesting the increase in IVI 16/TXNPx was due to protein production up regulation, and not
51 because parasi te number had drastically increased. The same membrane was stripped and re probed with anti actin antibody to confirm that equivalent protein amounts were loaded. IVI 16/TXNPx E xports from the PV in COP II Vesicles The method by which a protein would exit the PV and enter the host cell is unknown. We hypothesized that the export of IVI 16/TXNPx might utilize the host COP II vesicles for export. However, first it was necessary to verify that IVI 16/TXNPx trafficks within the host cell in vesicles, as oppo sed to being a free floating molecule. To address this, an experiment was designed which utilized a reagent called Streptolysin O. This reagent has been shown to selectively permeabilize the plasma membrane of RAW 264.7 cells, rendering molecules in the cytosol accessible to antibody labeling, whereas molecules protected by a vacuolar membrane would not be accessible (Beuzon et al., 2000). Considering this, Leishmania within RAW 264.7 cells processed for IFA should not label for IVI 16/TXNPx when Strepto lysin O permeabilization is utilized, if the molecule is contained in vesicles. Coverslips were collected at 24 hours post infection and an IFA utilizing Streptolysin O permeabilization was performed. The IVI 16/TXNPx antibody was labeled green, LAMP anti body as red, and DAPI labeled nuclei blue (Figure 3 5). The LAMP antibody which was used only recognizes the intra vacuolar portion of LAMP and should not label efficiently with Streptolysin O permeabilization. Saponin permeabilization, which permeabiliz es all membranes, was also used for identically processed coverslips to verify that the infection was productive and that IVI 16/TXNPx export into the host cell had occurred. Some cells were fully permeabilized by Streptolysin O, allowing all molecules t o be labeled. Infected cells in which the Streptolysin O permeabilization worked properly did
52 not contain parasites which labeled positive for IVI 16/TXNPx. This indicated proper Streptolysin O permeabilization becau se parasites usually label for IVI 16/ TXNPx, however if the PV is not permeabilized, the parasites should not be accessible to the IVI 16/TXNPx antibody. These infected cells were identified by spotting parasite nuclei in close association with a host nucleus. The cells which received saponi n permeabilization verified that IVI 16/TXNPx was exported from the PV during the infection. Cells which had been fully permeabilized by the Streptolysin O, indicated by labeling of the parasites within the PV, also showed that IVI 16/TXNPx labeled within the host cell. In cells which received proper Streptoly sin O permeabilization, labeling of IVI 16/TXNPx was absent within the host cell, the normal indication of IVI 16/TXNPx export. This suggests that IVI 16/TXNPx is contained within vesicular compartm ents after export into the host cell. It has been previously suggested that a parasite molecule, the acid phosphatase SAcP protein, which exits the PV into the host cell was contained within vesicles (McCall and Matlashewski, 2010). As mentioned in Chapte r 1, formation of the COP II coat requires the Sec23 protein. After the COP II vesicles buds, Sec23 will remain on the vesicle during transit of cargo (Zanetti et al., 2012). Considering this information, an IFA was performed where Sec23 was co labeled w ith IVI 16/TXNPx on coverslips collected 24 hours post infection (Figure 3 6). Sec23 was labeled with a green antibody, IVI 16/TXNPx with a red antibody, and DAPI labeled the nuclei blue. If IVI 16/TXNPx trafficks via COP II vesicles, it would be expecte d that the labeling of IVI 16/TXNPx would co localize with the labeling of Sec23 in some instances, producing a yellow color when merged. Indeed, it was found that IVI 16/TXNPx labeling frequently co localized with the labeling
53 of Sec23. It was found tha t the parasites also labeled for Sec23, which is possible, due to their eukaryotic nature. To address this question, an IFA was performed on parasites alone, in which Sec23 was labeled green and DAPI was used to identify parasite nuclei as blue (Figure 3 7). Indeed, it was found that Leishmania parasites label positive for the Sec23 protein. Although IFAs are a powerful tool, the technique has limitations. When it comes to co localization studies, the viewer never truly knows if two molecules are co loca lized due to the low resolution of light based microscopy. A technique which yields much higher resolution is electron microscopy (EM). EM sections were labeled with IVI 16/TXNPx and Sec23 within our lab (Figure 3 8). (The EM data presented here is pend ing publication and was performed by Johnathan Canton and Peter Kima.) Within the EM sections, it was discovered that IVI 16/TXNPx was localized to vesicles which also labeled for Sec23. This strengthened evidence that IVI 16/TXNPx trafficks within COP I I vesicles. It was also found that Sec23 labeled the PV membrane, which indicated COP II vesicles are recruited to the PV. As mentioned in Chapter 1, the compound CI 976 acts as an inhibitor of COP II vesicle budding. Considering this, if IVI 16/TXNPx is exported from the PV in COP II vesicles, treating infected cells with CI 976 should block IVI 16/TXNPx from entering the host cell. However, before we could address this question, we had to verify that CI 976 could block COP II vesicle budding within the RAW 264.7 host cells for 24 hours, as this is the time post infection when IVI 16/TXNPx export is the greatest. To address this question, we considered the fact that COP II vesicles are utilized in cargo sorting and vesicle transport for proteins destine d for the cell surface (Zanetti et al., 2012). A
54 cellular surface protein which is displayed on RAW 264.7 cells is the complement receptor type 3, which is recognize d by an antibody known as 5C6 (Cervia et al., 1993; Feng et al., 2004 ). Thus, treating RA W 264.7 cells with CI 976 should decrease the amount of 5C6 labeling at the cellular surface. An experiment was performed which treated RA W 264.7 cells with 10 M and 50 M concentrations of CI 976 for 12 and 24 hours. Coverslips were subjected to an IF A in which 5C6 was labeled red, Sec23 labeled green, and DAPI labeled the nuclei blue (Figure 3 9). RAW 264.7 cells which received no treatment had a uniform distribution of 5C6 labeling. Treat ment of RAW 264.7 cells with 10 M CI 976 decreased the label ing of 5C6 at the cellular surface both 12 and 24 hours post treatment. Treatment with 50 M CI 976 drastically reduced the labeling of 5C6 at the cellular surface both 12 and 24 hours post treatment, and some retention of the label could be seen in the c ell. Next, a quantification of the amount of 5C6 labeling at the cellular surface was performed on the CI 976 treated RAW 264.7 cells using images from an IFA and ImageJ software (Figure 3 10). It w as further verified that the 10 M CI 976 treatment part ially reduced the amount of CI 976 labeling at the cellular surface at both 12 and 2 4 hours post treatment. The 50 M treatment significantly reduced the amount of labeling of 5C6 at the cellular surface both 12 and 24 hours post treatment. Considering t he data gathered from these experiments it was determined that the CI 976 compound would be effective for treatments running until 24 hours post infection. To assess whether or not CI 976 would inhibit the export of IVI 16/TXNPx into the host cell, an exp eriment was designed where RAW 264.7 cells were infected with
55 Leishmania parasites for 4 hours. Parasites which did not infect cells were then washed away, and medium was applied which contained the CI 976 compound at either 10 M or 50 M concentrations. Coverslips were subsequently collected at 12 hour and 24 hours post infection. An IFA was performed in which IVI 16/TXNPx was labeled red, Sec23 was labeled green, and DAPI was utilized to label nuclei blue (Figure 3 11). At 12 hours, the 10 M CI 976 treatment reduced the amount of IVI 16/TXNPx labeling outside the PV, in comparison to the no treatment sample. Furthermore, co localization of the IVI 16/TXNPx with Sec23 labeling was still visible. The 12 hour 50 M CI 976 treatment significantly redu ced the amount of IVI 16/TXNPx labeling outside the PV. The scenario was the same for the 24 hour time point, 50 M CI 976 significantly reduced the amount of IVI 16/TXNPx labeling outside the PV in comparison to the no treatment sample. A quantificatio n of the amount of IVI 16/TXNPx labeling outside the PV with CI 976 treatment was performed (Figure 3 12). The quantification was performed in the same manner as the original IVI 16/TXNPx labeling outside the PV quantification. Measurements of the no tre atment sample revealed that IVI 16/TXNPx labeling outside the PV increased from 12 to 24 hours, which correlated with the original quantification of IVI 16/TXNPx labeling outside the PV. However, treati ng infected cells with 10 M CI 976 slightly decrease d the measured amount of IVI 16/TXNPx outside the PV at 12 and 24 hours, indicating that the compound does seem to affect the m olecules export. Treating with CI 976 at 50 M significantly reduced the amount of IVI 16/TXNPx labeling outside the PV at both 12 and 24 hours. Treating with either 10 M or 50 M CI 976 does not greatly affect the intensity of IVI 16/TXNPx labeling within the PV. This
56 suggested that the decrease of IVI 16/TXNPx labeling outside the PV was from decreased export of the protein, n ot from an overall decreased production of protein by the parasite. These results provide further evidence that COP II vesicles are implemented in export of molecules from the Leishmania PV. Blocking Endoplasmic Reticulum Recruitment Affects IVI 16/TXNPx Export It has been shown that the PV can actively recr uit host endoplasmic reticulum (ER) molecules to its membrane (Ndjamen et al., 2010). This ability provides a mechanism by which the PV could acquire host COP II coat proteins to utilize for PV export. Work within our laboratory has shown that a compound termed Retro 2 can inhibit the recruitment of ER to the PV membrane, causing shrinkage in the size of PVs and decreasing parasite proliferation. Considering this, it may also be possible for Retro 2 t o block the formation of COP II coat proteins from PVs. Such an effect would decrease the amount of IVI 16/TXNPx labeling outside the PV, if the molecules export via COP II vesicles. It was first necessary to verify that the Retro 2 compound was affecti ng the RAW 264.7 cellular host. As mentioned in Chapter 1, Retro 2 affects cells by perturbing the localization of Syntaxin 5, a SNARE [soluble NSF(N ethylmaleimide sensitive fusion protein) attachment protein receptor] that usually functions in the ER Go lgi intermediate region (Seaman and Peden, 2010). Therefore, an IFA was performed in which Syntaxin 5 was labeled so that its cellular localization could be analyzed. Under normal circumstances, Syntaxin 5 should label around the Golgi apparatus. The Gol gi apparatus can be viewed by labeling the protein GM130, which is tightly bound to Golgi membranes (Nakamura et al., 1995). After Retro 2 treatment, one would expect the Syntaxin 5 to label in small vesicles within the cytoplasm (Seaman and Peden, 2010).
57 The IFA was performed such that Syntaxin 5 was labeled green, GM130 as red, and DAPI labeled the nuclei blue (Figure 3 13). As expected, the no treatment sample revealed that Syntaxin 5 was localized to the region of the Golgi apparatus. Treatment with 100 M Retro 2 revealed a dispersed labeling pattern of Syntaxin 5 within the cytoplasm, which indicated that the drug properly affected the RAW 264.7 cells. Quantification was performed, using pictures from the previously described IFA and ImageJ softwar e, where the amount of Syntaxin 5 labeling in the Golgi region was measured (Figure 3 14). Also measured was the amount of Syntaxin 5 labeling outside the Golgi region. It wa s found that treatment with 100 M Retro 2 decreased the amount of Syntaxin 5 la beling at the Golgi region. Correspondingly, it was found that treatment with 100 M Retro 2 increased the amount of Syntaxin 5 labeling outside the Golgi. As mentioned earlier, Retro 2 has the ability to block ER recruitment to the PV. Considering thi s, an experiment was designed to assess whether or not Retro 2 could block recruitment of calnexin to the PV membrane. Calnexin is a transmembrane ER resident protein (Ndjamen et al., 2010). Thus, its recruitment to the PV is indicative of ER recruitment To set up the experiment, RAW 264.7 cells were transfected with a Calnexin GFP construct, which causes ER to fluoresce green. These cells were then infected for 4 hours before Retro 2 was added at a 100 M concentration. After 48 hours post infection, coverslips were collected and an IFA was performed (Figure 3 15). In the IFA, LAMP was labeled red and DAPI labeled nuclei blue. In the no treatment sample, it was found that the Calnexin GFP construct delineated the PV, which
58 indicated ER recruitment. The Calnexin GFP construct did not label the PV in Retro 2 treated samples, which indicated blockage of ER recruitment. Having verified that Retro 2 blocked the recruitment of ER to the PV, an experiment was designed to see if the export of IVI 16/TXNPx would be affected by the compound. After treatment with Retro 2 at a 100 M concentration, coverslips were collected at 24 and 48 hours post infection and an IFA was performed (Figure 3 16). For this IFA, IVI 16/TXNPx was labeled green, LAMP was labeled red, and DAPI labeled nuclei as blue. It wa s found that treatment with 100 M Retro 2 inhibited the labeling of IVI 16/TXNPx within the host cell at both 24 and 48 hour time points, which indicated blockage of export from the PV. Quantification of the a mount of IVI 16/TXNPx labeling outside the PV was performed using pictures from the above described IFA and ImageJ software (Figure 3 17). The no treatment sample revealed that the highest amount IVI 16/TXNPx labeling outside the PV occurred at 24 hours p ost infection, which corresponded with previous ob servations. Treatment with 100 M Retro 2 decreased the amount of IVI 16/TXNPx labeling outside the PV at both 24 and 48 hours post infection. The fluorescence intensity of IVI 16/TXNPx within the PV was not significantly affected by Retro 2 treatment at either time point, indicating that the Retro 2 affected IVI 16/TXNPx export rather than protein production. Overall, this data suggests that blocking ER recruitment may inhibit export from the PV. Leish mania Utilizes COP II Vesicles to Modulate Host Cell S ignaling Leishmania has the ability suppress a number of macrophage activities. The parasites control the host macrophage by modulating its signal pathways, the end result being repression of various c ytokines and microbicidal molecules. The cytokines which Leishmania suppresses are primarily those which are pro inflammatory (Gregory and
59 Olivier, 2005). When the parasite suppresses host signaling pathways, it is likely doing so through secreted effect or proteins. If these effector proteins export from the PV using COP II vesicles, blocking COP II vesicles with CI 976 should inhibit the parasites ability to suppress host signaling pathways. An Enzyme Linked Immunosorbent Assay (ELISA) was designed whi ch would test this hypothesis. The pathway w e choose to assess was the tumor necrosis factor (TNF ) pathway. Involved in inflammation, TNF initiates the production and secretion of a cascade of cytokines when activated (Aeberli et al., 2002). When macrophages are activated using compounds like lipopolysaccharid e (LPS) and interferon hey are induced to secrete TNF by Leishmania inhibits the secretion of TN F ier, 2005). Treatment of infected, activated macrophages with CI 976 should cause the levels of secreted TNF to increase, if COP II vesicles are used to secrete ef fectors which suppress the TNF pathway. To begin the experiment, RAW 264.7 macrophages were infected for 4 hours before parasites were washed away. After this, CI 976 was added at four diff erent concentrations: 7.5 M, 12.5 M, 25 M, and 50 M. After 2 hours post CI 976 treatment, the macrophages were activated using a combination of LPS and IFN Supernatants of the cell culture were then collected 24 hours later, and an ELISA was performe d to measure the levels of TNF 18). Activated macrophages that were infected but received no treatment served as the baseline for evaluation of t he effect of CI 976. It was found that treatment with CI 976 at all concentrati ons increased the level of TNF e baseline. The effect on TNF highest for the 12.5 M and 25 M CI 976 treatment. Levels of TNF 50 M CI 976
60 treatment decreased in comparison to the lower concentrations. This might be due to a cytotoxic effect CI 976 can have on RAW 264.7 cells at high concentrations, which caused a decrease in the number of cells available to secrete TNF experimentation is needed to verify that CI 976 do es not affect the level of TNF produced by uninfected RAW 264.7 cells.
61 Figure 3 1. Export of IVI 16/TXNPx outside the PV. Time points are indicative of the hours post infection and a re arranged as follows: A) 4 h, B) 12 h, C) 24 h, D) 48 h, E) 48 h. LAMP delineat es the PV The PV is outlined with a yellow at would be outlined is shown ( row E), with the same PV outlined ( r ow D). The host cell is also outlined with a white dotted line to provide cellular context. Arrows point to IVI 16/TXNPx labeling outside the PV.
62 Figure 3 2. cTXNPx is not exported outside the PV. The time points post infectio n are arranged as follo ws: A) 4 h, B) 12 h, C) 24 h, D) 48 h. There was no labeling of cTXNPx outside the parasite at any time point post infection.
63 Figure 3 3. Quantification of IVI 16/TXNPx labeling outside the PV. A) The measured fluorescence intensity outside the PV. B) The measured fluorescence intensity within the PV. The measured fluorescence intensity of IVI 16/TXNPx outside the PV, in comparison to that of cTXNPx, indicated that IVI 16/TXNPx was ex port ed from the PV. T he highest amount of IVI 16/TXNPx labeling outside the PV occurred at 24 h post infection, and was determined to be significantly different from that of the labeling of cTXNPx. Significance is indicated by a yellow star. Each time point was based on measurements from 45 cells. A.F.U. is Arbitrary Fluorescence Units.
64 Figure 3 4. Expression levels of IVI 16/TXNPx and cTXNPx. A) IVI 16/TXNPx protein expression. B) cTXNPx protein expression. C) Actin loading control. Time points are indicative of the hours post infection. IVI 16/TXNP x protein expression appeared to be induced over the course of the infection. In contrast, cTXNPx expression remained constant throughout the cour se of the infection. Actin serves as a loading control. The ladder is presented as orange bars with the cor responding molecular weight in kilo Daltons (kDA) written in yellow.
65 Figure 3 5. IVI 16/TXNPx resides in vesicles after PV export. A) Cells permeabilized with saponin. B) Cells permeabilized with Streptolysin O. C) Additional cells permeabili zed with Streptolysin O. Selective permeabilization with Streptolysin O revealed that IVI 16/TXNPx was contained within a vesicular membrane while in the host cell. Cells which receiv ed saponin permeabilization showed that IVI 16/TXNPx was exported durin g the infection. Arrowheads point to IVI 16/TXNPx outside the PV. The Strept olysin O treated cells that were fully permeabilized also revealed that IVI 16/TXNPx was exported outside the PV. Streptolysin O permeabilized cells in which only the host cell plasma membrane was permeabilized within the same field showed that IVI 16/TXNPx did not label outside the PV wit hin infected cells, which indicated the molecule was in a vesicle. Parasite nuclei are indicated by arrows.
66 Figure 3 6. IVI 16/TXNPx co localizes with Sec23. Co labeling of IVI 16/TXNPx with Sec23 revealed that the two molecules co localized after IVI 16/TXNPx was exported from the PV. The orange boxed areas indicate regions where labeling of IVI 16/TXNPx co localized with Sec23. Zoo med in pictures of these regions are featured in the co localization column. Three cells are presented to emphasize the reproduc ibility of these results
67 Figure 3 7. Parasites label for Sec23. DAPI stain revealed the parasite nuclei and kinetop last.
68 Figure 3 8. IVI 16/TXNPx and Sec23 co localize on vesicles. IVI 16/TXNPx was labeled with 5 nM gold particles while Sec23 was labeled with 15 nM gold particles. A) An EM section taken from a cell which was infected for 24 h Th e orange box highlights an area where the labeling of IVI 16/TXNPx co localized wi th Sec23 labeling. B) Magnification of the orange box from panel A The two molecules appeared to label on a vesicular structure. C) An EM section taken from a cell which was infected for 24 h The blue box highlights an area where Sec23 labeled the PV membrane. D) Magnification of the blue box from panel C The small black arrow points to IVI 16/TXNPx labeling while the large black arrows points to Sec23 labeling. (The EM data presented here is pending publication and was performed by Johnathan Canton and Peter Kima.)
69 Figure 3 9. CI 976 inhibits the labeling of 5C6 at the host cell surface. A) No t reatment. B) Treatment with 10 M CI 976 for 12 h C) Treatment w ith 10 M CI 976 for 24 h. D) Treatment with 50 M CI 976 f or 12 h. E) Treatment with 50 M CI 976 for 24 h. Treatment with 50 M CI 976 significantly decreased the amount of 5C6 labeling at the host cell surface at both 12 h and 24 h. Arrows point to areas where the 5C6 label was retained in the host cell.
70 Figure 3 10. Quantification of 5C6 labeling at the host cell surface. Treatment with CI 976 at 50 M significantly decreased the amount of 5C6 labeling at the host cell surface at 12 h and 2 4 h, as indicated by the star. Ten cells were measured for each condition at both time points. A.F.U. is Arbitrary Fluorescence Units.
71 Figure 3 11. CI 976 treatment inhibits export of IVI 16/TXNPx to the host cell. A) No treatment 12 h post i nf ection. B) Treatment with 10 M CI 976 12 h post treatment. C) Tre atment with 50 M CI 976 12 h post treatment. D) No treatment 24 h post i nfection. E) Treatment with 10 M CI 976 24 h post treatment. F) Treatment with 50 M CI 976 24 h post treatment Cells which received no treatment had prominent labeling of IVI 16/TXNPx within the host cell. White a rrows point to IVI 16/TXNPx which was exported to the host cell. Areas where IVI 16/TXNPx co localized with Sec23 are indicated by orange boxes. Cel ls that received 10 M CI 976 had reduced IVI 16/TXNPx labeling within the cell. Cells which received 50 M CI 976 had significantly reduced labeling of IVI 16/TXNPx within the host cell.
72 Figure 3 12. Quantification of the effect of CI 976 on IVI 16/TX NPx export. A) The fluorescence intensity of IVI 16/TXNPx outside the PV. B) The fluorescence intensity of IVI 16/TXNPx wi thin the PV. Treatment with 50 M CI 976 significantly decreased the amount of IVI 16/TXNPx export ed into host cell at 12 h and 24 h, as indicated by the star. The fluorescence intensity of the labeling of IVI 16/TXNPx within the PV was not significantly affected by the CI 976 treatment at either time point, indicating that CI 976 affected the export of IVI 16/TXNPx rather than prote in production. Twenty cells were measured for each condition at both time points. A.F.U. is Arbitrary Fluorescence Units.
73 Figure 3 13. Retro 2 mislocalizes Syntaxin 5. A) No tr eatment. B) Treatment with 100 M Retro 2. Syntaxin 5 was localized to the Golgi apparatus, labeled by GM130, unde r the no treatment condition Tr eatment with 100 M Retro 2 caused Syntaxin 5 to label with a dispersed cytosolic pattern.
74 Figure 3 14. Quantification of the localization of Syntaxin 5. Treatment with 100 M Retro 2 caused a decrease of Syntaxin 5 labeling at the Golgi and a corresponding increase in cytosolic labeling, termed Extra Golgi labeling. Ten cells were measured for each condition. A.F.U. is Arbitrary Fluorescence Units.
75 Figure 3 15. Retro 2 blocks ER recruitment to the PV. A) No treatment. B) Tr eatment with 100 M Retro 2. RAW 264.7 host cells w ere transfected with a Calnexin GFP construct before infection. U nder no treatment conditions calnexin was recruited to the PV mem brane. Tr eatment with 100 M Retro 2 inhibited ER recruitment to the PV. Arrows point to parasite nuclei.
76 Figure 3 16. Retro 2 inhibits the export of IVI 16/TXNPx to the host cell. A) No treatment 24 h post in fection. B) Treatment with 10 0 M Retro 2 24 h post treatment. C) No treatment 48 h post infection. D) Treatment with 100 M Retro 2 48 h post treatment. Cells which received no treatment had labeling of IVI 16/TXNPx within the host cell. Arrows point to IVI 16/TXNPx which was exp orted to the host cell. Cells which received Retro 2 at 100 M had significantly reduced labeling of IVI 16/TXNPx within the host cell.
77 Figure 3 17. Quantification of the effect of Retro 2 on IVI 16/TXNPx export to the host cell. A) Fluorescenc e intensity of IVI 16/TXNPx outside the PV. B) Fluorescence intensity of IVI 16/TXNPx within the PV. Treatment with Retro 2 at 100 M inhibited the export of IVI 16/TXNPx to the host cell at both 24 h and 48 h. The fluorescence intensity of IVI 16/TXNPx within the PV was not significantly affected by Retro 2 treatment at either time point, indicating that Retro 2 affected IVI 16/TXNPx export rather than protein production. Twenty cells were measured for each condition at each time point. A.F.U. is Arbit rary Fluorescence Units.
78 Figure 3 18. CI 976 blocks Leishmania from suppressing macrophage tumor necrosis factor The RAW 264.7 untreated sample showed that basal production of TNF Activa tion stands for RAW 264.7 cells that received activation by lipopolysachharide and interferon Amazonensis stands for RAW 264.7 cells which were infected with L. amazonensis parasites. Amazonensis + Activation stands for activated RAW 264.7 cells that were infected, which caused a suppression of TNF production in comparison to the activated RAW 264.7 cells alone. All CI 976 treatment samples shown were infected and activated in the same manner as mentioned above. Treatment with the CI 976 compound at all concentrations increased the amount of TNF activated sample which received no treatment (Amazonensis + Activation).
79 CHAPTER 4 DISCUSSION To date, there is little evidenc e of parasite molecules being secreted from the Leishmania PV. The results presented in this thesis suggest that the tryparedoxin peroxidase termed IVI 16/TXNPx is exported from the PV. It has already been shown that Leishmania secretes tryparedoxin pero xidase molecules into its extracellular environment (Cuervo et al., 2009). Thus, the thought that this molecule has functions outside the parasite is logical. The mechanism by which IVI 16/TXNPx would exit the PV is unknown. However, selective permeabil ization with Streptolysin O revealed that IVI 16/TXNPx likely exists within vesicular compartments during its export from the PV. When the PV acquires endoplasmic reticulum (ER) components (Ndjamen et al., 2010), it could also acquire COP II coat protein s, which normally function to form vesicles for transport from the ER (Dacks et al., 2009). The COP II protein coat could then acquire cargo from within the Leishmania PV, subsequently bud off, and transport the cargo in a vesicle to some target destinati on. The co localization of IVI 16/TXNPx molecules with host Sec23, an essential COP II coat protein (Barlowe, 2002), provided evidence for this mechanism. Furth ermore, the observation that treatment with the COP II inhibitor CI 976 diminished the amount of IVI 16/TXNPx that reached the host cell strengthens this hypothesis. If IVI 16/TXNPx is exported via COP II vesicles, there must be a mechanism by which it is selected as cargo for export from the PV. There are two main hypotheses which describe possi ble COP II vesicle soluble cargo specific selection process (Belden and Barlowe, 2001; Sato and Nakano, 2007 ). In this model, soluble cargo molecules export from the ER in vesicles at a concentration equal to that
80 found in the ER lumen (Charles, 2003). If IVI 16/TXNPx exits the PV via this mechanism, the parasite would only need to produce the protein and secrete it into the PV lumen at a sufficient concentration. Another COP II sol uble cargo selection mechanism is the receptor mediated export model, which proposes an active selection process that concentrates cargo into vesicles. This mechanism states that trans membrane cargo receptors are utilized to link lumenal cargo to the COP II coat proteins. A number of membrane proteins have been identified that play such roles, including Erv29, which is required for efficient ER export of certain secretory cargo in yeast (Charles, 2003). A region on the soluble protein gp residues which define the motif are I39, L42, and V52, and are suggested as being an L export motif. In agreement with this, database searches have identified other s ecretory proteins with such hydrophobic residues that have similar spacing and positioning (Otte and Barlowe, 2004). Intriguingly, the IVI 16/TXNPx sequence contains the I L V residues with identical spacing, specifically being I103, L106, and V116. Of even greater interest is that the V116 residue is one of the amino acids that is not conserved between IVI 16/TXNPx and a number of other Leishmania intracellular tryparedoxin peroxidase variants, in fact it is the only variant containing the residue in th at position (Figure 4 1). Employing SWISS Model (Guex and Peitsch, 1997; Schwede et al., 2003; Arnold et al., 2006 ), the structure of IVI 16/TXNPx was modeled using the crystal structure of a tryparedox in peroxidase from Trypanosoma c ruzi as a template (P ieyro et al., 2005), and subsequently visualized with PyMOL software (Schrodinger, LLC). The structure
81 reveals that I103 and V116 are placed on the exterior of the protein, making them easily accessible to a receptor protein (Figure 4 2). Further inspec tion of the model reveals that L106 exists within a small pocket and is still accessible from the exterior at the correct angle. Although these observations are intriguing, it must be stressed that they are merely speculative, and have no experimental bas is. The Retro 2 compound inhibits the retrograde pathway utilized by Ricin molecules via the mislocalization of Synt axin 5 (Seaman and Peden, 2010). Retro 2 was found to inhibit the export of IVI 16/TXNPx to the host cell. It was previously discovered th at the PV has the ability to recruit ER components to its membrane, including the ER resident protein calnexin (Ndjamen et al., 2010). Our results indicated that Retro 2 inhibited the recruitment of calnexin to the PV membrane, which suggested that additi onal ER components may also be blocked. Building off this concept, we propose that Retro 2 may also act to inhibit the recruitment of COP II coat proteins derived from the ER, resulting in a decrease of IVI 16/TXNPx reaching the host cell. As described in Chapter 1, tryparedoxin peroxidase enzymes are an essential component of the Leishmania organism, mostly known for their ability to combat oxidative stress (Singh and Dubey, 2009). Only recently has it been suggested that this type of enzyme may function outside the PV (Kima et al., 2010). However, it is unknown what this function may be, beyond that of the role already described. A recent study found that genetically deleting a mitochondrial tryparedoxin peroxidase variant (mTXNPx) resulted in parasite s which could not thrive in a murine model of infection. Of particular interest was that this avirulent phenotype was not due to the lack of peroxidase activity conferred by mTXNPx, as the parasites still behaved like wild type
82 organisms when subjected to a number of oxidative stress models. Based on their results, the authors proposed a novel function for mTXNPx, that of a molecular chaperone. They suggested this because the enzyme can suppress the thermal aggregation of citrate synthesis in vitro (Cast ro et al., 2011), a characteristic trait of chaperones (hrman et al., 2007). Furthermore, the mtxnpx knockout strain was more sensitive to temperature shifts from 25 to 37C, which resembles a phenotype of organisms deficient in chaperone genes. The re sults of this research change the model that tryparedoxin peroxidases function solely to detoxify peroxides, and also demonstrate that the molecule can be a determinant of pathogenicity (Castro et al., 2011). Even if IVI 16/TXNPx does not have an undisco vered function, its innate ability to detoxify peroxides could serve a purpose in the host cell. In eukaryotic cells, hydrogen peroxide has been established as an important regulator of signal transduction. Due to the cytotoxic nature of hydrogen peroxid e, generation of the molecule is highly regulated. In line with this, the localization and activities of host hydrogen peroxide detoxifying enzymes are also highly regulated. A number of pathways are regulated by hydrogen peroxide, including some involve d in immune cell activation (Veal et al., 2007). For instance, the molecule has been suggested to promote lymphocyte activation by controlling the activity of negative regulatory phosphatases within a cell (Reth, 2002). Furthermore, reactive oxygen inter mediates like hydrogen peroxide have been suggested to be the primary stimuli in activating the eukaryotic transcription factor NF kB (Schmidt et al., 1995). NF kB, considered the central mediator of immunity, promotes the expression of over 100 genes pri marily involved in the host immune
83 response. The genes expressed include a large number of cytokines, receptors for immune recognition, proteins for antigen presentation, and adhesion receptors for transmigration of blood vessel walls (Hiscott et al., 200 1). Thus, it is likely a primary target for an intracellular pathogen. Considering this, if IVI 16/TXNPx was delivered to the appropriate location within a host cell, it could disrupt the activation of pathways like NF kB by eliminating the hydrogen perox ide messengers. It was noted during CI 976 treatment infections that the CI 976 compound did not noticeably decrease parasite proliferation during infections of the RAW 264.7 host cells, at least from an observational standpoint. Perhaps this is because IVI 16/TXNPx does not serve to increase parasite survivability within an individual host cell. Rather, the molecule may serve to promote overall parasite proliferation within a mammalian host by subverting the activation of lymphocytes and leukocytes outs ide the immediate macrophage host cell. Indeed, an effect like this could not be elucidated using an in vitro infection model. Although IVI 16/TXNPx was the molecule of focus for this study, the proposed COP II PV export pathway would likely be utilized b y multiple proteins. Our results showed that blocking COP II export of the host inhibited Leishmania parasites from suppressing tumor necrosis factor provided evidence that the COP II export pathway is necessary for Leishmania to modulate host signaling, presumably because it is needed for parasite protein secretion. L signal utilized by IVI 16/TXNPx for COP II cargo selection, additional proteins could be identified by performing a search of the Leishmania proteome for proteins which include the motif. There are likely other pathways and mechanisms used by Leishmania t o secrete proteins from the PV.
84 However, we are the first provide to evidence that COP II vesicles are utilized for PV export by Leishmania
85 Figure 4 1. The IVI L uncated version of the sequen ce is presented so that individual residues are viewable. Red L 16/TXNPx sequence. The V of the motif is not conserved between the sequences, and is only present in IVI 16/TXNPx at this lo cation. [ Figure adapted from Kima, P.E., Bonilla, J.A., Cho, E., Ndjamen, B., Canton, J., Leal, N., and Handfield, M. (2010). Identification of Leishmania Proteins Preferentially Released in Infected Cells Using Change Mediated Antigen Technology (CMAT). PLoS Neglect. Trop. D. 4 e842. (Page e842, Figure 2).]
86 Figure 4 2. L 16/TXNPx model st ructure. The model of IVI 16/TXNPx structure indicated that the Valine (VAL 116) and Isoleucine (ILE L molecule. The Leucine (LEU 106) is located within an accessible pocket.
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94 BIOGRAPHICAL SKETCH Vikarma Wayne Brooks grew up in Plant City, FL, and graduated from Plant City High School in 2006. He then attended the University of Florida where he pursued a Bachelor of Science in Microbiology and Cell Science, which he completed in 2010. Following this trend, he attended graduate school at the University of Florida, where he completed a Master of Science in Microbiology and Cell Science in May of 2012.