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Regulation and Characterization of Nucleic Acid-Binding Proteins in Chlamydia Trachomatis

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Title:
Regulation and Characterization of Nucleic Acid-Binding Proteins in Chlamydia Trachomatis
Creator:
Runac, Justin M
Place of Publication:
[Gainesville, Fla.]
Florida
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University of Florida
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english
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1 online resource (11 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Biochemistry and Molecular Biology (IDP)
Committee Chair:
GRIESHABER,SCOTT STEPHEN
Committee Co-Chair:
RICE,KELLY C
Committee Members:
FLANEGAN,JAMES B
MCKENNA,ROBERT
KIMA,PETER EPEH
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Apoptosis ( jstor )
Cell death ( jstor )
Chlamydia ( jstor )
Chlamydia trachomatis ( jstor )
DNA ( jstor )
Histones ( jstor )
Infections ( jstor )
Nucleic acids ( jstor )
Proteins ( jstor )
RNA ( jstor )
Biochemistry and Molecular Biology (IDP) -- Dissertations, Academic -- UF
chlamydia -- regulation -- srna
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bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Medical Sciences thesis, Ph.D.

Notes

Abstract:
The obligate intracellular bacterium Chlamydia trachomatis is responsible for more than 3% of worldwide blindness, the most prevalent bacterial sexually transmitted disease, and is a common cause of pelvic inflammatory disease. C. trachomatis progresses through a biphasic development cycle alternating between an inactive, infectious form called an elementary body (EB) and a metabolically active form called a reticulate body (RB). EBs are characterized by a condensed nucleoid structure, minimal transcription, and no protein expression. The chlamydial chromosome becomes relaxed as the EB progresses into the RB phase, and nearly all genes within the chlamydial genome become transcribed at some time within the RB phase. Small non-coding RNAs (sRNA) have been detected in a variety of bacteria and play a significant role in the regulation of the proteome. Approximately 40 putative sRNAs have been identified in C. trachomatis. The majority of these putative sRNAs are transcribed from the antisense strand of an ORF. However, several sRNAs have been identified that are transcribed from intergenic regions, and are likely to have multiple mRNA targets. Previous work has identified the sRNA IhtA, which is critical to regulation of the development cycle of C. trachomatis. IhtA suppresses expression of Hc1, a histone-like protein that is responsible for condensing chlamydial chromatin upon differentiation from the RB to EB form. We hypothesize that IhtA interacts with several other transcripts, including CTL0322, whose expressed products are critical to the chlamydial development cycle. Our findings demonstrate that IhtA binds to the 5 prime region of the Hc1 and CTL0322 transcripts and prevents expression of each protein. We also show for the first time that Hc1 and CTL0322 proteins have a higher affinity for dsDNA over ssDNA and RNA, with a strong preference for GC-rich regions. Due to the chlamydial genome being very AT-rich, the GC-rich dsDNA preference is likely important for regulation of transcription in combination with sigma factors. These results contribute to the greater understanding of bacterial sRNA regulation and may allow for the development of designer sRNAs as a molecular biology technique. ( en )
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In the series University of Florida Digital Collections.
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Includes vita.
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Includes bibliographical references.
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Description based on online resource; title from PDF title page.
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This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: GRIESHABER,SCOTT STEPHEN.
Local:
Co-adviser: RICE,KELLY C.
Statement of Responsibility:
by Justin M Runac.

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LD1780 2014 ( lcc )

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REGULA TION AND CHARACTERIZATION OF NUC L EIC ACID BINDING PROTEINS IN CHLAMYDIA TRACHOMATIS By JUSTIN RUNAC A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014

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© 2014 Justin Runac

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To FORTRAN 77, you made learning engineering so boring that I decided to become a biochemist instead

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4 ACKNOWLEDGMENTS I thank my parents for giving me the opportunity to attend challenging schools and the support to allow me to focus on my education. I thank my mentors, Drs. Scott and Nicole Grieshaber, for allowing me to join their lab and teaching me how to use technology that I never encountered in my undergraduate studies. I thank my friend Kim, who helped me heal up after a shattered collarbone, and get back to the lab. I somehow managed to not miss a single passage of my cell culture even with my collarbone in 5 pieces. I thank my lab mate Andrea for always giving me encouragement to keep moving g well.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 17 Epidemiology of Chlamydia ................................ ................................ .................... 17 Ch lamydia Life Cycle ................................ ................................ .............................. 18 Role of Nucleic Acid Binding Proteins in Chlamydial Development ........................ 19 Regulation within Chlamydia ................................ ................................ ................... 20 Host Egress ................................ ................................ ................................ ............ 22 Summation ................................ ................................ ................................ .............. 23 2 MATERIALS AND METHODS ................................ ................................ ................ 25 Cell Culture ................................ ................................ ................................ ............. 25 Bacteria Cells ................................ ................................ ................................ .......... 25 Harvesting and Purification of Chlamydial EBs ................................ ....................... 25 Harvesting and Purification of Chlamydial RBs ................................ ....................... 26 Vector Construction ................................ ................................ ................................ 26 Kill assays and Growth Curves ................................ ................................ ............... 27 Microscope ................................ ................................ ................................ ............. 28 Live Cell Imaging ................................ ................................ ................................ .... 28 Microinjection ................................ ................................ ................................ .......... 28 Transfection ................................ ................................ ................................ ............ 29 Egress Diffusion Experiments ................................ ................................ ................. 29 Fluorescence Staining of E. coli ................................ ................................ .............. 30 Fluorescence Staining of Eukaryotic Cells ................................ .............................. 31 PAGE and Western Blot Analysis ................................ ................................ ........... 32 In gel Staining ................................ ................................ ................................ ......... 34 Densitometry ................................ ................................ ................................ ........... 34 DNA and RNA Electrophoresis ................................ ................................ ............... 34 Soft Agar Assay ................................ ................................ ................................ ...... 35 Recombinant Protein Purification ................................ ................................ ............ 36 Protein Concentration Measurements ................................ ................................ ..... 38 RNA Expression and purification ................................ ................................ ............ 38

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6 BLI (sRNA mRNA Interaction) ................................ ................................ ................ 38 Oligonucleotides for BLI (Protein NA Interaction) ................................ ................... 39 BLI (Protein NA Interaction) ................................ ................................ .................... 40 Enzyme Activity Assay ................................ ................................ ............................ 41 DNase Protection Assay ................................ ................................ ......................... 42 Preparation of Genomic DNA ................................ ................................ ................. 42 Nucleic Acid Isolation (for qPCR) ................................ ................................ ............ 43 qPCR ................................ ................................ ................................ ...................... 43 3 CHARACTERIZATION OF NU CLEOTIDE BINDING PROTEINS IN CHLAMYDIA ................................ ................................ ................................ ........... 45 Summary ................................ ................................ ................................ ................ 45 Background ................................ ................................ ................................ ............. 45 Nucleic Acid Bindi ng Proteins in Chlamydia ................................ ..................... 45 Ectopic Expression of Proteins in Other Organisms ................................ ......... 46 Functional analysis of HctA and HctB ................................ .............................. 47 Characteristics of the Hypothetical Protein CTL0322 ................................ ....... 48 Results ................................ ................................ ................................ .................... 49 CTL0322 Localizes to the Nucleus in Eukaryotic Cells ................................ .... 49 CTL0322 Is Toxic to Escherichia coli ................................ ............................... 50 Recombinant Expression of CTL0322 Induces Apparent SOS Response in E. coli ................................ ................................ ................................ ............ 50 CTL0322 Has Binding Preference to GC Rich dsDNA ................................ ..... 51 CTL0322 Degrades DNA ................................ ................................ .................. 52 CTL0322 Protects DNA from Endonuclease Activity ................................ ........ 52 Discussion ................................ ................................ ................................ .............. 53 4 INTERACTIONS OF THE SMALL RNA REGULATOR IhtA ................................ ... 64 Summary ................................ ................................ ................................ ................ 64 Background ................................ ................................ ................................ ............. 65 Regulation of Histone Expression in Chlamydia ................................ ............... 65 Correlation of IhtA and HctA Expression ................................ .......................... 65 IhtA Rescues E. coli from HctA induced Cell Death ................................ ......... 66 IhtA Is Conserved Among Species ................................ ................................ ... 66 Identification of Putative IhtA Targets ................................ ............................... 67 Hypothesis of CTL0322 and IhtA Interaction ................................ .................... 67 Results ................................ ................................ ................................ .................... 6 7 IhtA Interaction with HctA Conserved Across Species ................................ ..... 67 IhtA Interacts with the CTL0322 Transcript In Vitro ................................ .......... 68 IhtA Inhibits Expression of CTL0322 cheZ Fusion Protein in E. coli Surrogate System ................................ ................................ ......................... 69 CTL0322 Is Tempora lly Expressed During Development ................................ . 70 Discussion ................................ ................................ ................................ .............. 71

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7 5 HOST EGRESS ................................ ................................ ................................ ...... 79 Summary ................................ ................................ ................................ ................ 79 Background ................................ ................................ ................................ ............. 79 Mechanisms of Host Cell Exit ................................ ................................ ........... 79 Mac/perforin ................................ ................................ ................................ ..... 80 Hypothesized Role of Chlamydial Protein CT153 ................................ ............. 81 Results ................................ ................................ ................................ .................... 81 Inclusion Membrane Ruptures Prior to Host Lysis ................................ ............ 81 Lysis Likely Occurs Without Discrete Pore Formation ................................ ...... 82 CT153 Is an Early Gene in the Development Cycle ................................ ......... 82 CT153 Localizes to the Chlamydial Membrane ................................ ................ 83 Discussion ................................ ................................ ................................ .............. 84 6 CONCLUSION AND FUTURE DIRECTIONS ................................ ......................... 89 LIST OF REFERENCES ................................ ................................ ............................... 92 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 99

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8 LIST OF TABLES Table page 3 1 Kinetics for prote in oligonucleotide interactions . ................................ ................. 60 4 1 Putative IhtA targets ................................ ................................ ............................ 75

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9 LIST OF FIGURES Figure page 3 1 All putative nucleic acid binding proteins localize to the nucleus when ectopically expressed in HeLa cells ................................ ................................ .... 56 3 2 Growth Curve of E. coli during induced expression of recombinant proteins ....... 57 3 3 Expression of CTL0322 demonstrated SOS phenotype in E. coli ........................ 58 3 4 CTL0322 has a binding preference for GC rich dsDNA ................................ ....... 59 3 5 CTL0322 has modest in vitro binding affinity to all types of synthetic oligonucleotides. ................................ ................................ ................................ . 60 3 6 CTL0322 and HctB degrade plasmid DNA ................................ ........................... 62 3 7 CTL0322 protects plasmid DNA ................................ ................................ ........... 63 4 1 IhtA conserved amongst species ................................ ................................ ......... 73 4 2 IhtA of each species interacts with the cognate hctA target mRNA in vitro .......... 74 4 3 IhtA rescues kill phenotype across species ................................ .......................... 75 4 4 Predicted interactions with IhtA ................................ ................................ ............ 76 4 5 IhtA binds to hctA , CTL0097, and CTL0322 in vitro ................................ ............. 76 4 6 CheZ experimental constructs ................................ ................................ .............. 77 4 7 IhtA represses expression of HctA and ctl0322 in E. coli surrogate ..................... 78 4 8 Northern and we stern blots of IhtA and targets ................................ .................... 78 5 1 The inclusion becomes permeable to each fluorophore during egress events . .... 86 5 2 CT153 is an early onset gene and the protein is present throughout the infection cycle. ................................ ................................ ................................ .... 87 5 3 CT153 localizes to the c hlamydia l membrane. ................................ ..................... 88

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10 LIST OF ABBREVIATIONS A Amp APO APS AS ATCC ® ATP BLAST BLI C CB CDC ChIP Cm CPAF CT ddH 2 O DEPC DHS DI DMEM DNA DNase dNTP Adenine Ampicillin Apochromatic lens Ammonium persulfate Anti sense American Type Culture Collection Adenosine triphosphate Basic Local Alignment Search Tool Bio layer interferometry Cytosine Carbenicillin Centers for Disease Control and Prevention Chromatin precipitation Chloramphenicol Chlamydial protease like activity factor Chlamydia trachomatis Double distilled water Diethylpyrocarbonate DNase hypersensitive site Deionized D eoxyribonucleic acid Deoxyribonuclease Deoxynucleotide triphosphate

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11 dsDNA DTT EB Double stranded deoxyribonucleic acid Elementary body ECL EDTA ELISA EMCCD EmGFP FBS FITC g G GFP Gluc GST HBSS hctA hctB His IF IFU IhtA Incs IPTG IVT Enhanced chemiluminescent Ethylenediaminetetraacetic acid Enzyme linked immunosorbent assay Electron multiplying charge coupling device Emerald green fluorescent protein Fetal bovine serum Fluorescein isothiocyanate Gravitational acceleration Guanine G reen fluoresc ent protein Glucose Glutathione S transferase The gene encoding the protein HctA (also known as Hc1 ) The gene encoding the protein HctB (also known as Hc2 ) Histidine Immunofluorescent Inclusion forming unit Inhibitor of HctA translation Inclusion membrane proteins Isopropyl D 1 thiogalactopyranoside In vitro transcription

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12 Kan K D kDa k off k on LB LGV LPS M m Ab MAC MEP MFE miRNA mM MOI Kanamycin Dissociation constant (at equilibrium) Kilodalton Off rate (reverse) On rate (forward) Lysogeny broth Lymphogranuloma venerum Lipopolysaccharide Molar (moles per liter) Monoclonal antibody Membrane attack complex Methylerythritol phosphate Minimal free energy MicroRNA 10 3 molar Multiplicity of infection MOMP MoPn Major outer membrane protein Mouse Pneumonitis mRNA MS Mu ( µ) MW NA ncRNA Ni NTA Messenger RNA Mass spectrometry 10 6 Molecular weight Nucleic acid Non coding RNA Nickel nitrilotriacetic acid

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13 NIH NTP OD O/N ORF Ori PAGE PBK PBS National Institutes of Health Nucleotide triphosphate Optical density Overnight Open reading frame Origin of replication Polyacrylamide gel electrophoresis Phosphate buffered potassium P hosphate buffered saline PCR Polymerase Chain Reaction PF PI qPCR RB Perforin Post infection Quantitative PCR Reticulate body RBS RCF RNA RNase RPMI RT SA SD SDS siRNA sRNA Ribosome binding site Relative centrifugal force Ribonucleic acid Ribonuclease Roswell Park Memorial Institute media Reverse transcriptase Streptavidin Shine Dalgarno sequence Sodium dodecyl sulfate Small interfering RNA Small non coding RNA

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14 Spec SPG Spect inomycin Sodium phosphate glutamate buffer ssDNA STI Single stranded deoxyribonucleic acid S exually transmitted infection T T3SS TAE TARP TBE TBS TEMED tRNA TSS U UV V Thymine Type III secretion system Tris base acetic acid EDTA Translocated actin recruiting p hosphoprotein Tris base boric acid EDTA Tris buffered saline Tetramethylethylenediamine Transfer RNA Transcription start site Uracil Ultraviolet Vol t

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15 Abstract of Dissertation Presented to the Graduate School of the University of Florid a in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REGULA TION AND CHARACTERIZATION OF NUCLEIC ACID BINDING PROTEINS IN CHLAMYDIA TRACHOMATIS By Justin Runac August 2014 Chair: Scott Grieshaber Major: Medical Sciences Biochemistry and Molecular Biology The obligate intracellular bacterium Chlamydia trachomatis is responsible for more than 3% of worldwide blindness , the most prevalent bacterial sexually transmitted disease, and is a common cause o f pelv ic inflammatory disease. C. trachomatis progresses through a biphasic development cycle alternating between a n inac tive, infectious form called an elementary body (EB) and a m etabolically active form called a reticulate body (RB) . EBs are characterized by a condensed nucleoid structure, minimal transcripti on, and no protein expression. The chlamydial chromosome becomes relaxed by the removal of nucleosome structure as the EB progresses into the RB phase, and nearly all genes within the chlamydial genome bec ome transcri ptionally active while in the RB phase. S mall non coding RNA s ( sRNA ) have been detected in a variety of bacteria and play a significant role in t he regulation of the proteome. Approximately 40 putative sRNAs have been identified in C. trachomat is . The majority of these putative sRNAs are transcribed from the antisense strand of an ORF . However, several sRNAs have been identified that are transcribed from intergenic regions, and are likely to have multiple mRNA targets. Previous work has identified the sRNA IhtA , which is critical to regulation

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16 of the development cycle of C. trachomatis . IhtA suppress es expression of HctA , a histone like protein that is responsible for condensing chlamydial chromatin upon differentiation from the RB to EB form . We hypothesize that IhtA interacts with several other transcripts, including CTL0322, whose expressed products are critical to the chlamydial development cycle. of the HctA and CTL03 22 transcripts and prevent s expression of each protein . We also show for the first time that HctA and CTL0322 protein s have a higher affinity for dsDNA over ssDNA and RNA, with a strong preference for GC rich regions . Due to the chlamydial genome being ver y AT rich, the GC rich dsDNA preference is likely important for regulation of transcription in combination with sigma factors. These results contribute to the greater understanding of ba cterial sRNA regulation and may a llow for the development of designer sRNAs as a molecular biology technique.

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17 CHAPTER 1 INTRODUCTION Epidemiology of Chlamydia Chlamydia trachomatis is an obligate intracellular pathogen that is the causative agent of trachoma, which affects 84 million people in 56 countries, and has resulted in 1 3 million current cases of worldwide blindness (Wright, Turner, & Taylor, 2008) . In 2010, C. trachomatis infections were by far the most commonly reported notifiable infectious disease in the United States with a total of more than 1.3 million cases (CDC, 2010) . Because 70 75% of women infected with C . trachomatis are symptom free (World Health Organization, 2001) , it is expected that the number of cases is highly underreported . Chlamydia infections are a common cause pelvic inflammatory disease (PID), which can lead to sc arring inside the female reproductive system and subsequent infertility, ectopic pregnancy, and potentially cervical cancer with other complicating factors (Workowski & Berman, 2010) . C. trachomatis consists of 3 biovars, dist inguishable by the type of infection caused, and 15 major serovars that are distinguished by the proteins found in the outer membrane (Yuan, Zhang, Watkins, & Caldwell, 1989) . Serovars A C are responsible for trachoma, a leading cause of blindness . Serovars D K are responsible for sexually transmitted infections (STIs) that can lead to PID in females, urethritis, and several other diseases . Although urethritis can occur in both females and males, it is difficult to diagnos e in females since the purulent discharge that is symptomatic of urethritis is often lacking in female infections . Serovars L1, L2, and L3 are responsible for the STI lymphogranuloma venerum (LGV) . The serovars that cause LGV are much more invasive than th e C. trachomatis serovars . These LGV causing pathogens attack the lymphatic system via breaks in the skin or

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18 though mucous membranes which results in large swellings that are characteristic of bubonic diseases (Thomson, 2008) . Prior to 2003, cases of LGV in the developed world were extremely rare (Richardson & Goldmeier, 2007) . However recent outbreaks have occurred among gay men in Europe and these outbreaks are often associated with HIV co infecti on (Nieuwenhuis, Ossewaarde, Meijden, & Neumann, 2003) . Chlamydia Life Cycle C. trachomatis undergoes a biphasic development cycle during an infection . A metabolically inactive elementary body (EB) enters the host cell by phago cytosis, which is dependent on host presentation of protein disulfide isomerase (Abromaitis & Stephens, 2009) and the translocation of TARP into the host mediated by the chlamydial type III secretion system (Lane, Mutchler, Khodor, Grieshaber, & Carabeo, 2008) . In the early stages of infection (1 3 hours), C. trachomatis modifies the phospholipid contents of its vacuole, termed an inclusion, to prevent entry into the lysosomal pathway (Hackstadt, Rockey, Heinzen, & Scidmore, 1996) . Within the first 6 hours post infection, EBs differentiate into the metabolically active, but non infectious reticulate body (RB) (Nicholson, Olinger, Chong, Schoolnik, & Stephens, 2003) . RBs are able to increase in number by performing binary fission . This process generally continues until the inclusion fills much of the volume within the host cytoplasm . RBs undergo secondary differentiation asynchronously back to th e infectious EB form as the inclusion becomes filled with RBs, notably along the inclusion inner membrane . The step is characterized by a recondensation of the chromosome to form the nucleoid structure. This step is also accompanied by a reduction in the s ize of the cell from 1 linking by disulphide bonds in the outer membrane proteins to impart a structural rigidity to the outer membrane. This

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19 structural rigidity is believed to play a role in mainta ining osmotic homeostasis in EBs and allowing EBs to survive outside of the host cell in conditions that may otherwise be stressful for the membrane. At 48 72 hours post infection, the inclusion ruptures, and shortly thereafter the host membrane is lysed t o allow C. trachomatis to egress from the host . The chlamydial EBs are then able to restart the development cycle upon infection of a new host. Role of Nucleic Acid Binding Proteins in Chlamydial Development Developmentally regulated nucleic acid binding p roteins were discovered in Chlamydia by use of south western blotting (Wagar & Stephens, 1988) . This technique involves applying oligonucleotide probes to proteins affixed to a nitrocellulose membrane to detect nucleic acid bin ding proteins. The th ree major bands found in the EB south western blot were proteins that migrated to 17 kDa, 25.7 kDa, and 58 kDa. None of these bands were found in the south western blot of RB proteins. The 17 kDa and 25.7 kDa bands were proposed as pos sible nucleoprotein since they were also detected in isolated chromosomal preps, in addition to whole cell lysate preparations. It wa s later determined that the H1 like protein HctA was the 17 kDa protein identified in this earlier stud y (Hackstadt, Baehr, & Ying, 1991) , and the H1 like protein HctB was the 25.7 kDa protein (Brickman, Barry III, & Hackstadt, 1993) . HctA and HctB share significant sequence homology with the eukar yotic histone, H1. The eukaryotic histone H1 is not part of the nucleosome bead, but instead has been shown to interact with the linker DNA between the nucleosomes. The chlamydial histone like proteins, HctA and HctB are present in EBs and are major consti tuents of the condensed chromatin and help form the nucleoid structure. The molecular structure of this condensed chromatin is

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20 unknown. Several other DNA binding proteins were noted in the Wagar south western experiments, but only HctA and HctB have been i dentified and characterized. Regulation within Chlamydia Because C. trachomatis genome and resources . C. trachomatis has a small genome comprising of 889 920 coding sequences, 846 of which are common to all serovars (Thomson, 2008) , with a plasmid encoding 8 ORFs . Although nearly all genes become active sometime during a typical infection cy cle, the process is well regulated to ensure that expression of genes is occurring at the correct time in development . Traditionally, most credit for this regulation was given at the level of transcription with the use of the histone like protein HctA (Hackstadt, Baehr, & Ying, 1991) . More recently, however, attention h as been focused on regulation of expression via small non coding RNA (sRNA) regulators (Grieshaber, Grieshaber, Fischer, & Hackstadt, 2006) (AbdelRahman, Rose, & Belland, 2011) . sRNA regulators are classified based on their mode of action . Some of the earliest identified sRNA regulators belong to the categories of those that modify protein activity and those with intrinsic activities . The former includes 6S sRNA, which has been found to inhibit promoters with weak 35 regions (Cavanagh, Klocko, Liu, & Wassarman, 2008) . An example of the latter processing of tRNA precursors (Ellis & Brown, 2009) . Another class of sRNA regulators is cis encoded RNAs that are true antisense of a portion of an mRNA . While these sRNA reg ulators are limited in scope due to having a single target, they comprised a large population of the total number of unique sRNA regulators in bacteria . Some examples of these antisense sRNA regulators have been found to repress the

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21 expression of toxic pro teins (Fozo, Hemm, & Storz, 2008) . Of more relevance, due to the ability to impact the expression of multiple transcripts, is the category of base pairing sRNA regulators with limited complementarity . This category of sRNA regu lators is often expressed under stress conditions (Gottesman & Storz, 2010) or at critical points in a development cycle . This mechanism of limited complementarity sRNA regulation appears to be similar to that of miRNAs in euka ryotes, but there are significant differences . While eukaryotic miRNAs begin as large transcripts and are later processed by dicer and other RNA modifying enzymes, bacterial sRNA regulators are generally not processed after transcription . Eukaryotic miRNAs transcript . Trans encoded sRNA regulators also typically have very limited complementarity (6 8 bp seed lengths) to their target versus that of miRNAs (Vogel & Wagner, 2007) . Because sRNA regulation in C. trachomatis has only recently been studied, very little is known about the targets of these sRNA regulators . Although sRNA regulators from enteric bacteria often require the chaperone Hfq to be to fully effective (Gottesman & Storz, 2010) , chlamydial sRNAs thus far have not been found to require co factors for their function . One well studied sRNA mRNA interaction in Chlamydia is that of IhtA and its target, hctA . HctA is the gene that encodes for the histone like protein HctA . HctA as mentioned previously, condenses the EB chromosome and is involved in the maintenance of the developme nt cycle . HctA is expressed late in the infection cycle concurrent with the RB to EB transition .

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22 While small RNA regulation in other bacteria often involves base pairing at the ribosome binding site to block expression, similar to what is found with the Ih tA hctA interaction, there are several other regulatory outcomes that have been discovered . Some sRNA regulators bind downstream of the RBS of the target transcript, and this activity results in RNase E dependent decay of the mRNA ( Pfeiffer, Lucchini, Hinton, & Vogel, 2009) . There are also several examples of sRNA regulation in which translation is activated instead of being repressed (Prevost, Salvail, Desnoyers, Jacques, Phaneuf, & Masse, 2007) (Huntzinger, et al., 2005) . Host Egress One of the primary reasons for such tight regulation of the transcri ptome and proteome in Chlamydia is to prepare the cell for successful egress from its eukaryotic host . Because the environment outside of a host cell does not allow an RB to survive and infect a new host, regulation of the development cycle must ensure that most of the chlamydial cells are redifferentiated to EBs prior to egress from the host, and any factors necessary to escape the host and infect new hosts are produced in necessary quantities. Although several attempts have been made to characterize chlamydial egress from the host (Todd & Caldwell, 1985) (Perfettini , Hospital, Stahl, Jungas, Verbeke, & Ojcius, 2003) (Hybiske & Stephens, 2007) , most of these studies have resulted in inconsistent findings or have suffered from lack of detail. Perfettini et al. have suggested that Chlam ydia , in the early stages of infection , blocks apoptosis of the host cell in order to complete a successful infection cycle. Previous research had found that protein synthesis by Chlamydia is responsible for this apoptosis inhibition (Fan, et al., 1998) . The method of inhibition is a blockage of mitochondrial cyctochrome c release, which is one of the primary pathways that results

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23 in apoptosis. As the infection progress es , the inclusion begins to take up a majority of the volume within the host cell . It is during this stage of the infection cycle that the mechanisms for egress or chlamydial persistence are poorly understood. While some cells infected by Chlamydia trachomatis proceed towards a persistent infection and do not relea se the bacteria (Dean & Powers, 2001) , most host cells proceed toward egress and release chlamydial EBs, which allows the infection cycle to begin again in another host cell. The two egress pathways that have been described for Chlamydia trachomatis are the extrusion pathway (Beatty, 2007) , characterized by an unlysed inclusion budding from its host, and a lysis pathway, characterized by sequential membrane permeabilizations. While the sequence of ev ents in each of the egress pathways is poorly understood, identification of the factors involved in each pathway has been almost entirely neglected by Chlamydia researchers . Other intracellular bacteria have been found to use proteases, lipases, and pore f orming proteins in order to facilitate their exit from the host (Paz, et al., 2010) (Gao, Guo, McLaughlin, Morisaki, Engel, & Brown, 2004) (Smith, Marquis, Jones, Johnston, P ortnoy, & Goldfine, 1995) , but due to the additional barrier, the inclusion, which Chlamydia trachomatis must also escape from, a more complicated strategy, is likely necessary for a successful egress event. Summation While several of the steps involved in the Chlamydia trachomatis development cycle have been studied extensively, many of these steps, such as R B E B transition and host egress, have been mostly neglected by Chlamydia researchers. The entry of Chlamydia tra chomatis into its host, facilitated by secretion of the TARP protein, is well studied, and the timing of most of the events of the develop cycle are well known, but

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24 the regulation of those events has not been described fully enough to provide a good unders tanding of the process. In this study, it will be show n that the small non coding RNA IhtA, which is critical in the progression through the EB RB transition, interacts with more than one mRNA and is likely a global regulator of the chlamydial infection cy cle. Ad ditional details will also be provided on the progression of chlamydial egress that has be en lacking in previous studies.

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25 CHAPTER 2 MATERIALS AND METHODS Cell C ulture All cell lines were obtained from A McCoy cells (C RL 1696) , HeLa 229 cells (CCL 2.1) , and COS 7 cells (CRL 1651) were grown in RPMI 1640 , supplemented with 10% FBS and 10 ® ). Bacteria Cells Recombinant protein expression, kill assays, and growth curves were performed using BL21 AI TM E. coli from Invitrogen TM . Soft agar experiments were performed in both JW1870 2 (CGSC Strain # 9556) and E. coli K 12 MG1655. T he JW1870 2 strain features a knockout of the cheZ gene, and the Grieshaber lab performed the knockout of cheZ in MG16 55 to allow more robust motility during rescue of the cheZ knockout . Harvesting and Purification of Chlamydial EB s McCoy cells were grown in Corning ® tissue culture treated, filtered, polystyrene flasks to 70% confluence. McCoy cell s were infected with Chl amydia trachomatis serovar L2 (LGV 434) suspended in 1X HBSS media at an MOI of 5 and rocked at room temperature for one hour. Infected McCoy cells were then incubated in RPMI 1640 mL gentamicin for 48 hours. The culture media was decanted, and 10 mL of cold HBSS (4 ° C) was added to each 225 cm 2 flask. The infected cells were scraped into centrifuge bottles. Infected cells were sonicated at 100 watts for 20 seconds (2x). The suspension was centri fuged at 50 0 x g for 15 minutes, and the supernatant was transferred to a new tube and centrifuged at 30,00 0 x g for 30 minutes. The pellet was resuspended in SPG ( 220mM sucrose, 14mM Na 2 HPO 4 , 3mM NaH 2 PO 4 , 5mM L glutamic acid , pH 7.4 ; 4 mL per 225 cm 2 flas k) and dispe rse d with an

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26 18 gauge cannula. The suspension was gently layered over a 30% Renografin TM solution in K 36 buffer (50mM KH 2 PO 4 , 100mM KCl, 150mM NaCl, pH 7.0) and centrifuged at 40,00 0 x g for 30 minutes. Each pellet was resuspended in 2 mL SPG and gently layered over a Renografin density gradient consisting of 5 mL 40%, 12 mL 44%, and 8 mL 54%. The gradients were centrifuged at 40,00 0 x g for 60 minutes, and the EB band as collected at the 54% interface. The EBs were mixed with 10 volumes of SPG and centrifuged at 30,00 0 x g for 30 minutes. The pellet was resuspended in 1 mL SPG per 225 cm 2 flask and frozen at 70 ° C in aliquots. Harvestin g and Purification of Chlamydial RBs Infection was carried out using the same method as that of the EB harvesting and purification . At 24 hours post infection , cells were scraped from the flask, and lysing was performed using a dounce homogenizer (20 instead of sonication. The remainder of the EB purification was followed after this step to purify the RBs. Vector Construction Invitrogen TM Gateway ® Technology was used for maintenance and expression of the nucleic acid binding pr oteins. To prevent leaky expression of these putative toxic proteins that bind DNA and interfere with cell division , pENTR TM /D TOPO ® was used for the initial cloning due to the absence of a RBS. Each gene was subcloned into pEXP1 DEST to express N terminal 6x his tagged proteins and into pET104 Bioease to express N terminal biotin tagged proteins. Each gene was also subcloned into the destination vector pcDNA TM 6.2/EmGFP for expression of chlamydial proteins in eukaryotic cells. The N EmGFP DEST vector was u sed for eukaryotic expression of N terminal GFP tagged proteins, and the C EmGFP DEST vector was used for expression

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27 of C terminal GFP tagged proteins within the mammalian cells. Other than removing the stop codon for the C terminal tagged proteins, no add itional modifications were made. The leading CACCATG sequence was used as the Kozak translation initiation sequence for all C terminally tagged proteins in the mammalian cells (Xia, 2007) . The two compatible vectors pLac and pTet were used for the cheZ/soft agar experiments (Grieshaber, Fischer, Mead, Dooley, & Hackstadt, 2004) . pLac contains a ColE1 origin of replication and ampicillin resistance. pTet contains a p15A origin of replication and ch loramphenicol resistance. An L2 genomic library ® pET 41a(+) vector. A RsaI digestion of C. trachomatis L2 genomic DNA and electrophore sis were performed to purify 500 b p fragments. These fragments were ele ctroeluted and liga ted into pET 41a(+) and include an N terminal GST t ag and a C terminal 6x his tag when in proper frame . Kill assay s and Growth Curves A kill assay was performed t o determine toxicity of the recombinant nucleic acid binding proteins . BL21 AI TM cells were tr ansformed with pEX P1 including each of the genes in the study. Cells were grown overnight at 37 ° C on 0.1% glucose LB Amp 100 1% agar plates. Glucose is used to reduce leaky expression since it prevents lactose from inducing the lac promoter . Colonies were picked on day 2 and grown overnight at 37 ° C in 0.1% glucose LB Amp 100 . On day 3, the overnight culture was diluted 1:2000 into 4 mL LB Amp 100 and switched to at 30 ° C, and protein expression was induced with 0.4% L arabinose and 2mm IPTG i mmediately following dilution . OD at 600 nm was performed every hour for 7 hours to estimate culture growth.

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28 Microscope Images were obtained from a VISITECH QLC 100 spinning disk confocal system connected to a LEICA TM DMIRB microscope equipped with a LEICA TM HCX PL APO 63x oil immersion objective. The open source software package µ Manager ( https://www.micro manager.org/ ) was used to control the microscope and camera. Images were processed and analyze with the use of ImageJ ( http://imagej.nih.gov/ij/ ) and OM ERO ( http://www.openmicroscopy.org/site/products/omero ). Live Cell Imaging COS 7 and HeLa cells were grown on poly L lysine coated #1.5 micro coverglass in Corning ® tissue culture treated polystyrene cell culture cluster s in RPMI supplemented with 10% FBS and 10 mL gentamicin . After any necessary infections or transfections, coverslips were transferred to a perfusion chamber in a POCmini TM cell cultivation system. CO 2 input to the media was regulated with the use of a gas diffuser. And the temperature was maintained at 37 ° C with the use of a PECON TempControl 37 2 digital control unit. In order to maintain the same temperature in the objective a nd perfusion chamber, a BIOPTECHS TM objective heater control system was use d. Images were captured with a Photometrics TM Cascade II controlled EMCCD camera . Microinjection Microinjections were performed with the use of an Eppendorf ® FemtoJet ® system . Eppendorf ® Femtotips ® and homemade capillary tubes that were used for the inject ion of COS microinjection buffer used was PBK . All microinjections were performed prior to the five hour point in the infection cycle to prevent rupture of the inclusion. Cells were

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29 micro injected while plated on coverslips in a temperature controlled (37 ° C ), CO 2 controlled (5%) perfusion chamber. Cells were overlaid with HBSS. The injection point on the COS 7 cells was near the Golgi apparatus. Transfection All transfections of eukaryotic cells were performed with Invitrogen TM Lipofectamine® 2000 Transfection Reagent. For the egress diffusion experiments involving Em GFP, COS 7 cells were grown to 90% confluence and transfected with Invitrogen TM pcDNA TM 6.2/N E mGFP/GW/CAT . At 24 hours post transfection, the COS 7 cells were passaged into fresh growth media to prepare for the egress diffusion experiments. For the experiments involving ectopically expressing chlamydial proteins in eukaryotic cells, HeLa 229 cells were grown to 70% confluence and transfected with pcDNA TM 6.2/N EmGFP DEST with each of the genes of interest cloned into that destination vector. At 24 hours post transfection, the HeLa cells were prepared for fixation and staining. Egress Diffusion Experi ments COS 7 cells were grown to 70% confluence on poly L lysine coated coverglass, and then infected with Chlamydia trachomatis L2 serovar at an MOI of 5. At 5 hours post infection, the COS 7 cells on the coverglass were transferred to a perfusion chamber . The COS 7 cells were then overlaid with HBSS and the temperature was maintained at 37 ° C. Selected cells were microinjected with a fluorophore conjugated to various sizes of dextran molecules in the method describe in the microinjection section. The foll owing dyes were used in these diffusion experiments: Life Technologies Alexa

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30 Fluor ® 488 Dextran 3000 MW , Alexa Fluor ® 568 Dextran 10,000 MW, Alexa Fluor ® 647 Dextran 40,000 MW, and Fluorescein Dextran 500,000 MW. Unconjugated fluorescein was also used. The conjugated fluorophores were dissolved in phosphate buffered potassium (PBK) microinjection buffer at a concentration of 1 mg/ mL . The the capillary. The me dia was exchanged from HBSS to complete media, and the cells were incubated overnight at 37 ° C and 5% CO 2 . At 3 5 hours post infection (also 30 hours post injection) the cells on the coverglass were transferred back to the perfusion chamber and overlaid wit h complete media containing 25 g/ mL propidium iodide. The COS 7 cells were maintained at 37 ° C and 5% CO 2 . Live cell imaging was performed as described above for the duration of the infection cycle. Multiple cells were selected for imaging, and 4 z limits were set for each COS 7 that was found to be successfully infected and contained the microinjected fluorophore. Images were taken approximately every 1 minute , and were continued until after completion of chlamydial egress from the host cell. F luorescence Staining of E. coli In order to prepare E. coli for imaging, cells were tra nsformed with the Invitrogen TM pEXP1 expression vector carrying each gene of interest preceded by an N terminal 6x his tag. After overnight growth at 37 ° C on LB AMP100 0.1% Gluc pl ates, colonies were picked and grown in LB AMP100 liquid media at 37 ° C until OD 600 reached 0.2. IPTG was added to the concentration of 2mm, L arabinose was added to 0.4% , and the temperature was reduced to 30 ° C to induce protein expression while reducing cell division . Expression was induced for only 2 hours to reduce the risk of any impacts from

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31 an unintended mutation of the putative toxic genes that were being expressed. Cells were immediately centrifuged a 300 0 x g for 5 minutes and resuspended in 5 µ M Thermo Scientific ® D raq5, 10 µ g/ mL Hoechst TM 33342, 6 µ g/ mL ac ridine orange for 1 hour. After staining, the cells were resuspended in PBS for 5 minutes and then centrifuged again for 300 0 x g for 5 minutes. Thi s wash step was repeated 3 times. The cells were resuspended in a volume of PBS that was based on the original optical density in the ratio of 1 mL PBS:1 unit of OD 600 . The suspension was then mixed 1:1 with a MOWIOL ® mounting solution ( 100 mg/ mL MOWIOL® 4 88, 25% glycerol, 0.1 M Tris pH 8.5) and then the cells were mounted O/N on a microscope slide with a #1.5 coverglass and imaged. Fluorescence Staining of Eukaryotic Cells The HeLa cells used in the ectopic chlamydial protein expression studies were fixed for 10 minutes with methanol 24 hours post transfection. The cells were washed 2 times with PBS. Monoclonal Anti tubulin I (Sigma Aldrich® ) was use to show cell shape . And the far red fluorescent DNA dye Draq5 ( Thermo Scient ific ® ) was used to stain the nucleus. For the CT153/MACPF studies, monoclonal antibodies were produced against the GST tagged CT153 /MACPF by the Interdisciplinary Center for Biotechnology Research (University of Florida) Hybridoma Core Lab. The initial screening of the monoclonal serum was done with ELISA, and the best candidates were screened by staining infected and non infected HeLa cells. The monoclonal serum used for imaging in these studies had no background staining on uninfected HeLa cells and demonstrated consistent localization with the vast majority of other strong candidate

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32 monoclonal sera. An incA mAb was used to visualize the inclusion in the infected cells. Human serum from male AB plasma (Sigma Aldrich® ) was used to visualize Chlamydia trachomatis . Secondary antibodies conjugated to Alexa Fluor ® 488, 568, or 647 were used to visualize the primary antibodies. PAGE and Western Blot Analysis Polyacrylamide gels were either 4 20% gradient gels for electrophoresis with a large numbe r of proteins or 12% polyacrylamide resolving gel with a 4% stacking gel for most single protein visualizations. A Tris Glycine SDS buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS, pH 8.6) was used for each electrophoresis. Thermo Scientific® Non Reducing Lan e Marker Sample Buffer was using in most application. For smaller proteins, where the dye front would interfere with visualization of the bands, a loading buffer was used with bromophenol blue as the only dye (62 mM Tris HCl pH 6.8, 2% SDS, 0.01% bromophen ol blue, 10% glycerol). Transfers of protein from the polyacrylamide gel to the membrane were done in 1 hour at 100V in a Bio Rad ® mini cell with Tris Glycine SDS buffer and 10% methanol . The proteins were transferred to Bio Rad® Trans Blot Transfer Medium (Pure Nitrocellulose Membrane, 0.45 µ m). Blocking of western blots was done in TBST and 3% BSA (50 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween 20 , 3% BSA ). Primary antibodies were diluted in TBST and 1% BSA and incubation was performed overnight at 4 ° C. Monoclonal Anti tubulin I was used for normalization in protein preps involving eukaryotic cells. Anti MOMP (major outer membrane protein) was used for

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33 normalization in Chlamydia protein preps. Washes were performed in TBST for 10 minutes (3x). Secondary antibodies were diluted in TBST and incubation was performed at room temperature for 1 hour. For primary antibodies created in mice, Thermo Scientific ® Stabilized Peroxidase Conjugated Goat Anti Mouse (H + L) secondary was used for visualization of the pr oteins . For western blots involving streptavidin tagged proteins, the primary antibody incubation step was skipped, and Pierce ® High Sensitivity Streptavidin HRP was used for visualization of the proteins . For western blots involving His tagged proteins , the primary antibody incubation step was skipped, and Thermo Scientific ® SuperSignal TM West HisProbe Kit was used for visualization of the proteins. Precision Plus Protein TM Kaleidoscope TM Standards (Bio Rad ® ) were used to approximate the molecular wei ght of proteins . For his tagged proteins, the BenchMark ® His tagged Protein Standard (novex TM ) was used for an additional standard to estimate protein molecular weight following imaging. The HRP conjugates on each secondary antibody were visualized with Su perSignal TM West Dura Extended Duration Substrate (Thermo Scientific ® ). For low abundance proteins or weak primary antibodies, SuperSignal TM West Femto Maximum Sensitivity Substrate (Thermo Scientific ® ) was used in the electrogenerated chemiluminescence st ep. For the CT153/MACPF experiments, images from western blots were obtained using an Amersham Biosciences TM Hypercassette ® and Kodak TM BioMax ® Light Film

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34 Scientific Imaging Film. All other experiments were imaged using the Bio Rad ® Gel Doc TM XR+ system . Quantity One TM 1 D analysis software was used for image processing and basic image analysis. In gel Staining For non specific protein visualization in polyacrylamide gels, coomassie staining was used (45% methanol, 10% glacial acetic acid, 3g/L Coomassie B rilliant Blue R250). Staining was performed for 2 hours, then gels were rinsed with diH 2 0 prior to 2 4 hours of destaining (40% methanol, 10% acetic acid). Images were obtained with the Bio Rad ® Gel Doc TM using the white light transilluminating platform an d an amber filter, or for better color maintenance of the standards, a standard cell phone camera was used. In gel staining of his tagged proteins was performed using the InVision TM His tag In gel Stain kit (novex TM ). Images were obtained with the Bio Rad ® Gel Doc TM using 302 nm UV light and a CCD camera with a band pass filter. Densitometry ImageJ was used to estimate protein concentrations on western blots. Density was measured to ensure all bands were under the saturation range. The thickest band was use d to determine the area for the measurement of band density. Background from an unused portion of the membrane was subtracted from all density measurements of protein bands. DNA and RNA Electrophoresis For visualization of DNA, 1% agarose gels were used in 1x TBE buffer. For DNA less than 200 nucleotides, 1.8% agarose gels were used. 1x SYBR ® Safe DNA Gel ( Invitrogen TM ) was included in each gel to eliminate the need for ethidium bromide use or soaking after electrophoresis. Fermentas TM 1 kb Plus DNA Ladder was

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35 used to estimate DNA migration. 6x DNA Loading Dye (Thermo Scientific ® ) was used for larger DNA fragments. A homemade 5x dye less loading buffer containing only glycerol for smaller DNA fragments in which the dye would interfere w ith the visualization of the DNA. For visualization of small RNA (less than 200 nucleotides), polyacrylamide gel electrophoresis was performed instead of agarose gel electrophoresis. Urea TBE polyacrylamide gels (15% acrylamide/bis acrylamide, 89 mM Tris B orate, 2 mM EDTA, 7 M Urea) were run for 1 hour at 100V. RiboRuler TM Low Range RNA Ladder, ready to use (Thermo Scientific ® ) was used to estimate migration of RNA. After electrophoresis, the gels were removed from the cassette a nd soaked in 1x TBE and 1x S YBR ® Safe DNA Gel ( Invitrogen TM ). Although the SYBR ® Safe is not optimized for RNA, it was used for safety considerations. Images for both agarose electrophoresis and polyacrylamide electrophoresis were obtained using a Safe Imager TM Blue Light Transillumi nator ( Invitrogen TM ) and a Canon TM PowerShot TM A620 camera. Images were processed and analyzed with Abode ® Photoshop TM CS software. Soft Agar Assay Electrocompetent E. coli cheZ MG1655 wer e transform ed with the pLac vector carrying either a constitutively expressed wild type ihtA small RNA or ihtA with a The cells were grown on LB CB100 Kan25 overnight at 37 ° C . The transformed cells were made electrocompetent again, and t hen were transf orm ed with the second vector, pTet carrying addition to the first 30 nucleotides of each known or putative target gene followed by the

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36 cheZ reporter gene. After the 2 nd electroporation, cells were grown on LB CB100 Cm3 4 overnigh t at 37 ° C . Colonie s were transferred to 100 µ l tryptone media , and incubated at 37 ° C for 6 hours. 2 µ l of each culture was then stabbed onto soft agar tryptone plates (1% Tryptone, 0.5% NaCl, 0.25% agar , 25 µ g/ mL Kan, 100 µ g/ mL CB, 34 µ g/ mL Cm ) using a pipette. The inoculated soft agar plates were then incubated upside down at room temperature overnight, and any migration was imaged and measured. Recombinant Protein Purification All recombinant proteins were expressed from BL21 AI TM E. coli (Invitrogen TM ) . The bacterial expression vector pEXP1 ( Invitrogen TM ) was used to express N terminal his tagged proteins. And the bacterial expression vector pET104 ( Invitrogen TM ) was used to express N terminal biotin tagged proteins. Cells transformed with each vector were grown overnight at 37 ° C on LB CB100 plates and then transferred to LB CB100 liquid media and grown at 37 ° C until OD 600 reached 0.4. Expression of recombinant proteins was induced with 0.4% L arabinose and 2 mm IPTG. Cells were induced for 2 hours at 37 ° C to reduce to likelihood of unplanned mutation of any genes whose products are toxic to E. coli . Cells were then chilled to 4 ° C and centrifuged at 500 0 x g for 10 minutes. The media was removed and the cells pellets were frozen to enhance lysis for the protein purification step. Frozen cells were resuspended in buffer appropriate for the purification step ( E. coli containing his tagged proteins were suspended in 20 mM sodium phosphate, 300 mM sodium chloride, and 10 mM imidazole at pH 7.4; E. coli containing biotin tagged proteins were suspended in 50 mM citrate phosphate at pH 4.0) . cOmplete Protease Inhibitor Cocktail Tablets (Roche) were added to prevent degradation of recombinant

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37 protein (1 pellet per 50 mL of lysis buffer) . Nuclease supplementation was avoided to prevent carry over impacts in future experiments where DNase activity was being measured for the recombinant protein. Cells were lysed by either mechanical lysis with the use of a French Press or by chemical lysis w ith the use of Sigma Aldrich® CelLytic TM B . Lysate was centrifuged at 16,00 0 x g for 10 minutes to pellet insoluble material. The supernatant was then applied to a column for purification of the tagged recombinant proteins. HisPur TM Ni NTA Resin (Thermo Sc ientific ® ) was used to purify the his tagged proteins. Upon completion of the elution step, the eluted proteins were applied to Amicon ® Ultracel ® Centrifugal Filters to concentrate the purified protein and remove excess imidazole. Imidazole concentration was reduced from 250 mM to approximately 10 mM and stored in PBS (0.1 M phosphate, 0.15 M sodium chloride, pH 7.2) at 4 ° C to maintain any potential enzymatic activity of the recombinant proteins. CaptAvidin TM Agarose Sedimented Bead Suspension (Molecular Probes ® ) was used to purify to biotin tagged proteins. Elution buffer was supplemented with 2 mM biotin to improve elution efficiency. Purified recombinant proteins were dialyzed in PBS and stored in 4 ° C. To purify tagless recombinant proteins, Pierce ® St reptavidin Agarose Resins (Thermo Scientific ® ) were used for their superior binding capabilities to bind the biotin tagged proteins used in this study. Elution was performed with EK Max TM (Invitrogen TM ), which cleaves the linker between the biotin tag and the protein of interest. The tagless protein was then further purified with EK Away Resin to remove the residual EK Max enzymes. The tagless protein was then dialyzed with PBS.

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38 Protein Concentration Measurements Concentration for each recombinantly express ed protein was estimated by using the Bradford assay. A nanodrop measurement was performed on each protein, and the extinction coefficient was estimated based on the Bradford assay results. For protein preparations that indicated imperfect purification bas ed on coomassie staining, contaminating protein was estimated based on band intensity, and this percentage was subtracted from the Bradford or nanodrop results. RNA Expression and purification Sense IhtA and hctA transcripts were synthesized from the T7 pr omoter of PCR amplified fragments generated from serovars L2 and D, C. pneumoniae, C. muridarum and C. caviae genomic DNA using t he primers containing the T7 recognition sequence . Antisense IhtA (scrambled co ntrol) was synthesized from the T7 promoter of a PCR amplified fragment generated from serovar L2. The hctA transcr ipts were designed to UTR starting at the tra nscription start site (TSS) (Fahr, Douglas, Xia, & Hatch, 1995) and an addition 21 nucleotide poly A tai l used to bind the transcript to the streptavidin biosensor tips. Run off transcripts were prepared using the MEGAshortscript TM T7 kit as described by the manufacturer (Ambion® ). RNA products were purified by performing an acid phenol chloroform extraction at pH 4.2 followed by an ethanol precipitation. After the ethanol precipitation, RNA was resuspended in DEPC treated nuclease free water. RNA concentration was determined by using the nanodrop. BLI ( s RNA m RNA Interaction ) Biolaye r interferometry studies o f s RNA m RNA interactions were performed using the Octet QKe TM ( fortéBIO TM , Menlo Park, CA). To anneal the ligand (hctA

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39 message) to the streptavidin biosensor tips, 1 biotinylated poly A tail) , RNA binding buffer (10mM Tris HCl pH 8, 125mM NaCl, 125mM KCl, 25mM MgCl 2 in DEPC treated water) were combined, heated for 1 min at 90 ° C and allowed to cool slowly. During this time, SA biosensor tips were equilibrated in RNA binding buffer for 15 min. RNA annealed to biotinylated oligo was loaded onto the SA tips for 15 min or until saturation. RNA loaded tips were then soake d in RNA binding buffer for 5 min prior to incubation with 1500 nM I htA which had been heated at 90 ° C for 1 min and allowed to cool to RT. The change in internally refl ected light attributable to s RNA m RNA interactions was collected in real time for 20 minutes using the software provided with the Octet QKe TM . Oligonucleotides for BLI (Protein NA Interaction) Customized ssDNA oligo nucleotides were ordered from Integrated DNA Technologies. Each sequence is 100 nucleotides in length and contains a T7 promoter in vitro transcription. The remaining s equence is random nucleotides that are 100% G C or 100% A T ssDNA. dsDNA was synthesized from the custo mized ssDNA nucleotides. Sigma Aldrich® REDTaq ® DNA Polymerase was incubated with the ssDNA, T7 terminator complement primers, and NTPs in polymerase buf fer. After heat activation of the polymerase, primers were annealed at 55 ° C. Extension was done at 72 ° C for 30 minutes. The annealing and extension cycles were repeated 4 times, and the temperature was maintained at a maximum of 72 ° C to prevent denatura tion of synthesized dsDNA. The synthesized dsDNA was purified using the QIAGEN®

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40 QIAquick ® PCR purification kit to remove all unincorporated primers and dNTPs. Ethanol precipitation with sodium acetate was used to further purify the dsDNA. Successful synthe sis of dsDNA was verified by noting the change in OD at 260nm/280nm. RNA was also synthesized from the customized ssDNA oligonucleotides. Ambion® MEGAshortscript TM with T7 RNA polymerase was used to synthesize 100 base pair transcripts of the same randomiz ed format of the ssDNA oligonucleotides. Ambion® TURBO DNase TM was applied after the IVT reaction to degrade template ssDNA, and an ethanol precipitation with ammonium acetate was performed to purify the synthesize RNA transcripts. Bio Rad ® Micro Bio Spin T M P 6 Gel Chromatography Columns were used to remove unincorporated nucleotides and further purify the RNA transcripts. BLI (Protein NA Interaction ) Biolaye r interferometry studies of sRNA m RNA interactions were performed using the Octet QKe TM ( fortéBIO TM , Menlo Park, CA). Ni NTA Dip and Read Sensors were equilibrated in binding buffer (0.1 M phosphate, 0.15 M sodium chloride, pH 7.2) for 15 minutes prior to starting the experiment. To anneal the ligand ( putative nucleic acid binding protein ) to the Ni NTA Dip and Read Senso rs, his tagged proteins were expressed as described earlier and incubated in the Octet Qke TM platform with the sensors for 15 minutes. The sensors were incubated again in binding buffer to wash off any unbound protein and establish a base line for kinetics measurement. The sensors were then incubated for two minutes in either GC rich or AT/AU rich nucleic acid preparations of the following concentrations: 720 nM, 240 nM, and 80 nM. Due to the limited capabilities of the Octet QKe TM platform , measurements of biolayer thickness

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41 were taken every 3 1/3 seconds for each sensor. Following the association step with the nucleic acids, the sensors were returned to binding buffer, and a change in thickness of the biolayer was measured for 15 minutes t o determine dissociation . The Octet Qke TM software was used for kinetics analysis. A reference well was used with each concentration of nucleic acid, and the biosensor had no measurable change in thickness, so no subtractions were made from the experimenta l biosensor. The association curve was aligned to the final five seconds of the second baseline step. An interstep correction was made to baseline. Full local fitting fitting was done for association and dissociation steps. The steady state response was de termine between 105 and 115 seconds of the two minute association step, and was used to estimate a K D . k on and k off were also determined from the local fitting, and a projected K D was calculated by dividing the initial k off by the initial k on (Edwards & Leatherbarrow, 1997) . Enzyme Activity Assay Prior to performing DNase protection assays, potential enzymatic activity was assayed for the recombinantly expressed proteins to determine if it plays a role in any nucleic acid degradation in other experiments . The recombinant his tagged proteins ( HctA , HctB , CTL0322, and the his tagged library) were incubated with plasmid DNA (pcDNA 6.2/N EmGFP/GW/CAT) at various dilutions (1 protein : 100 bp DNA, 1 protein : 1000 bp DNA, 1 prot ein : 10,000 bp DNA) in PBS . A duplicate of the 1 protein : 100 bp DNA reaction was duplicated with EDTA applied to the protein portion for 5 minutes prior to incubation with the plasmid DNA. Recombinant protein was incubated with DNA for 5 minutes at 37 ° C, and then EDTA was immediately added. Reaction mixes were then heated to 65 ° C for 10 minutes as a deactivation step. Reaction mixes were then

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42 incubated with 10 proteinase K for 2 hours at 50 ° C. DNA was purified with a phenol (isoamyl alcohol) chl oroform extraction. Gel electrophoresis was then performed on a 1.8% agarose gel. DNA incubated with 2 units of DNase I in the first step was used as a positive control for this experiment, and DNase incubated with no protein in the first step was used as a neg ative control. DNase Protection Assay To assay DNase protection conferred by protein binding, each his tagged recombinant protein (CTL0322, HctA , HctB , and the his tagged library) were incubated with plasmid DNA in PBS at a ratio of 1 protein : 100 bp DNA for two minutes at 37 ° C to allow binding. The two minute time period was determine based on the duration of time indicated for saturation binding in BLI experiments. The total amount of DNA used in each reactio n was 1.5 . The protein/DNA mixture was then incubated with five fold dilutions of DNAse I (1/5 unit, 1/25 unit, 1/125 unit, and 1/625 unit) for five minutes at 37 ° C. EDTA was immediately added to stop the DNase I enzyme activity, and the reactions were incubated at 65 ° C for ten minutes. Reaction mixes were then incubated with 10 mL proteinase K for two hours at 50 ° C. DNA was purified with a phenol (isoamyl alcohol) chloroform extraction. Gel electrophoresis was then performed on a 1.8% agarose gel. Preparation of Genomic DNA Genomic preparations of Chlamydia genomes were performed using the DNeasy ® Blood & Tissue Kit (QIAGEN ® ) following the protocol for gram negative bacteria . Large scale preparations of E. coli genomic DNA were performed following the protocol in Current Protocols in Molecular Biology (Wilson, 1997) .

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43 Nucleic Acid Isolation (for qPCR) RNA and DNA preps for CT153 qPCR were obtained using the MagMAX TM Total Nucleic Acid Isolation Kit (Ambion ® ). HeLa 229 cells were grown in 6 well cell culture clusters to 70 80% confluence and infected with Chlamydia trachomatis L2 at an MOI of five. Lysis buffer containing 3 carrier NA was added to each infection at various time point s during the infection cycle for the 0 time point to 56 hours post infection. Cells were scraped upon application of lysis buffer and transferred to MagMAX TM bead tube. The cells were lysed by with the use of a bead beater for 30 seconds (two times with in cubations on ice between repeats). Lysates were centrifuged at 30,00 0 x g for 3 minutes, and 115 of the supernatant was mixed with 65 isopropanol. The remaining steps are as described by Ambion ® in the instructions. Two aliquots were made from each t otal nucleic acid. 1 of TURBO DNase TM (Ambion ® ) was added to each 15 g of total nucleic acid and incubated at 37 ° C for 30 minutes. DNase inactivation reagent was then added. The RNA samples were centrifuged at 10,00 0 x g for 90 seconds, and the supernatant was collected and stored at 20 ° C. The total nucleic acid aliquot was also stored at 20 ° C. qPCR In order to provide a standard curve for the CT153 qPCR experiments, primers were designed to provide at 133 bp amplicon for CT153. The control gene, 16S rRNA, had primers designed that would provide an amplicon size at 146. Dilutions of each of the amplicon preps were made to provide a standard curve from 10 2 to 10 7 copies. qPCR was performed with SYBR ® green 1 step RT PCR kit. Reverse transcriptase was used in the qPCR for the RNA only preps. Reverse transcriptase was

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44 not used for either the standards, negative controls, or the DNA quantification qPCR. qPCR was performed on the total nucleic acid prep to estimate original cell count. These samples would be expected to have 1 2 copies of the CT153 gene in the genome, but the RNA copies would not impact these DNA qPCR experiments since reverse transcriptase was not used to create cDNA from the mRNA. Triplicate samples from ea ch preparation were used for to detect equipment standard error for qPCR. The negative controls used to determine false amplicons were total nucleic preps that had undergone DNase I digestion. During qPCR of the negative controls , reverse transcriptase was not use, and therefore no DNA would be expected to be present in the reaction. This experiment was performed three times, but due to high equipment variations between runs, as well as qPCR enzyme variations, statistics were no t done f o r the results. A rep resentative sample from one experiment was used to demonstrate the trends that were found in all experiments.

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45 CHAPTER 3 CHARACTERIZATION OF NUCLEOTIDE BINDING PROTEINS IN CHLAMYDIA Summary Nucleotide binding proteins are known to play a large role in regulation of transcription and translation in eukaryotes, but only recently have researchers begun to study these types of proteins in prokaryotes. The two most studied are the HU and H NS proteins in E. coli . HU protein is associated with the bacteria l nucleoid in E. coli , and may participate in the unwinding of oriC (Bahloul, Boubrik, & Rouviere Yaniv, 2001) , while H NS is implicated in transcription activation and repression by binding to AT rich regions (Singh & Grainger, 2013) . While Chlamydia trachomatis does not have a homolog to H NS, two highly abundant nucleotide binding proteins, HctA and HctB , have been found in Chlamydia . A few studies have been done to determine the binding preference for each of these proteins, but much work remains in this area. Other putative nucleotide binding proteins have been mostly neglected by Chlamydia resear chers. This study will provide additional insight into the binding preferences of Hc tA and HctB , as well as that of the recently hypothesized nucleo tide binding protein CTL0322. This research also suggests that CTL0322 has an enzymatic activity of DNA degradation. Background Nucleic Acid Binding Proteins in Chlamydia Although DNA binding proteins were detected in Chlamydia trachomatis in the late 1980s (Wagar & Stephens, 1988) (Hackstadt, Baehr, & Ying, 1991) (Perara, Ganem, & Engel, 1992) , little has been done since to characterize this binding. This initial experiment used south western blotting to detect three highly abundant

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46 chlamydial proteins which bound DNA on the membrane in the denatured form of each protein. In addition to the thr ee abundant proteins at 18 kDa, 25.7 kDa, and 56 kDa, several other proteins with DNA binding capability were noted. Without the use of mass spectrometry, one of the difficulties encountered in identifying these proteins is that highly charged proteins oft en migrate more slowly on a polyacrylamide gel than a neutral protein with a similar molecular weight. It was later determined that the 18 kDa and 25.7 kDa proteins were the histone like proteins HctA (13.7 kDa actual MW) and HctB (23.8 kDa actual MW) resp ectively (Tao, Kaul, & Wenman, 1991) . The other low abundance DNA binding proteins from this initial experiment have not been identified. Because of the sequence similarity between eukaryotic H1 and chlamydial HctA , it was hypo thesized that HctA is a histone like protein that is responsible for the nucleoid formation seen in chlamydial EBs. Ectopic Expression of Proteins in Other Organisms Because active Chlamydia are found only inside host cells and are further surrounded by another membrane termed an inclusion, i t has proven difficult to elucidate the function of many chlamydial proteins in vivo. The typical genetic tricks of knocking down or overexpressing prote in to suggest function in Chlamydia are not available. Because of this, most research has involved expressing chlamydial protein within other organisms. Re combinant expression of HctA in E. coli has been demonstrated to cause the same phenotype , condensed chromatin, that is manifested in chlamydial EBs (Barry III, Hayes, & Hackstadt, 1992) . This phenotype in E. coli , condensed c hromatin, results in cell death .

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47 Functional analysis of HctA and HctB Chlamyd ial proteins HctA and Hct B have relatively low molecular weight s and are both very basic proteins. The estimated pI of HctA is 10.7, and that of HctB is 12.6. The high isoelectric point is due to the abundance of arginine and lysine in both proteins , which is typical in nucleic ac id binding proteins . HctA has an N terminal region with a somewhat lower charge that is believed to be responsible for dimerization of the protein (Pedersen, Birkelund, Holm, Ostergaard, & Christiansen, 1996) . This portion of the protein has also shown the most common amino acid sequence identity between species. T he C terminal region, showing less sequence identity between C hlamydia species, is believed to be responsible for nucleotide binding (Remacha, Kaul, & Wenman, 1996) . Petersen et al. tested the interaction of both HctA and HctB to DNA by using gel retardation assays. Both proteins were found to retard the migration of DNA. Supercoiled, nicked circular, and linear DNA were tested in these experiments, and HctA demonstrated a particular preference to supercoiled DNA. In later expe riments, HctB was not found to have a preference between the three variations of dsDNA, but it was found to retard DNA with a lower protein:DNA ratio (Pedersen, Birkelund, & Christiansen, 1996) . These later experiments als o tested to see if HctA and HctB demonstrate cooperative binding to DNA, but none was found. Several experiments have been performed to determine the gene or sequence specificity of HctA and HctB , but these have all had mixed or inconsistent results. Much of the difficulty with these experiments originates from performing in vitro experiments without knowing the microenvironments that are present within a chlamydial cell during the infection cycle. Concentrations of the DNA binding proteins, competition for more than one sequence of DNA, and potential cofactors that facilitate or inhibit binding can

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48 all play a role in vivo, and are likely absent during an in vitro experiment. Barry et al. have suggested a dual role for HctA based on experiments with varying HctA and DNA concentrations (Barry III, Brickman, & Hackstadt, 1993) . At low concentrations, HctA was found to act more like a helicase by unwinding supercoiled DNA. While at high concentrations (one HctA molecule per 85 b ase pairs), HctA was found to condense DNA dramatically and prevent the DNA from leaving the well during a gel mobility shift assay. It is not clear if this points towards a dual role for HctA at different periods of the chlamydial development cycle, or wh ether this is just an intriguing observation in an in vitro study. Transcription and translation assays have demonstrated that HctA represses both processes in vitro (Pedersen, Birkelund, & Christiansen, 1994) , but again, these experiments do not take into account the potential microenvironments in Chlamydia during the development cell, nor do they account for the changes in HctA :DNA ratios during this cycle. One of the more physiologically relevant studies of HctA binding involved performing immunoprecipitation of crosslinked HctA DNA complexes from infected HeLa cells (30 hours PI). Because high throughput sequencing was not available to these researchers, the crosslinking was reversed from the precipitated HctA D NA complex, and the fragments were analyzed by southern blot (Kaul, Allen, Bradbury, & Wenman, 1996) . HctA was found to exhibit selective binding to a regi on just upstream of the hctA gene, suggestin g a potential feedback inhib ition. Characteristics of the Hypothetical Protein CTL0322 Like HctA and HctB , the hypothetical protein CTL0 322 has a low molecular weight and a high isoelectric point; pI is estimated at 10.0. The relatively high abundance of the basic amino acids arginin e and lysine are most responsible for this

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49 isoelectric point. A protein BLAST search for CTL0322 showed a high degree of homology within the Chlamydia species, as well as to that of other bacteria in the Chlamydiales order (i.e. Waddlia chondrophila and Pr otochlamydia amoebophila ). The next best hit outside of the Chlamydiae is the UV DNA damage endon uclease, UvsE with low confidence (E value= 2.4). Nucleotide blast screening by the Gupta lab had similar results with the next best hit outside the Chlamydiae being a Vibrio parahaemolyticus gene with a low E value of 1.8 (Griffiths, Ventresca, & Gupta, 2006) . A large scale study was done by the Valdivia lab to recombinantly express a full chlamydial library in yeast cells to analy ze the localization and characterize the yeast phenotype post induction. CTL0322 was found to localize to the nucleus in yeast (Sisko, Spaeth, Kumar, & Valdivia, 2006) . Because CTL0322 was one of only 10 chlamydial proteins that were nucleotropic, CTL0322 was ectopically expressed in Hep2 cells , and the nucleotropism was found to occur in these cells also. CTL0322 does not have a nuclear localization sequence, so the localization to the nucleus is likely due to a binding pref erence rather than transport. Results CTL0322 Localizes to the Nucleus in Eukaryotic Cells Because GFP shows no specific localization when expressed in eukaryotic cells, tagging ectopically expressed proteins with GFP allows the identification of any local ization that is a result of the protein in question. The control protein, GFP tagged GW/CAT (chloramphenicol acetyltransferase) shows general diffusion throughout HeLa cells when ectopically expressed (Figure 3 1A) . HctA and HctB tagged with GFP localized to the nucleus, as would b e expected for known DNA binding molecules with very positive charges throughout each protein. GFP tagged CTL0322 demonstrated the

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50 same nucleotropism in HeLa cells as HctA and HctB , which is consistent with previously published re sults by the Valdivia lab when CTL0322 was expressed in Hep2 cells. Even without the nuclear envelope present, CTL0322 still localized to the chromosomes in the mitotic cell (Figure 3 1B) . While a non mitotic nucleus has many other biomolecules present, su ch as lamins, the chromosome in a dividing cell that has reached metaphase has less of these other biomolecules present and provides further evidence that CTL0322 is localizing to the DNA. CTL0322 Is Toxic to Escherichia coli Previous studies have shown th at expressing the recombinant protein HctA in E. coli results in chromatin condensation, similar to the phenotype displayed by Chlamydia trachomatis in the EB stage of development (Barry III, Hayes, & Hackstadt, 1992) . Thi s phenotype in E. coli results in cell death as E. coli does not have a mechanism to remove these histones from its genome. Expression of the other chlamydial histone, HctB , results in cell death in E. coli as well (Figure 3 2A). Similar to these findings of HctA and HctB , recombinant expression of CTL0322 in E. coli results in cell death. Cell growth and division, as indicated by OD 600 , was halted more quickly following induction by CTL0322 than by that of either HctA or HctB . Western blots of the his tagged recombinant proteins (Figure 3 2B) show that each protein was successfully expressed in E. coli , and that the migration distance matched what would be expected from the full length protein and 6x his tag . Recombinant Expression of CTL0322 Induces Apparent SOS Response in E. coli The phenotype of E. coli following expression of HctA matched that of previous studies (Figure 3 3A). While a similar phenotype was expected from expression of CTL0322 based on th e kill assay, the phenotype of E. coli expressing CTL0322 was

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51 quite unique (Figure 3 3B) . These bacteria displayed filamentation and nodules from a large number of the cells. Filamentation is normally a result of E. coli continuing to elongate without sept a formation, a phenotype often associated with the bacterial SOS response. Expression of HctB in E. coli resulted in a similar phenotype as that of HctA , although not as consistent with the presence of nucleoid formation in all cells (Figure 3 3C). And uni nduced E. coli carrying the hctA gene served as a control and demonstrated a typical phenotype in a non stress condition (Figure 3 3D). CTL0322 Has Binding Preference to GC Rich dsDNA BLI analysis of in vitro binding between each of the nucleotide binding proteins and various synthetic oligonucleotides showed some general tr ends of preference to GC rich DNA oligonucleotides and AT rich RNA oligonucleotides, but each protein had a unique profile for binding preference (Figure 3 4). The chlamydial protein lib rary, as expected, demonstrated no interaction with any of the oligonucleotides. The proteins from the library were purified with the tag on the C terminal end of any expressed protein that did not include a stop codon in the 500 bp cloned fragment. Becaus e most chlamydial nucleic acid binding proteins are encoded from genes less than 500 bp, the library is not expected to contain the major chlamydial nucleic acid binding proteins . HctA showed high affinity for GC rich dsDNA, but surprisingly higher affinit y for AU rich RNA. HctB showed preference for GC rich ssDNA and GC rich dsDNA, but little interaction with the other oligonucleotides. CTL0322 showed a clear preference for GC rich dsDNA, but interacted relatively strongly with each type of oligonucleotide . For the opposite perspective of looking at which proteins showed the highest affinity for each of the oligonucleotides, HctA demonstrated the strongest affinity in each case (Figure 3 5) . Of not e , only HctA and CTL0322 demonstrated a clear binding affini ty

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52 for each of the synthetic RNA oligonucleot ides. These were also the only two proteins to show much binding affinity of AT rich dsDNA and ssDNA. The estimated K D of each of the binding reaction s is shown in Table 3 1. A low response for the interaction b etween HctB and most of the oligonucleotides prevented K D from being estimated. CTL0322 Degrades DNA A simple way to detect DNA binding by a protein is by demonstrating its protection of DNA from DNase I. Prior to performing this experiment, an important f irst step is to ensure that the protein of interest does not have an endonuclease or exonuclease activity by itself. As expected, neither the chlamydial protein library nor HctA caused any DNA degradation (Figure 3 6) . While previous research has suggested HctA may be capable of helicase activity (Barry III, Brickman, & Hackstadt, 1993) , no previous research has suggested a nuclease activity for HctA . Surprisingly, HctB demonstrate a limited amount of DNA degradation when i ncubated at a ratio a 1 HctB protein : 100 bp of DNA. This enzymatic activity was inhibited by pre soaking the HctB protein with the Mg++ chelating agent EDTA. CTL0322 also showed the ability to degrade plasmid DNA at the same protein : DNA ratios at HctB , and again this activity was inhibited with EDTA. CTL0322 Protects DNA from Endonuclease Activity Each of the nucleotide binding proteins when incubated with DNA demonstrated an ability to offer protection from DNase I degradation compared to both the chlamydial protein library and unadorned DNA (Figure 3 7) . Each of the proteins was incubated with DNA at a ratio of 1 protein : 100 bp plasmid DNA with varying concentrations of DNase I applied. HctA demonstrated the greatest degree of protection by prote cting plasmid DNA up to 1/25 units of DNase I, while none of the other proteins offered

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53 protection in that range. CTL0322 and HctB offered protection to DNA up to 1/125 units of DNase I, and the chlamydial protein library offer no protection to DNA. Discus sion When ectopically expressed in HeLa 229 cells, CTL0322, HctA , and HctB all localized to the nucleus . Since none of these proteins has a nuclear localization sequence, this localization is more likely a result of binding preference than a result of tran sport. Each of these proteins has a high isoelectric point, which is consistent with DNA binding proteins. While the nucleus in a non dividing cell contains a large number of fibrous lamin proteins and lipids in the nuclear envelop in addition to the DNA p resent in the chromosomes, C TL0322 was found to co locali ze with mitotic chromosomes after the nuclear envelope had broken down , providing further evidence of DNA bi nding preference . Although CTL0322 is toxic to E. coli , as has been shown with HctA and Hct B , the phenotype is significantly different and suggests that CTL0322 has a different function than those histone like proteins. Overexpression of CTL0322 resulted in filamentous E. coli that appear to be deficient in septa. Many of the cells also displaye d large nodules. This phenotype is consistent with the SOS response in E. coli . DNA damage is one of the more typical causative agents for the SOS response . Since no functional domains have been identified in CTL0322, we are unable to describe the mechanis m behind this phenotype . CTL0322 showed a remarkable binding affinity to each type of oligonucleotide tested in the BLI experiment (dsDNA, ssDNA, and RNA), but showed a preference to GC ofactors, and could only limitedly attempt to create the environment present within Chlamydia , these

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54 results would need to be supported with additional evidence from in vivo experiments like immunoprecipitation of crosslinked CTL0322 DNA from an infection with a CTL0322 antibody. One of the surprising findings of this study was that CTL0322 could degrade DNA in vitro . Al though it is possible that a contaminant nuclease was present as a result of non specific binding during the protein purification, neither HctA nor chlamydial protein library preparations displayed DNA degrading abilities. All protein purifications were performed with the same affinity slurry and methods. This purification was repeated several times , including purifications with different tags, and the same DNA degradation activity appeared each time. The coomassie staining of each protein preparation did indicate additional proteins present, so a contaminating nuclease cannot be ruled out. While the CTL0322 protein preparation had activit y that degraded DNA , it also demonstrated the ability to protect DNA from DNase I activity. Although the protection activity did not match that of HctA , it does appear to lend itself to future DNA footprinting experiments in order to determine specific bin ding preference of CTL0322. While the BLI experiments suggested a GC rich dsDNA binding preference, an in vivo experiment during an infection is important to both verify the BLI results and potentially identify specific site on the chlamydial genome that C TL0322 binds. P revious research described different functions of HctA based on concentration, and it is possible the same will be found for CTL0322. High concentration of CTL0322 and HctA were found to aggregate DNA during a DNase protection assay, but und er

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55 lower concentrations, each was able t o protect DNA as compared to a c hlamydial protein library. In summary, this study helped characterize the DNA binding preferences of the previously identified histones HctA and HctB , while demonstrating the binding o f the recently hypothesized nucleotide binding protein CTL0322. The phenotype of E. coli expressing CTL0322 is remarkably different to that of HctA and suggests a much different role for CTL0322 in the Chlamydia development cycle. Because no true homologs exist to CTL0322, further study is needed to determine if t he nuclease activity found in this experiment is indicative of its function in the Chlamydia development cycle.

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56 Figure 3 1. All putative nucleic acid binding proteins localize to the nucleus wh en ectopically expressed in HeLa cells . A) EmGFP tagged Hc1, Hc2, CTL0322, and control GW/CAT expressed in HeLa 229 cells. HeLa cells stained with anti tubulin I to show general cell features and Draq5 to visualize nucleus. B) CTL0322 GFP expressed in mi totic HeLa cell; Draq5 allows visualization of chromosome even after nuclear envelope breakdown.

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57 Figure 3 2. Growth Curve of E. coli during induced expression of recombinant proteins . A) Recombinant CTL0322, HctA , and HctB each killed E. coli when expression induced with IPTG and L arabinose. Uninduced bacteria demonstrated standard growth curves. B) Western blots with HisProbe verified successful induction.

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58 Figure 3 3. Expression of CTL0322 demonstrated SOS phenotype in E. coli . A) Expression of recombinant HctA resulted in E. coli with condensed chromatin B) Expression of recombinant CTL0322 resulted in likely SOS response (nodular, filamentous chains of bacteria) in E. coli . C) HctB had a similar phenotype as that of HctA , alt hough not as pronounced. D) Uninduced cells had typical non stressed E. coli phenotype

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59 Figure 3 4. CTL0322 has a binding preference for GC rich dsDNA . A) The chlamydial protein library demonstrated no binding affinity to any nucleic acids. H ctA has highest binding affinity for AU rich RNA and GC rich dsDNA. HctB demonstrates less in vitro binding that the other putative nucleic acid binding proteins, bu t has notable affinity to GC rich ssDNA. CTL0322 shows a similar binding profile to that of HctA , but greatest affinity is for GC rich dsDNA.

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60 Figure 3 5. CTL0322 has modest in vitro binding affinity to all types of synthetic oligonucleotides.

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61 Table 3 1 . Kinetics for protein oligo nucleotide interactions . K D determined by division of initial k off by initial k on . Low response rates prevented analysis where noted.

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62 Figure 3 6. CTL0322 and HctB degrade plasmid DNA . At moderate protein to DNA ratios, CTL0322 and HctB both demonstrated the ability to degrade plasmid DNA in PBS buffer with no additional enzymes. Presoaking each protein with the Mg++ chelating agent EDTA inhibited this degradation. Neither HctA nor the Chlamydia protein library demonstrated this nuclease activity.

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63 Figure 3 7 . CTL0322 protects plasmid DNA . HctA , HctB , and CTL0322 each demonstrated the ability to DNA at moderate protein to DNA ratios (1:100) and low DNase activity. Neither control, the Chlamydia protein library nor naked DNA showed protection of DNA at any concentration of DNase.

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64 CHAPTER 4 INTERACTIONS OF THE SMALL RNA REGULATOR IhtA Summary Noncoding RNAs were discovered and characterized as early as 1965 , but it regulation was fully appreciated (Lee, Feinbaum, & Ambros, 1993) . And it was much later before the role of noncoding RNAs was recognized in bacterial regulation of protein expression. Traditionally, protein expression in bacteria was believed to have been regulate d at the transcription level by (sigma) factors , since translation was expected to immediately follow transcription in bacteria . Recently, small regulatory RNAs have been found in several bacteria, and the large number of potential regulatory RNAs found in screens suggests that research is in its infancy in this area. Microarray data suggests that more than 35 intergenic sRNA mole cules are expressed in late cycle Chlamydia trachomatis , and approximately half of these have been confirmed by northern blots (AbdelRahman, Rose, & Belland, 2011) . One sRNA that has gathered particular attention is IhtA, which has been found to regulate the expression of the histone like protein HctA in Chlamydia trachomatis serovar L2 (Grieshaber, Grieshaber, Fischer, & Hackstadt, 2006) . These studies will demonstrate that the regulation of HctA by IhtA is conserved across Chlamydia . The results from these studies also suggest that IhtA regulates expression of other chlamydial proteins and is a global regulator of the chlamydial development cycle.

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65 Background Regulation of Histone Expression in Ch lamydia The development cycle of Chlamydia trachomatis consists of an alternation between infectious , non replicating EB particle s and replicate, but non infectious RB particles. The EB phase is characterized by the condensation of the chromosome into nucl eoids, primarily facilitated by the histone like protein HctA (Hackstadt, Baehr, & Ying, 1991) . A less cha racterized histone like protein HctB , is also involved in the process of nucleoid formation (Perara, Ganem, & Engel, 1992) . HctA is regulated at the translation level by the small regulatory RNA IhtA (Grieshaber, Grieshaber, Fischer, & Hackstadt, 2006) 28 (Yu & Tan, 2003) (Shen, et al., 2006) . 28 are able to regulate the expression of HctA and HctB , respectively, neither is able to remove HctA or HctB from nucleosomes. An alternate regulatory pathway plays a role in the removal of histone like proteins 28 prevent additional histone studied, expression of IspE, part of the MEP pathway, results in the removal of HctA and HctB from the nucleoid structure (Grieshaber, Fischer, Mead, Dooley, & Hackstadt, 2004) (Grieshaber, Sager, Dooley, Hayes, & Hackstadt, 2006) . Correlation of IhtA and HctA Expression Grieshaber et al. were able to detect IhtA in C. trachomatis by northern blot as early as 4 hours post infection (Figure 4 8 A ), coinciding with the conversion from the EB to RB form. It is also during this period that chlamydial chromatin begins to decondense.

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66 IhtA northern blot intensity hits its peak at approximately 12 hours PI, a period when nearly all chlamydial cells have differentiated to RBs, and have not begun the asynchronous redfferentia tion back to EBs. IhtA presence begins to diminish after this point. HctA abundance, shown by western blot in figure 4 8A is inversely correlated to IhtA abundance. IhtA Rescues E. coli f rom HctA induced Cell Death When recombinantly expressed in E. coli , HctA causes the chromosome to condense, similar to the phenotype of chlamydial EBs, and results in cell death (Barry III, Hayes, & Hackstadt, 1992) . Ho wever, co expression of IhtA within those same cells results in the repression of expression of HctA and a rescue from cell death (Grieshaber, Grieshaber, Fischer, & Hackstadt, 2006) . IhtA Is Conserved Among Species While the i nitial studies of the IhtA hc tA interaction were carried out with C. trachomatis serovar L2 genome sequences, IhtA is well conserved across species within Chlamydiaceae (figure 4 1A). Using RNAfold for structure prediction (Figure 4 1B), IhtA structure is also fairly conserved among species with the exception of C. pneumonia in the centroid structure . The MFE structures are predicted by a summation of the free energies o f each of the loops at 37 ° C to find the minimal free energy structure (Zuker & Stiegler, 1981) . The centroid structures are predicted by minimizing the base pair distance in the Boltzmann weighted ensemble. Loop 1 has a series of seven nucleotides that are perfectly complementary to seven nucleotides, including the AUG start codon, in the hctA transcript. The interaction of these seven nucleotides is hypothesized to block ribosome formation on the transcript to repress expressi on.

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67 Identification of Putative IhtA Targets The GC rich region surrounding the AUG start codon of hctA is complementary to loop 1 found in the predicted secondary structure of IhtA, and is also required for repression of HctA expression by IhtA . To identif y other potential targets of IhtA, we search through the chlamydial genome to find ORFs with six nucleotides around the start codon that are complementary to loop 1 of IhtA . The online tool TargetRNA was also used to identify other potential targets . The p arameters use d in the TargetRNA search were six nucleotide seed length and binding of loop 1 of IhtA . Nine potential targets (including hctA ) were identified (Table 4 1) ; CTL0322 and CTL0097 were identified with both screens . Four of the potential targets are ribosomal proteins , which likely had an alternate regulatory system , while the others potential targets have unknown function. The hypothesized interaction between IhtA and the two putative targets that will be investigated in this study are shown in f igure 4 4. Hypothesis of CTL0322 and IhtA Interaction Because the CTL0322 protein shares many characteristics with HctA (low molecular weight, highly basic charge, and arginine/lysine rich) and the RNA transcript of CTL0322 shares a similar sequence around the start codon as that of hctA , it presented itself as perfect candidate to research as a target of the small RNA regulator IhtA . We hypothesize that IhtA interacts with several transcripts involved in the progression through the development cycle, and s erves as a global regulator. Results IhtA Interaction with HctA Conserved Across Species Biolayer interferometry can be used to measure in vitro interaction between biomolecules by affixing one biomolecule to a sensor and detecting a change in the

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68 interference pattern of white light when incubated with a potential ligand. The slope of the binding curve during the association step indicates the on rate for the association between the two molecules. Since IhtA functions without the requirement of a ch aperone or helper protein, BLI is a useful technique to estimate its binding preferences. hctA run off transcripts of each species were annealed to a biotinylated poly A oligonucleotide by synthesizing a poly transcript The biotinylated target transcripts were affixed to the streptavidin sensor, and then incubated with species specific IhtA transcripts. The data was normalized to percent max imum change in reflected light over time and compared to antisense L2 IhtA binding. The measurements demonstrate that IhtA binding to its cognate hctA target transcript is conserved among Chlamydiaceae (Figure 4 2 ) . The conservation of IhtA hctA across species was also verified in the surrogate E. coli kill assay (Tattersall, Rao, Runac, Hackstadt, Grieshaber, & Grieshaber, 2012) . While serovar L2 demonstrated the greatest percentage change in rescue upon induction of the cognate IhtA, each species showed statistically significant rescue when IhtA was induced (figure 4 3). IhtA Interacts with the CTL0322 Transcript I n Vitro The biolayer interferometry experiment discussed previously was used to investigate the in vitro interaction between IhtA and several of its putative target transcripts . The targets, CTL0097, CTL0322, and hctA , were biotinylated and affixed to the streptavidin biosensor. The hctB transcript was used a negative control in this experiment as it does no hctA transcript that is implicated in its interaction with IhtA. The change in biolayer thickness

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69 (indicated RNA : RNA binding) was measured over time (Figure 5 3). Interaction between each transcript and a ntisense IhtA was subtracted from each time point to account for any non specific RNA : RNA interactions. Each of the putative targets demonstrated binding affinity to IhtA, while hctB showed only negligible affinity to IhtA. IhtA Inhibits Expression of CT L0322 cheZ Fusion Protein in E. coli Surrogate System Since the product of the hctA transcript has a detectable phenotype in E. coli ( DNA condensation and cell death) , studying the interaction between IhtA and hctA within an E. coli surrogate system was ma de possible by cloning the entire hctA gene . Because the other potential IhtA targets do not have a know n function or detectable phenotype, this option is not available for this s tudy . The Grieshaber lab developed a surrogate system to detect chlamydial mRNA sRNA interactions in E. coli with the use of a reporter gene . end of the potential targeted transcript (from the transcription start site to +30 in the ORF) was cloned into pTet in front of the reporter gene , cheZ (Figure 4 6A) . The ATG start codon from the reporter gene is not included in the fusion product to prevent potential translation originating from a cryptic ribosomal binding site that could be present downstream of the start codon o f the potential target sequence . Co expressing the IhtA transcript from the pLac expression ve ctor allows for the determination of IhtA repress ing the expression of the fusion product by detection of the reporter gene . Comparing the phenotype of E. coli co ntaining recombinant pTet and IhtA expressing pLac versus the same culture with empty pLac will allow us to identify repression of expression of the reporter gene by IhtA.

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70 CheZ is a phosphatase that is responsible for removing the phosphate from phosphoryl ated CheY, which in turn signals the flagellar motor to rotate counter clockwise (Parkinson, Ames, & Studdert, 2005) . In the absence of CheZ, the flagellar motor remains spinning in the clockwise direction, which leads to rando m directional changes while swimming (Parkinson, Ames, & Studdert, 2005) . C hemotaxis is eliminated in E. coli when cheZ is knocked out . Recombinant expression of CheZ from the pTet plasmid rescue s chemotaxis in cheZ E. coli . R estored chemotaxis can be observed by injecting the transformed E. coli on 0.25% soft agar and observing movement away from point of injection. In order to validate our surrogate system , hctA was cloned in front of cheZ within the pTet ve ctor to create a fusion transcript (Figure 4 6B) . Co expression of the HctA CheZ fusion protein and scrambled IhtA resulted in restored chemotaxis (Figure 4 7, top middle ). Co expression of the fusion transcript with IhtA resulted in a lack of rescue. Beca use co expression of IhtA with the non fusion cheZ cheZ E. coli does not inhibit repression (Figure 4 7, bottom right) , this of CheZ expression. The c andidate target transcript ctl0322 cheZ demonstrated repression of the rescue of chemotaxis, which indicated interaction with IhtA in the surrogate system, while ctl0097 did not . A s expected, co expression of hctA cheZ and IhtA did not result in repressio n of rescue . CTL0322 I s Temporally Expressed During Development Previous research has demonstrated that HctA abundance is inversely correlated with IhtA abundance over a time course of the chlamydial development cycle (Figure 4 -

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71 8A) . Northern blot analysis o n indicates that IhtA is not detectable in purified EBs, while western blots indicate the presence of HctA in EBs. The reverse is found for RBs, and offers a clearer look at the pattern displayed over a time course that involves mixed EB/RB populations at most of the time points (Grieshaber, Grieshaber, Fischer, & Hackstadt, 2006) . Western blot analysis of CTL0322 in EBs (0/48 hours) and RBs (24 hours) does not show the same pattern as that found for HctA (Figure 4 8B). CTL0322 was only faintly detectable in EBs, while it was much more abundant at 24 hours post infection. MOMP was used as a loading control. Although MOMP is the chlamydial protein that is present in the largest quantity in both EBs and RBs, the abundance is not eq ual nor is the structure the same in each form (Newhall & Jones, 1983) (Hatch, Allan, & Pearce, 1984) . Therefore, c oomassie staining of whole cell extracts was used to indicate total protein content (Figure 4 7B) . Discussion Even though IhtA is located in an intergenic region of the chlamydial genome, the conservation of its sequence amongst species within the Chlamydiaceae indicates its importance to Chlamydia . The detection of IhtA by northern blots demonstrates its relative abundance, and its temporal expression during the RB phase indicates it role as a small RNA regulator throughout this phase. The interaction between IhtA and the hctA transcript was the fi rst example demonstrated of small RNA regulation in Chlamydia . Since there are at least 18 additional unique intergenic small RNAs detectable by microarray and northern blot analysis, it is likely that small RNA regulation plays a large role in the chlamyd ial development c ycle, and the IhtA hctA interaction is just the beginning of these studies. Not only was IhtA found to be highly conserved among

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72 species, but its regulatory interaction with hctA was also found to be conserved in each species. Regulation b y trans encoded small RNA generally involves only short and imperfect complementarity to their targets, and normally allows a single small RNA to regulate the expression from several different transcript s. These studies demonstrate that IhtA interacts with both CTL00 97 and CTL0322 in vitro, and is hypothesized to play a role in their regulation in vivo. The IhtA CTL0322 t ranscript interaction within the E. coli surrogate system helped to prov ide additional evidence of this regulation within Chlamydia . Altho ugh the western blot analysis of CTL0322 indicated an inverse correlation to HctA expression, it does not imply that IhtA does not play a role in the regulation of CTL0322 expression. Previously studies have indicated mixed results regarding the temporal e xpression of CTL0322. A shotgun proteomic analysis of C. trachomatis, using 1 D SDS PAGE coupled with GeLC MS/MS to identify chlamydial peptides, found CTL0322 only present in EBs and did not detect it in RBs at 15 hours post infection (Skipp, Robinson, O'Connor, & Clarke, 2005) . A more recent study, using LC LC/MS MS, found the opposite as CTL0322 was detected in high levels in RBs and not detected in EBs (Saka, et al., 2011) . Relative abundance of transcripts, binding affinity, and presence of co factors would all be expected to play a role in the regulation of expression by small RNAs such as IhtA. While the BLI and CheZ experiments offer a good start, further research is needed to characterize the interaction between IhtA and the CTL0322 transcript, and its impact on the regulation of expression.

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73 Figure 4 1. IhtA conserved amongst species . A) The sequence of IhtA B) Structure predictions of IhtA were made using RNAfold server. Minimal free energy (MFE) and centroid models are the same for four species, and only differed with C. pneumonia . Colors of each nucleotide indicate base pair probability; legend on bottom ri ght of figure.

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74 Figure 4 2. IhtA of each species interacts with the cognate hctA target mRNA in vitro. Run off hctA transcripts made from species specific PCR fragments were annealed to biotinylated oligo T and bound to BLI sensor tips. hctA bound tips were incubated with native IhtA or antisense IhtA and the change in thickness on the sensor, indicating RNA:RNA binding was measured over time.

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75 Figure 4 3. IhtA rescues kill phenotype across species . Representative viability assay of E . coli expressing either hctA alone (+pLac) or coexpressing species specific hctA and IhtA (+cognate ihtA). Each condition was performed in triplicate with a minimum of three repeats. The bars represent the S.E.M. of each triplicate. The * indicates p valu e <0.001 and # indicates p value = 0.003. (Grieshaber, Grieshaber, Fischer, & Hackstadt, 2006) Table 4 1 . Putative IhtA targets.

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76 Figure 4 4. Predicted interactions with IhtA. IhtA is predicted to interact with the hctA transcript in the GC rich region around the start codon. Hypothetic IhtA recognition sequences in CTL0097 and CTL0322 are shown below hctA . Figure 4 5. IhtA binds to hctA , CTL0097, and CTL0322 in vitro. Biolayer interferometry demonstrates binding by IhtA to each of the hypothetical targets, while interaction with the negative control transcript hctB was negligible, as expected since it does not contain IhtA recognition se quences.

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77 Figure 4 6. CheZ experimental constructs. A) Schematic of the experimental design for using the cheZ the fusion putative target transcript. The pTet plasmid carries the cheZ repo plasmid carries either wild type IhtA or scrambled IhtA as a negative control. B) This schematic shows the portion of each putative target cloned into the pTet vector to create the fusion tr anscript with cheZ .

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78 Figure 4 7. IhtA represses expression of HctA and ctl0322 in E. coli surrogate . The fusion cheZ transcripts of the putative IhtA targets were induced in cheZ deficient E. coli and with either coexpression of IhtA or a scrambl ed version of IhtA. ctl0322 and hctA fusion products exhibited repression when co expressed with IhtA, whereas ctl0097 and the two controls ( hctB and cheZ non fused) did not. Figure 4 8. Northern and western blots of IhtA and targets. A) HctA presence is inversely correlated with IhtA presence during the chlamydial infection cycle (left). Because of mixed EB/RB populations during the infection cycle, purifications for each phase were done to show a clearer picture of the correlation between Hc tA and IhtA (right). (Grieshaber, Grieshaber, Fischer, & Hackstadt, 2006) B) In a separate experiment, western blots were done on EBs and 24 hour post infection (primarily RB) for HctA , MOMP, and CTL0322 (left). A coomassie sta in was performed to show general protein abundan ce for each preparation. The middle lane was loaded with 10 µ TM .

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79 CHAPTER 5 HOST EGRESS Summary While chlamydial entry into host cells has been very well studied, the mechanisms involved in chlamydial exit from its host is poorly understood. Because the replicative form of Chlamydia trachomatis is unable to infect cells, a clear first step in the pro cess of egress is for Chlamydia to redifferentiate from RBs to EBs prior to release from the host. The conversion to EBs is characterized by the condensation of the chromatin, which is mediated by the histone like protein HctA . From this point, researchers have suggested various methods of egress; some descriptions appear to be in conflict with each other, and some may be different options depending on the environment or immune response from the host. The study presented here w ill provide additional details on the lysis form of egress. We will also provide conclusive evidence that the hypothesized pore forming molecule CT153/MACPF is not directly involved in egress. Background Mechanisms of Host Cell Exit An asynchronous conver sion to the EB form can be detected as early as 18 hours post infection, and this conversion progresses in earnest from 24 hours until release from the host at approximately 48 hours. Although persistence is one option for chlamydial infections, the majori ty of infections result in release from the cell. The earliest characterizations of chlamydial egress describe it as either an exocytosis event (Todd & Caldwell, 1985) or a lysis event (Campbell, Richmo nd, & Yates, 1989) , while a more recent study has described the exit as an apoptotic event (Perfettini, Hospital,

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80 Stahl, Jungas, Verbeke, & Ojcius, 2003) . A recent study used a GFP based approach to observe egress in an at tempt to resolve some of the differences in prior studies (Hybiske & Stephens, 2007) . The GFP based approach resulted in a finding that C. trachomatis uses two mutually exclusive egress pathways, lysis and extrusion. Extrusion was described as a slow process of a pinching of the inclusion resulting in detachment from the host cell. Mac/perforin Pore forming toxins (PFTs) are often associated with pathogenesis in bacteria and protozoa. Two families of globular PFTs are the c hole sterol dependent cytolysins (CDCs) and membrane a ttack complex/perforin (MACPF) . While these distinct families do not share sequence homology, recent studies have suggested that proteins from these families share some structural similarity. While CDCs app ear to be produced only by gram positive bacteria, it is notable that the MACPF domain has been found in both prokaryotic and eukaryotic organisms. The MACPF domain is generally associated with proteins of the immune system, including several components o f the complement system and perforin. Although there is significant homology between the MACPF domains, there is quite a wide variety of targets for the proteins that carry this domain. Several of the proteins with MACPF domain in the complement system i nsert into the membranes of gram negative bacteria, while perforins can be used to cause lysis of viral infected eukaryotic cells. There is little understanding of the target of bacterial produced proteins containing the MACPF domain, which has led to a r enewed interest in these proteins.

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81 Hypothesized Role of Chlamydial Protein CT153 Under microtubule polymerizing co nditions, we have identified a c hlamydial protein that contains a MACPF domain. CT153 shares much sequence homology with a number of p ro tozoan MACPF proteins . CT153 is also located in a region of the chlamydial genome termed the plasticity zone (Thomson, et al., 2008) (Taylor, Nelson, Dorward, Whitmire, & Caldwell, 2010) . The PZ is a region of the genome that is responsible for pathogen host interactions and often contains virulence factors. We hypothesize that C . trachomatis produces CT153 in order to assist with egress from the host cell at the end of the infection cycle . Results Inclusion Membrane Ruptures Prior to Host Lysis In order to study the progression of events during chlamydial egress, we improved upon the GFP based experimental design of Hybiske by surrounding the infected cells with propidium iodide . Propidium iodide st ains nucleic acids, but the COS7 membrane is impermeable to propidium iodine, so only upon lysis of the host membrane will it stain the nucleus and any other nucleic acids present. And instead of relying on GFP to observe diffusion, this study in volved microinjection of fluorophores linked to dextran into the COS7 cells. Fluorescent molecules linked to various sizes of dextran molecules allows for different sized molecules to be tested for permeability. No evidence was found of the extrusion metho d of egress described by Hybiske, and nearly all infected cells proceeded to lyse the host cell. Permeabilization of the inclusion membrane was the first step in the egress mechanism, as was evidenced from the diffusion of the fluorophore into the inclusio n prior to staining of the host by propidium

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82 iodide (Figure 5 1A C). This step was followed by lysis of the host membrane, which normally occurred within 3 5 minutes of the lysis of the inclusion. Lysis Likely Occurs Without Discr et e Pore Formation In orde r to test for pore formation during lysis events, fluorophores of various size were microinjected into the c ytoplasm of infected COS7 cells. One of the common roles of proteins containing MACPF domains is to for m pores in membranes, and C. trachomatis expresses a protein that contains a MACPF domain. Although the size of range of 1 to 50 nm radius (Aroian & van der Goot, 2007) . The fluoro phores that were injected into the COS7 radius, and non permeability of the inclusion was maintained, indicating a lack MACPF pore formation . In each case imaged, the lysis event appeared to be a complete rupture of the membrane, not a slow leak of fluorophores. CT153 Is an Early Gene in the Development Cycle In order to study any temporal expression of CT153/MACPF, qPCR and western blot analysis was performed. The quantity of the CT153 transcrip t was normalized in three different ways to offer the best understanding of the abundance of the transcript during each segment of the development cycle (Figure 5 2A) . CT153 transcript was normalized to total nucleic acid, and was found to reach its peak l evel at approximately 16 hours post infection. It must be noted that this includes host cell nucleic acids in addition to those of C hlamydia . Since EBs are non active forms of Chlamydia have any ideal reference transcripts available, we normalize d CT153 against genome copy to reflect a per cell copy number. CT153 was detectable as early as 2 hours PI, and reached its peak abundance per cell at 8 hours PI. CT153 transcript continued to

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83 be present for the remainder of the development cycle, but decl ined in abundance on a per cell basis steadily after the 8 hour time point. This pattern is most indicative of an early gene. Finally, CT153 was normalized against 16S rRNA, as this would indicate abundance per active cell (RB form). As expected, this infl ated the copy number in the later time periods. The peak shifted to 10 hours PI, and the decline following the peak was less notable. Due to the high abundance of dormant EBs late in the infection cycle, normalization against 16S rRNA during the latter par t of the infection cycle provides a better understanding of the temporal expression of CT153 in active cells. Although RNA copy number is often indicative of the expression level of the protein, we performed western blots to determine protein abundance. Wh en normalized against host tubulin, CT153 protein was detectable very early in the infection (8 hours PI) and continued to accumulate throughout the infection cycle (Figure 5 2B) . MOMP was used a control for this western blot as it is the most abundant p rotein in both EBs and RBs. MOMP has alternate structure in EBs and RBs, and this is likely the reason for the lack of detection in the EB enriched time periods (8 hours and 50 hours PI). CT153 Localizes to the Chlamydial Membrane Immunofluorescence was pe rformed to identify the localization of CT153 during a typical chlamydial infection . As was the case with western blot analysis, immunofluorescence detected CT153 early in the infection cycle. At each time point CT153 was detected, it was found to co local ize in the area of the chlamydial membrane, and never localized to either the inclusion membrane or the host membrane (Figure 5 3).

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84 Discussion Hybiske had previously demonstrated the sequence of events in host egress via the lysis mechanism by expressing G FP in infected cells and imaging every 5 minutes . We were able to get a more complete pictur e of these events by i mag ing with hig her frequency/frame rate, using fluorophores of various sizes , and including propidium iodide in the media surrounding the cells . The order of events determined by Hybiske match ed the results from our studies, and the propidium iodide allowed us to demonstrate that the host membrane maintained its integrity for several minutes following inclusion membrane lysis. Although we hypothesized that C. trachomatis expresses a factor that forms indicated by the diffusion occurring instantaneous without any prior leaking no matter the size of the fluorophore. Although this does not rule out the use of a MACPF, this does appear to indicate a lack of discrete pores. Although we did not observe the extrusion method of chlamydial egress in any of our experiments, it is likely that this was a res ult of our studies maintaining c alcium in the growth media. Calcium was sh own to be required for the lytic egress pathway, and our inclusion of calcium in each experiment could explain why we only witnessed the lytic pathway . Hybiske used calcium free ring ers solution to prevent intracellular calcium signaling and found a much higher rate of ex trusion that we did. Although the transcription and expression profile of CT153 did not suggest a role in chlamydial egress since C. trachomatis uses tight regulation of protein expression to ensure proteins are available only when needed by the cell , we still co nsidered the possibility that the CT153 protein was being sequestered in an enzymatically inactive

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85 state early in the infection , and then was activated for the egress process. However, CT153 was found to co localize with the chlamydial membrane and never found at the inclusion or host membrane, thus indicating it is unlikely to play a role in egress. Due to its localization, expression profile, and predicted str ucture, it may play a role in secretion within the chlamydial membrane. Additional research is necessa ry to provide a more detailed new hypothesis for the role of CT153/MACPF.

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86 Figure 5 1. The inclusion becomes permeable to each fluorophore during egress events . A) COS7 cells were infected with C. trachomatis serovar L2 and microinjected with Dextran Alexa conjugate molecules of the size indicated in each figure. Images were captured each minute, and frame 4 21 were omitted from the figure due to space requirements, but showed a gradual increase in propidium i odide staining. B) Microinjected fluorophores were replaced with GFP, and the results matched that found with the fluorophores. C) A large molecular weight fluorophore was microinjected into the infected COS7 cells, and the frame rate was improved to 1 fra me/10 seconds.

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87 Figure 5 2. CT153 is an early onset gene and the protein is present throughout the infection cycle. A) Abundance of CT153 transcript was normalized to either total nucleic acid, genome copy of CT153, or 16S rRNA. B) Western blot analysi s of HeLa cells infected with C. trachomatis serovar L at an MOI of 5. tubulin was used as a loading control, and MOMP was used as a control since it is the protein with the highest abundance in both EBs and RBs.

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88 Figure 5 3 . CT153 localizes to the chl amydia l membrane . EmGFP tagged CT153 shared a similar localization pattern as that of human serum, which identifies the location of Chlamydia . A void of EmGFP in the middle of each chlamydial cell indicates a localization to the membrane of each cell.

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89 CHA PTER 6 CONCLUSION AND FUTURE DIRECTIONS As an obligate intracellular bacterium, C. trachomatis must practice economy of its genome and resources in order to survive in the nutr ient limiting environments that it face s . While some of the regulatory mechanisms that help C. trachomatis accomplish this are partially characterized, much more work is left to be done in this area. C. trachomatis uses histone like proteins to silence its genome while in t he EB phase, but this s ilencing activity is absent in RBs . The nucleotropic protein CTL0322 is a more likely candidate to be involved in transcriptional or translational regulation during the RB phase. CTL0322 was found in our studies to be much more abundant in RBs than EBs, an d showed binding affinity to dsDNA, ssDNA, and RNA. CTL0322 also demonstrated preference for GC rich oligonucleotides, and because the chlamydial genome is AT rich, this may indicate an important characteristic that allows for site specific binding within the chlamydial genome. Western blot analysis of CTL0322 coupled with immunofluorescence staining during the entire infection cycle are critical future experiments that would be helpful in identifying the role of CTL0322 within C. trachomatis . DNase seq or ChIP seq would be valuable experiments to better characterize the binding preference of CTL0322. Since research into small RNA regulation in Chlamydia is in its early stages, we expect that there may be interactions between sRNAs and mRNAs that may not f it the same model as the interaction between IhtA and hctA . In other bacteria, it has been demonstrated that sRNAs can have both a positive and negative impact on expression level from a target transcript. Several sRNAs have the ability to alter secondary structure of mRNAs and make the Shine Delgarno sequence more accessible to

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90 ribosomes, thus increasing expression. Even with our current understanding of the interaction between IhtA and hctA , bioinformatic approaches to identifying targets have their limit rich region surrounding the AUG start codon as being critical for interaction with IhtA, it is possible that other sequences in undiscovered target transcripts could allow interaction with IhtA. We were able to demonstra te in vitro interaction between IhtA and two of its putative target transcripts, CTL0097 and CTL0322. And while CTL0097 showed inconsistent results of being regulated in our E. coli surrogate system, CTL0322 expression was found to be repressed by IhtA. Al though using CheZ as a reporter g ene in our su rrogate system may not appear to be the ide al choice for detecting interaction between IhtA and individual bioinformatical ly identified transcripts due to the time requirement necessary to witness motility , it lends itself to being used in high throughput screening for s RNA fusion transcript reg ulation. And the second benefit of CheZ screening is that it can be used for finding targets whose expression is either positively or negatively regulated by a sRNA . Alth ough CTL0322 was found to have a different expression profile as that of HctA , it does not necessarily imply that CTL0322 is not regulated by IhtA. Stoichiometry and binding affinity of IhtA to other transcripts would be expected to impact the regulatory e ffects of IhtA expression. S ince C. trachomatis is currently a genetically intractable organism, most studies of sRNA regulator will continue to occur in surrogate organisms and in vitro experiment s . Site directed mutagenesis of IhtA and hctA have been performed to help characterize the interaction between those molecules, and the same could be done with

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91 CTL0097 and CTL0322 with both BLI and the E. coli surrogate system. Understanding how nucleotide changes in IhtA impacts its binding affinity with its targets could allow researchers to design small RNAs to regulate specific genes. Designer sRNAs could become an invaluable molecular biology tool if they can be transformed into C. trachomatis and used to knockdown expression of specific gene.

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94 Gottesman, S., & Storz, G. ( 2010). Bacterial Small RNA Regulators: Versatile Roles and Rapidly Evolving Variations. Cold Spring Harbor Prospectives in Biology , 1 16. Grainger, D. C., Overton, T. W., Reppas, N., Wade, J. T., Tamai, E., Hobman, J. L., . . . Busby, S. J. (2004). Genomic Studies with Escherichia coli MelR Protein: Applications of Chromatin Immunoprecipitation and Microarrays. Journal of Bacteriology, 186 (2 0), 6938 6943. Grieshaber, N. A., Fischer, E. R., Mead, D. J., Dooley, C. A., & Hackstadt, T. (2004). Chlamydial histone DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis. PNAS , 7451 7456. G rieshaber, N. A., Grieshaber, S. S., Fischer, E. R., & Hackstadt, T. (2006). A small RNA inhibits translation of the histone like protein Hc1 in Chlamydia trachomatis. Molecular Microbiology, 59 (2), 541 550. Grieshaber, N. A., Sager, J. B., Dooley, C. A., Hayes, S. F., & Hackstadt, T. (2006). Regulation of the Chlamydia trachomatis Histone H1 Like Protein Hc2 Is IspE Dependent and IhtA Independent. Journal of Bacteriology , 5289 5292. Griffiths, E., Ventresca, M. S., & Gupta, R. S. (2006). BLAST screening of chlamydial genomes to identify signature proteins that are unique for the Chlamydiales, Chlamydiaceae, Chlamydophila and Chlamydia groups of species. BMC Genomics , 1 20. Hackstadt, T., Baehr, W., & Ying, Y. (1991, May). Chlamydia trachomatis developmentally regulated protein is homologous to eukaryotic histone H1. Proceedings of the National Academies of Sciences, 88 , 3937 3941. Hackstadt, T., Rockey, D. D., Heinzen, R. A., & Scidmore, M. A. (1996). Chlamydia trachomatis interrupts an exocytic pathway to acquire endogenously synthesized sphingomyelin in transit from the Golgi apparatus to the plasma membrane. The EMBO Journal, 15 (5), 964 977. Hatch, T. P., Allan, I., & Pearce, J. H. (1984). Structura l and Polypeptide Differences Between Envelopes of Infective and Reproductive Life Cycle Forms of Chlamydia spp. Journal of Bacteriology , 13 20. Huntzinger, E., Boisset, S., Saveanu, C., Benito, Y., Geissmann, T., Namane, A., . . . Romby, P. (2005). Staphy lococcus aureus RNAIII and the endoribonuclease III coordinately regulate spa gene expression. The EMBO Journal, 24 (4), 824 835.

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95 Hybiske, K., & Stephens, R. S. (2007). Mechanisms of Host Cell Exit by the Intracellular Bacterium Chlamydia. PNAS , 11430 11435 . Kaul, R., Allen, M., Bradbury, E. M., & Wenman, W. M. (1996). Sequence specific binding of chlamydial histone H1 like protein. Nucleic Acids Research , 2981 2989. Lane, B. J., Mutchler, C., Khodor, S., Grieshaber, S. S., & Carabeo, R. A. (2008). Chlamydia Entry Involves TARP Binding of Guanine Nucleotide Exchange Factors. PLoS Pathogens, 4 (3), 1 11. Lee, R. C., Feinbaum, R. L., & Ambros, V. (1993). The C. elegans Heterochronic Gene lin 4 Encodes Small RNAs with Antisense Complementarity to lin 14. Cell , 84 3 854. Newhall, W. J., & Jones, R. B. (1983). Disulfide Linked Oligomers of the Major Outer Membrane Protein of Chlamydiae. Journal of Bacteriology , 998 1001. Nicholson, T. L., Olinger, L., Chong, K., Schoolnik, G., & Stephens, R. (2003, May). Globa Stage Specific Gene Regulation during the Developmental Cycle of Chlamydia trachomatis. Journal of Bacteriology, 185 (10), 3179 3189. Nieuwenhuis, R. F., Ossewaarde, J. M., Meijden, W. I., & Neumann, H. A. (2003). Unusual presentation of early lymphogranuloma ven ereum in an HIV 1 infected patient: effective treatment with 1 g azithromycin. Sexual Transmitted Infections, 79 , 454 455. Parkinson, J. S., Ames, P., & Studdert, C. A. (2005). Collaborative signaling by bacterial chemoreceptors. Current Opinion in Microbi ology, 8 , 116 121. Paz, I., Sachse, M., Dupont, N., Mounier, J., Cederfur, C., Enninga, J., . . . Sansonetti, P. (2010). Galectin 3, a Marker for Vacuole Lysis by Invasive Pathogens. Cellular Microbiology , 530 544. Pedersen, L. B., Birkelund, S., & Christi ansen, G. (1994). Interaction of the Chlamydia trachomatis histone H1 like protein (Hc1) with DNA and RNA causes repression of transcription and translation in vitro. Molecular Microbiology , 1085 1098. Pedersen, L. B., Birkelund, S., & Christiansen, G. (19 96). Purification of recombinant Chlamydia trachomatis histone H1 like protein Hc2, and comparative functional analysis of Hc2 and Hc1. Molecular Microbiology , 295 311.

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96 Pedersen, L. B., Birkelund, S., Holm, A., Ostergaard, S., & Christiansen, G. (1996). T he 18 Kilodalton Chlamydia trachomatis Histone H1 Like Protein (Hc1) Contains a Potential N Terminal Dimerization Site and a C Terminal Nucleic Acid Binding Domain. Journal of Bacteriology , 994 1002. Perara, E., Ganem, D., & Engel, J. N. (1992). A developm entally regulated chlamydial gene with apparent homology to eukaryotic histone H1. Proceedings of the National Academy of Sciences , 2125 2129. Perfettini, J. L., Hospital, V., Stahl, L., Jungas, T., Verbeke, P., & Ojcius, D. M. (2003). Cell Death and Inflammation During Infection with the Obligate Intracellular Pathogen, Chlamydia. Biochimie , 763 769. Pfeiffer, V., Lucchini, S., Hinton, J., & Vogel, J. (2009, August). Coding sequence targeting by MicC RNA reveals b acterial mRNA silencing downstream of translational initiation. Nature Structural & Molecular Biology, 16 (8), 840 846. Prevost, K., Salvail, H., Desnoyers, G., Jacques, J. F., Phaneuf, E., & Masse, E. (2007). The small RNA RyhB activates the translation of shiA mRNA encoding a permease of shikimate, a compound involved in siderophore synthesis. Molecular Microbiology, 64 (5), 1260 1273. Remacha, M., Kaul, R., & Wenman, W. M. (1996). Functional domains of chlamydial histone H1 like protein. Biochemical Journa l , 481 486. Richardson, D., & Goldmeier, D. (2007, January). Lymphogranuloma venereum: an emerging cause of proctitis in men who have sex with men. International journal of STD & AIDS, 18 (1), 11 16. Saka, H. A., Thompson, J. W., Chen, Y. S., Kumar, Y., Dubois, L. G., Moseley, M. A., & Valdivia, R. H. (2011). Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms. Molecular Microbiology , 1185 12 03. Shen, L., Feng, X., Yuan, Y., Luo, X., Hatch, T. P., Hughes, K. T., . . . Zhang, Y. x. Journal of Bacteriology , 7364 7377. Singh, S. S., & Grainger, D. C. (2013). H NS Can Facilitate Specific DNA binding by RNA Polymerase in AT rch Gene Regulatory Regions. PLOS Genetics , e1 10. Sisko, J. L., Spaeth, K., Kumar, Y., & Valdivia, R. H. (2006). Multifunctional analysis of Chlamydia specific genes in a yeast expression system. Molecular Micr obiology, 60 (1), 51 66.

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97 Skipp, P., Robinson, J., O'Connor, C. D., & Clarke, I. N. (2005). Shotgun proteomic analysis of Chlamydia trachomatis. Proteomics , 1558 1573. Smith, G. A., Marquis, H., Jones, S., Johnston, N. C., Portnoy, D. A., & Goldfine, H. (199 5). The Two Distinct Phospholipases C of Listeria monocytogenes Have Overlapping Roles in Escape from a Vacuole and Cell to Cell Spread. Infection and Immunity , 4231 4237. Tao, S., Kaul, R., & Wenman, W. M. (1991). Identification and Nucleotide Sequence of a Developmentally Regulated Gene Encoding a Eukaryotic Histone H1 Like Protein from Chlamydia trachomatis. Journal of Bacteriology , 2818 2822. Tattersall, J., Rao, G. V., Runac, J., Hackstadt, T., Grieshaber, S. S., & Grieshaber, N. A. (2012). Translation Inhibition of the Developmental Cycle Protein HctA by the Small RNA IhtA Is Conserved across Chlamydia. PLOS One , e1 9. Taylor, L. D., Nelson, D. E., Dorward, D. W., Whitmire, W. M., & Caldwell, H. D. (2010). Biological Characterization of Chlamydia trach omatis Plasticity Zone MACPF Domain Family Protein CT153. Infection and Immunity , 2691 2699. Thomson, N. R. (2008). Chlamydia trachomatis: Genome sequence analysis of lymphogranuloma venereum isolates. Genome Research, 18 , 161 171. Thomson, N. R., Holden, M. T., Carder, C., Lennard, N., Lockey, S. J., Marsh, P., . . . Clarke, I. N. (2008). Chlamydia trachomatis: Genome sequence analysis of lymphogranuloma venereum isolates. Genome Research , 161 171. Todd, W. J., & Caldwell, H. D. (1985). The interaction of Chlamydia trachomatis with host cells: ultrastructural studies of the mechanism of release of a biovar II strain from HeLa 200 cells. Journal of Infectious Diseases , 1037 1044. Todd, W. J., & Caldwell, H. D. (1985). The Interaction of Chlamydia trachomatis with Host Cells; Ultrastructural Studies of the Mechanisms of Release of a Biovar II Strain from HeLa 229 Cells. Journal of Infectious Diseases , 1037 1044. Vogel, J., & Wagner, E. G. (2007). Target identification of small noncoding RNAs in bacteria. Current Opinion in Microbiology, 10 , 262 270. Wagar, E. A., & Stephens, R. S. (1988). Developmental form specific DNA binding proteins in Chlamydia spp. Infection and Immunity , 1678 1684. Wilson, K. (1997). Prepartion of Genomic DNA from Bacteria. Current Protocols in Molecular Biology , 2.4.1 2.4.5.

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98 Workowski, K. A., & Berman, S. (2010, December 17). Sexually Transmitted Diseases Treatment Guidelines, 2010. MMWR, 59 (RR12), 1 110. World Health Organization. (2001). Global Prevalence and Incidence of Selected Curable Sexually Transmitted Infections. Geneva, Switzerland. Wright, H. R., Turner, A., & Taylor, H. R. (2008, June 7 13). Trachoma. The Lancet, 371 (9628), 1945 1954. Xia, X. (2007). The +4G Site in Kozak Consensus Is Not Related to the Efficiency of Tra nslation Initiation. PLoS ONE , e188. gene in Chlamydia. Molecular Microbiology , 577 584. Yuan, Y., Zhang, Y. x., Watkins, N. G., & Caldwell, H. D. (1989, April). Nucleotid e and Deduced Amino Acid Sequences for the Four Variable Domains of the Major Outer Membrane Proteins of the 15 Chlamydia trachomatis Serovars. Infection and Immunity, 57 (4), 1040 1049.

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99 BIOGRAPHICAL SKETCH Justin was born to Pam and Mark Runac in Pittsburgh, PA. From an early age, Justin was always interested in science and mathematics. While preparing for a career as a chemical engineer, Justin found that his interests lie more in the chemistry related parts of th e field and less on the engineering aspect, and decided to pursue a path in biochemistry and molecular biology. The pathway from undergraduate student to Ph . D . was somewhat of an endurance trek which gave Justin an opportunity to serve in the U . S . Army and live overseas for several years. Justin earned his Ph . D. f rom the University of Florida in the summer of 2014. Justin hopes to combine his love of science with his experience in public service and allow more of the population around the world to benefit f rom the great advance s made in biomedical sciences during the past few decades.



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THEJOURNALOFCELLBIOLOGYJCB: ARTICLE © 2008 Paschen et al. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 182 No. 1 117…127 www.jcb.org/cgi/doi/10.1083/jcb.200804023 JCB 117 Correspondence to Georg H ä cker: hacker@lrz.tum.de Abbreviations used in this paper: AHT, anhydrotetracycline; CHX, cycloheximide; CK8, cytokeratin 8; CM, coumermycin; CPAF, chlamydial protease-like activity factor; gyrB-CPAF, N-FLAG-3xgyrB-CPAF construct; NB, novobiocin; PARP, poly (ADP-ribose) polymerase; TET, tetracycline; TNF, tumor necrosis factor; Tsp, tail-speci“ c protease; zVAD, zVAD-fmk. The online version of this paper contains supplemental material. Introduction Chlamydia trachomatis , an obligate intracellular bacterium, is the leading cause of bacterially sexually transmitted disease and a main cause of preventable blindness. C. trachomatis has a biphasic developmental cycle. The infectious elementary body (EB) infects primarily epithelial cells where it develops within a membrane-bound vacuole (called an inclusion) into a replicating noninfectious reticulate body (RB). Within two days, the RB redifferentiates into an EB and is released from the infected cell. Chlamydiae thus develop in a compartment that is separate from the rest of the human or animal host cell. However, the bacteria impact on a number of signaling pathways in the host cell, and cause substantial changes to cellular transcription as well as cell damage ( McClarty, 1994 ; Wyrick, 2000 ; Fields and Hackstadt, 2002 ). How Chlamydia achieves this is not known in great detail. Several chlamydial species have been found to possess the components of a functional type III secretion system, which likely enables the bacteria to inject effector proteins into the host cytosol, and a number of such candidate proteins have been identi ed (for review see Peters et al., 2007 ). Infection with Chlamydia causes massive stress to the host cell, and cytolytic activity associated with Chlamydia infection has been described for more than 30 years ( Friis, 1972 ; Todd and Storz, 1975 ; Chang and Moulder, 1978 ; Wyrick et al., 1978 ). By electron microscopy, massive changes to organelles were noticed at later stages of infection, such as dilation and vacuolation of ER, distortion of mitochondria, and nuclear condensation ( Todd and Storz, 1975 ; Todd et al., 1976 ). Although some of these changes resemble features of apoptosis ( Ojcius et al., 1998 ; Perfettini et al., 2002 ; Ying et al., 2006 ), further characterization of signaling pathways indicates that the apoptotic pathway is not activated by Chlamydia and the cytopathic changes observed are nonapoptotic ( Ying et al., 2006 ). Cytopathicity and cell death may be a defense mechanism of the cell to block bacterial replication or may aid bacterial spreading and cause infection-associated in ammation. It has been entirely unclear how Chlamydia induces cell death. The gene of a potential cytotoxin has been identi ed in the C. trachomatis genome ( Belland et al., 2001 ), but was later found to be nonfunctional in many serovars ( Carlson et al., 2004 ). Chlamydiae replicate in a vacuole within epithelial cells and commonly induce cell damage and a deleterious in” ammatory response of unknown molecular pathogenesis. The chlamydial protease-like activity factor (CPAF) translocates from the vacuole to the cytosol, where it cleaves several cellular proteins. CPAF is synthesized as an inactive precursor that is processed and activated during infection. Here, we show that CPAF can be activated in uninfected cells by experimentally induced oligomerization, reminiscent of the activation mode of initiator caspases. CPAF activity induces proteolysis of cellular substrates including two novel targets, cyclin B1 and PARP, and indirectly results in the processing of proapoptotic BH3-only proteins. CPAF activation induces striking morphological changes in the cell and, later, cell death. Biochemical and ultrastructural analysis of the cell death pathway identify the mechanism of cell death as nonapoptotic. Active CPAF in uninfected human cells thus mimics many features of chlamydial infection, implicating CPAF as a major factor of chlamydial pathogenicity, Chlamydia -associated cell damage, and in” ammation. Cytopathicity of Chlamydia is largely reproduced by expression of a single chlamydial protease Stefan A. Paschen , 1 Jan G. Christian , 1 Juliane Vier , 1 Franziska Schmidt , 1 Axel Walch , 2 David M. Ojcius , 3 and Georg H ä cker 1 1 Institute for Medical Microbiology, Immunology and Hygiene, Technische Universit ä t M ü nchen, D-81675 Munich, Germany 2 Institute of Pathology, Helmholtz Zentrum M ü nchen, German Research Center for Environmental Health (GmbH), D-85764 Neuherberg, Germany 3 School of Natural Sciences, University of California, Merced, Merced, CA 95344

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JCB € VOLUME 182 € NUMBER 1 € 2008 118sors, both are proteolytically processed into two subunits during their physiological activation, and in both cases the active enzyme is made up of a complex of both subunits. We therefore a imed at achieving experimental oligomerization of CPAF to model the physiological clustering of caspase-9 after its recruitment into the apoptosome during apoptosis ( Bao and Shi, 2007 ). The open reading frame of C. trachomatis CPAF was fused to an N-terminal partner consisting of the FLAG antibody epitope and a triple repeat of a fragment of bacterial gyrase (gyr) B. GyrB binds the cell-permeable synthetic ligand coumermycin (CM). Because CM has two gyrB binding sites, it can bind two gyrB proteins simultaneously, inducing dimerization of the fusion partner of gyrB ( Fig. 1 A ). A triple repeat rather than an individual gyrB molecule was chosen to enhance complex formation upon CM addition. We and others have used this system in the past for conditional complex formation of intracellular signaling proteins ( Farrar et al., 1996 ; Hacker et al., 2006 ). Because transient transfections indicated that the protein was toxic to human cells (unpublished data), the fusion construct was placed under the control of a tetracycline (TET)-inducible promoter, where expression was silenced in cells carrying the tet-repressor but induced upon addition of tetracycline (  tet-on Ž ). When the gyrB-CPAF construct was transfected into 293T cells stably carrying the tet-repressor (T-REx-293), the addition of tetracycline or its analogue anhydrotetracycline (AHT) induced the appearance of a protein of the expected size of the full-length protein as well as a smaller product, which ran at the expected size of a protein containing the N-terminal tag fused to the N-terminal CPAF-fragment, indicating processing of the protein ( Fig. 1 B ). T-REx-293 cells were then transfected and clones were selected that stably carried the TET-inducible gyrB-CPAF construct. Five clones were identi ed, in which addition of TET/AHT induced expression of a protein of the same size as the smaller product during transient transfection, again very likely corresponding to the fragment of gyrB and the N-terminal fragment of CPAF ( Fig. 1 B and unpublished data). No band corresponding to the intact protein was detected in these clones, suggesting more ef cient processing. All of the clones showed the same phenotype upon induction of gyrB-CPAF (see following paragraph). Expression of gyrB-CPAF thus leads to spontaneous processing of the protein. To test for CM-mediated oligomerization, cell extracts from the stable clone K6 expressing gyrB-CPAF were analyzed by size exclusion chromatography. GyrB-N-CPAF was detectable with a peak around the expected molecular weight of the protein (120 kD). Addition of CM caused a shift in the protein to higher molecular weight fractions ( Fig. 1 C ). Because of the low expression of wild-type gyrB-CPAF, we performed additional experiments with a cleavage-de cient mutant of gyrB-CPAF (CPAFmut1 [see following paragraph], which is expressed at considerably higher levels), expressed by transient transfection and induction in T-REx-293 cells. As shown in Fig. 1 C , most of the protein eluted at about the predicted size of the unprocessed monomer (150 kD), although easily detectable amounts appeared in fractions containing higher molecular weight proteins. Addition of CM caused a shift of the protein peak, which then eluted One chlamydial protein has been directly puri ed from the cytosol of infected cells. Named chlamydial protease-like activity factor (CPAF), this protein was isolated as a factor that can degrade two host transcription factors, RFX5 and USF-1 ( Zhong et al., 2001 ). Because these transcription factors are involved in the expression of major histocompatibility complex (MHC) molecules, it has been speculated that CPAF may contribute to immune evasion of Chlamydia ( Zhong et al., 2001 ). Similarly, it has been suggested that CPAF plays a role in the loss of expression of the MHC-like protein CD1d during infection, which may also enhance escape from host immune surveillance ( Kawana et al., 2007 ). The cytoskeletal protein cytokeratin (CK) 8, a component of intermediate laments, has also been identi ed as a substrate of CPAF proteolysis ( Dong et al., 2004c ), and recently it has been proposed that the pro-apoptotic BH3-only proteins, which are degraded during chlamydial infection ( Fischer et al., 2004 ; Ying et al., 2005 ), are CPAF substrates ( Pirbhai et al., 2006 ). Because Chlamydia cannot be genetically modi ed, direct proof of the role of CPAF has been dif cult. CPAF is synthesized in the chlamydial inclusion as one polypeptide, but is rapidly processed into two subunits that assemble into heterodimers and are proteolytically active in the host cytosol ( Dong et al., 2004a,b ). Expression of the CPAF precursor in human cells did not induce CPAF processing and yielded no proteolytic activity ( Dong et al., 2004a ). Although potential functions had thus been assigned to CPAF, we reasoned that an active protease, free in the cytosol of a human cell, may be expected to damage the cell, and that CPAF therefore should be a candidate factor for cytopathic activity. However, until now it has not been possible to express active CPAF in human cells in the absence of infection, and meaningful analysis of CPAF effects in infected cells is very dif cult due to the presence of numerous bacterial components in the cell. We here show that CPAF can be activated by  induced proximity Ž in the absence of infection. The induced proximity model was proposed to explain the activation of initiator caspases during induction of apoptosis ( Salvesen and Dixit, 1999 ; Pop et al., 2006 ; Bao and Shi, 2007 ). According to this model, the dimerization and activation of initiator caspases requires their adaptor-mediated clustering, followed by caspase processing. Similarly, we describe that forced clustering of CPAF leads to its processing and activation in human cells. Activation of CPAF caused massive morphological changes and nonapoptotic death of human host-cells, strongly resembling the changes and the form of cell death induced by chlamydial infection. CPAF should therefore be regarded as a major factor of chlamydial pathogenicity. CPAF activity may help Chlamydia at earlier stages to establish the growing inclusion in the cell and at later stages may facilitate release of newly replicated bacteria. Results Activation of CPAF by induced proximity To study the role of CPAF in cytopathicity, a model had to be developed to express active CPAF. We had noticed certain parallels of caspase-9 and CPAF. Both are synthesized as inactive precur-

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119 CYTOPATHICITY OF CPAF € Paschen et al. CPAF activity requires its processing site and an intact protease motif CPAF activity in lysates from infected cells was inhibited by an inhibitor of the cellular proteasome, lactacystin, but not another one, MG-132 ( Zhong et al., 2001 ). We used CPAF K6 cell lysate as a source of CPAF and lysate from cells transfected with a construct encoding myc-tagged CK8 as a substrate, to test for the effects of standard protease inhibitors on CPAF. Lactacystin but not MG-132, nor any other tested inhibitor, prevented degradation of CK8 ( Fig. 2 A and unpublished data). (The following inhibitors were tested: E64, pepstatin A, PMSF, TPCK, and a standard mix of protease inhibitors [Roche].) These results con rm the inhibitory activity of lactacystin previously reported in lysates from infected cells and identify an unusual inhibitor pro le for CPAF. It has been shown that a small amount of CPAF is cleaved when expressed in Escherichia coli , and the processing site has been mapped; a cleavage site mutant was not processed and was inactive when expressed in E. coli ( Dong et al., 2004a ). It has further been noted that CPAF contains a domain characteristic of bacterial Tail-speci c proteases (Tsp) ( Shaw et al., 2002 ), a class of serine proteases originally described in E. coli ( Silber et al., 1992 ). We therefore generated gyrB-CPAF constructs with around 500 kD and might correspond to a trimer or tetramer of the protein. A substantial fraction of the protein appeared to be engaged in formation of even higher molecular weight complexes. Most of these CM-induced complexes could be disrupted by addition of an excess of the monomeric ligand of gyrase B, novobiocin (NB) ( Fig. 1 C ). These results show the expected oligomerization of gyrB-CPAF by CM, as well as substantial spontaneous complex formation. The spontaneous oligomerization was probably due to the gyrase B domains because novobiocin could reduce the extent of complex formation (compare lanes AHT and AHT/CM/NB in gyrB-CPAFmut1; Fig. 1 C ). We next measured the proteolytic activity of gyrB-CPAF, using cleavage of CK8, one of the reported cellular CPAF substrates, as a read-out. As shown in Fig. 1 D , titration of AHT on the CPAF K6 clone caused increasing cleavage of CK8 even in the absence of CM-induced oligomerization, suggesting that spontaneous aggregation was suf cient for activation of gyrBCPAF. Addition of CM to cells induced with a lower concentration of AHT caused a higher level of CK8 degradation, which could be partly blocked by increasing concentrations of novobiocin ( Fig. 1 D ). GyrB-CPAF thus shows some spontaneous aggregation and activity, which can be enhanced by CM; the effect of CM can be prevented by NB. Figure 1. CPAF is activated by induced proximity. (A) Schematic representation of the gyrB-CPAF construct. CPAF was placed under the control of a tetracycline-inducible promoter. FLAG, FLAG tag; 3xgyrB, three consecutive copies of an N-terminal fragment of gyrase B from Escherichia coli ; CPAF, CPAF from Chlamydia trachomatis (amino acid residues 18 … 601). (B) CPAF expression in T-Rex-293 cells. Expression of CPAF was induced by tetracycline (TET) either in CPAF K6 cells stably expressing gyrB-CPAF or T-REx-293 cells transfected with the gyrB-CPAF construct. CPAF-N indicat es an N-terminal fragment of gyrB-CPAF after proteolytic activation. Triton X-100 cell extracts were analyzed by Western blotting with an antibody speci“ c for the FLAG tag. Asterisk, unspeci“ c signal. Molecular size markers (in kD) are indicated. (C) Size exclusion chromatography of CPAF. Cell extracts of either CPA F K6 cells (top) or T-REx-293 cells transfected with the CPAFmut1 (carrying the S491A active-site mutation; bottom) were separated on a Superose 20 0 gel “ ltration column. Anhydrotetracycline (AHT), coumermycin (CM), or novobiocin (NB) were used as indicated. The elution fractions and eluted molecu lar size markers are indicated. (D) Activation of CPAF by induced proximity. CPAF expression was induced in CPAF K6 cells with increasing amounts (t op) of AHT or using 0.5 ng/ml (bottom). Before addition of CM, cells were preincubated with indicated amounts of NB. Samples were analyzed by Weste rn blotting using anti-CK8 antibodies. The arrowhead indicates a cleavage product of CK8. Detection of actin served as a loading control.

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JCB € VOLUME 182 € NUMBER 1 € 2008 120CPAF. A number of control proteins (Bak, Bcl-2, actin) were not degraded ( Fig. 3 ; see following paragraph regarding cleavage of BH3-only proteins). The expression of active CPAF thus recapitulates the known proteolytic activities observed during infection with whole chlamydiae. Based on the levels of substrate cleavage, the amount of active gyrB-CPAF produced corresponds to the levels of CPAF expressed relatively early in the developmental cycle, at least in T-REx-293 cells. The amounts generated during later stages of infection are probably substantially higher and cause more complete degradation of cellular substrates ( Fig. 3 ). Degradation of anti-apoptotic BH3-only proteins is likely an indirect consequence of CPAF expression BH3-only proteins are essential mediators of mitochondrial apoptosis ( Hacker and Weber, 2007 ). These proteins are degraded during chlamydial infection ( Fischer et al., 2004 ), which can account for the protection against apoptosis of infected cells. Re-expression of active BH3-only proteins overcomes the Chlamydia -imposed block of apoptosis ( Fischer et al., 2004 ), indicating that this loss is functionally relevant. It has recently been reported that CPAF can degrade BH3-only proteins in cell lysates ( Pirbhai et al., 2006 ). In our initial analyses, we failed to see degradation of the BH3-only proteins Bim and Puma (unpublished data), which were easily detectable in CPAF K6 cells, point mutations either in the active site of the Tsp domain (S491A, CPAFmut1) or in the processing site (L273G, S275V, CPAFmut2). When expressed transiently in T-REx-293 cells, neither mutant was processed ( Fig. 2 B , top) and neither was able to cleave endogenous CK8 ( Fig. 2 B , bottom) or myctagged CK8 in a cell-free system using lysate from transfected cells ( Fig. 2 C ). This suggests that CPAF is cleaved autocatalytically and the Tsp domain containing the active-site serine residue is indeed required for its proteolytic activity. A number of host cell proteins have been reported to be cleaved by CPAF during chlamydial infection, including CK8, the transcription factors RFX5 and USF-1, and the BH3-only proteins Bim, Puma, and Bik ( Zhong et al., 2001 ; Dong et al., 2004c ). Another protein, the component of intermediate laments, vimentin, was also recently shown to be cleaved by CPAF (Valdivia, R., personal communication). We therefore tested the cleavage of these proteins upon CPAF activation in K6 cells. Cleavage of CK8, RFX5, and vimentin occurred upon CPAF activation in K6 cells and yielded fragments of the same sizes as during infection with C. trachomatis ( Fig. 3 ), whereas USF1 was not detectable by Western blotting in these cells. The cell cycle protein cyclin B1 is also degraded during chlamydial infection ( Balsara et al., 2006 ). Degradation products of the same sizes as during infection were generated upon activation of CPAF, suggesting that cleavage of cyclin B1 during infection is mediated by Figure 2. Analysis of proteolytic activity of CPAF. (A) Inhibition of CPAF activity by proteasome inhibitors. Cell extracts of CPAF K6 cells (gyrB-CPAF) or T-REx-293 cells expressing myc-tagged cytokeratin 8 (CK8-myc) were combined in the presence of various amounts of the proteasom e inhibitors lactacystin (LC; top) or MG-132 (bottom) as indicated, and analyzed by Western blotting using an antibody speci“ c for the myc tag. The arrowhead indicates a cleavage product of CK8-myc (asterisk marks an unspeci“ c signal). (B) Analysis of the proteolytic activity of CPAF mutants in vivo. T-REx-293 cells were transfected with either the gyrB-CPAF construct or one of two CPAF mutants. In CPAFmut1, the Tsp-active site was mutated by the replacement S491A. In mutant 2 (CPAFmut2), two amino acid residues (L273G, S275V) were exchanged to prevent autocatalytic cleavage of CPAF. CPAF e xpression was induced by AHT as indicated. Cell extracts were analyzed by Western blotting using either FLAG tag (top) or CK8 antibodies (bot tom). The arrowhead in the top panel shows the cleavage product very likely corresponding to gyrB-CPAF-N; the arrowhead in the bottom panel indicates a cleavage product of CK8. An unspeci“ c signal is marked by an asterisk. (C) Analysis of the proteolytic activity of CPAF mutants in vitro. T-REx-293 cells were tra nsfected with the CPAF constructs as indicated, and cell lysates were incubated with extracts containing myc-tagged CK8, and analyzed as describe d in A (top). Expression of the CPAF constructs was con“ rmed by Western blotting using a FLAG tag antibody (bottom).

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121 CYTOPATHICITY OF CPAF € Paschen et al. question of the cellular consequences of CPAF activity. On the one hand, CPAF induced the degradation of BH3-only proteins and is therefore an anti-apoptotic effector. On the other hand, free proteases in the cytosol have the potential to cause cell death. This has been shown not only for the specialized caspases, but also, for example, for lysosomal peptidases ( Lockshin and Zak eri, 2004 ) and even the promiscuous protease proteinase-K ( Wilhelm and Hacker, 1999 ). Chlamydial infection causes massive morphological changes to the host cell as well as nonapoptotic cell death ( Ojcius et al., 1998 ; Belland et al., 2001 ; Perfettini et al., 2002 ; Ying et al., 2006 ). We therefore asked whether CPAF might contribute to this cytopathicity. The expression and activation of CPAF in K6 cells ( Fig. 5 A ) caused striking morphological changes in the cells. The cells rounded up and began to detach from the culture dish. They formed clusters and at later stages, smaller vesicular fragments appeared ( Fig. 5 A ). Similar albeit less pronounced changes were observed upon transient transfection and activation of g yrBCPAF in T-REx-293 or T-REx-HeLa cells (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200804023/DC1). Indeed, the morphology resembled the changes observed during chlamydial infection of T-REx-293 cells (Fig. S2). Although not conclusive, this similarity suggests that CPAF is involved in at least some of the morphological changes induced during chlamydial infection. AHT on its own caused less dramatic changes in CPAF K6 cells than when CM was included, and an excess of novobiocin could reduce the phenotypical changes ( Fig. 5 B ). Cleavage of cellular proteins by CPAF may therefore be one mechanism by which Chlamydia induces the morphological changes observed in the host cell. The dramatic changes as observed by microscopy suggested that K6 cells expressing active CPAF were dying. Cell death induction in cell culture by chlamydial infection has been previously documented, and our recent analysis suggests that this cell death occurs by a nonapoptotic process ( Ying et al., 2006 ). When cell death was measured as loss of cellular metabolic activity by MTT assay, it became apparent that most cells were dead after 20 h of expression of active CPAF. No decrease in viability was measured at 7 h, although very clear morphological changes were already apparent ( Fig. 6 A and unpubl ished data). Cell death in this assay was not inhibited by the caspase inhibitor zVAD-fmk ( Fig. 6 A ), consistent with the lack of effect of zVAD-fmk in host cell death induced by chlamydial infection ( Ojcius et al., 1998 ; Perfettini et al., 2002 ; Ying et al., 2006 ). Because caspase activity is required for apoptotic cell death, this is suggestive of a nonapoptotic form of cell death. Plasma membrane integrity was relatively well maintained, with only 20% of cells taking up the vital dye propidium iodi de, despite a reduction of metabolic activity of 80% at 20 h of treatment with AHT/CM (Fig. S3, available at http://www.jcb .org/cgi/content/full/jcb.200804023/DC1). During apoptosis, the chromatin condenses and the nuclei are fragmented. As these changes in nuclear morphology are a good marker of apoptosis, we next analyzed the dying cells for changes in nuclear morphology. CPAF activation caused nuclear condensation in some cells; however, the morphology was not quite typical for apoptosis and distinct from the appearance although both proteins were degraded during infection with C. trachomatis ( Fig. 4 A ). However, longer periods of CPAF induction in K6 cells did lead to the degradation of Bim and Puma ( Fig. 4 A ). More extensive time-course studies revealed that Bim degradation occurred later than cleavage of the other substrates ( Fig. 4 B ). Although vimentin was already cleaved at 10 h after CPAF induction/activation with AHT/CM, the Bim levels were unchanged or even increased up to 15 h of CPAF activation, after which point they began to decrease. At 18 h after activation, Bim levels were clearly reduced. No smaller fragments of Bim were detected ( Fig. 4 B ; vimentin cleavage starts around 4 h under this protocol; unpublished data). This suggested that the degradation of Bim was not mediated directly by CPAF but by subsequent proteolytic events that had been initiated by CPAF. This interpretation is supported by another nding: the degradation of Bim by prolonged activation of CPAF was blocked by lactacystin and MG-132 ( Fig. 4 C ). The proteolytic activity of CPAF, however, is only inhibited by lactacystin but not MG-132 ( Zhong et al., 2001 ; Fig. 2 A ). It is thus unlikely that CPAF directly degrades BH3-only proteins. Nevertheless, the CPAF-induced degradation of BH3-only proteins is the main reason for apoptosis inhibition in infected cells, and CPAF is therefore the main anti-apoptotic factor of Chlamydia . CPAF expression leads to nonapoptotic cell death These results showed that gyrB-CPAF could reproduce the known proteolytic events of chlamydial infection. We then turned to the Figure 3. Infection with C. trachomatis or expression of CPAF causes cleavage of host cell proteins. (Left) Infection with C. trachomatis . T-REx-293 cells were infected with C. trachomatis for indicated periods of time. RIPA buffer extracts of the cells were prepared. (Right) CPAF K6 cells were treated with either 5 ng/ µ l AHT, CM, or both for 12 … 14 h as indicated. Triton X-100 cell extracts were prepared and all extracts were analyzed by Western blotting using indicated antibodies. The arrowheads indicate speci“ c cleavage products.

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JCB € VOLUME 182 € NUMBER 1 € 2008 122prominent cleavage band was different from the one generated during apoptosis; a very light band corresponding to apoptotic cleavage appears to be visible on the blot, although the appearance of this band was not sensitive to caspase inhibition ( Fig. 6 E ). Induction of apoptosis by treatment of the cells with TNF/CHX yielded the typical caspase-dependent PARP fragment ( Fig. 6 E ). Although PARP cleavage is thus seen upon CPAF activation, it is not mediated by caspase-3 but probably by CPAF itself. The relevance of the cleavage of this new CPAF substrate for the infection remains to be seen. We nally used electron microscopy for the analysis of structural changes in cells containing active CPAF. At earlier stages (after 7 h of CPAF expression/activity), although the cells had already rounded up and detached from the plate, no ultrastructural changes were observed (unpublished data). This is perhaps somewhat surprising given the strong morphological changes, but is in accordance with the unaffected metabolic activity at this time. At 20 h, however, only relatively few cells were still morphologically intact while the majority were in a process of disintegration ( Fig. 7 A, B ). Only few apoptotic cells were detected in this analysis but many necrotic cells, con rming that CPAF induced nonapoptotic (necrotic) cell death in CPAF K6 cells. At higher magni cation, dying cells showed numerous lamellar structures of unknown composition as well as many mitochondria that still seemed only slightly affected ( Fig. 7 C ). Ectopic expression and activation of CPAF thus causes a nonapoptotic form of cell death that appears indistinguishable from cell death induced during infection with C. trachomatis . of nuclei in the same cells undergoing apoptosis upon treatment with TNF/cycloheximide (TNF/CHX) ( Fig. 6 B ). Higher magni cation photographs of the nuclear morphology of cells dying due to treatment with TNF/CHX, upon activation of CPAF or upon chlamydial infection, are shown in Fig. S4 (available at http://www.jcb.org/cgi/content/full/jcb.200804023/DC1). Furthermore, unlike the changes induced by the apoptosisinducing protocol with TNF/CHX, the CPAF-induced nuclear morphological changes were not prevented by the caspase inhibitor zVAD-fmk ( Fig. 6 B ). These results reproduced the features of Chlamydia -induced cell death and suggested that CPAF induced cell death through a nonapoptotic mechanism. Apoptosis is the result of the activation of the apoptotic signal transduction pathway, and we therefore tested directly whether this pathway was activated. Caspase-3 is a central protease in the apoptotic pathway, and caspase-3 is regularly activated proteolytically during apoptosis. An antibody directed against an active caspase-3 fragment showed only few positive cells upon activation of CPAF (treatment with AHT/CM) ( Fig. 6 C ). Western blotting further failed to detect the cleaved form of caspase-3 seen during apoptosis in cells expressing active CPAF ( Fig. 6 D ), in agreement with the absence of caspase-3 activation in cells infected with C. caviae or C. trachomatis ( Ojcius et al., 1998 ; Ying et al., 2006 ). During apoptosis, caspase-3 cleaves the nuclear enzyme poly (ADP-ribose) polymerase (PARP) ( Nicholson et al., 1995 ), and PARP cleavage can thus be used as a marker of apoptosis. Surprisingly, PARP was degraded both during chlamydial infection and upon CPAF activation ( Fig. 6 E ). However, the Figure 4. BH3-only proteins are degraded by infection with C. trachomatis or upon prolonged expression of active CPAF. (A) (Left) T-REx-293 cells were infected with C. trachomatis for indicated time points and RIPA buffer extracts were prepared. (Right) CPAF K6 cells were treated with either 5 ng/ µ l AHT, CM, or both as indicated. Cell extracts were analyzed by Western blotting using antibodies speci“ c for Bim, Puma, or actin as loading control. (B) Time course of cleavage of CPAF substrates. CPAF K6 cells were treated with 5 ng/ µ l AHT and CM to induce CPAF expression for the indicated time periods. Cell extracts were analyzed by Western blotting. The arrowheads indicate cleavage product of vimentin. (C) Inhibition of Bim de gradation by proteasome inhibitors. CPAF K6 cells were treated with 5 ng/ml AHT or 5 ng/ml AHT plus CM. 6 h before cell harvesting, either 40 µ M MG-132 or 5 µ M clastolactacystin -lactone (LC) were added. Cell extracts were analyzed by Western blotting.

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123 CYTOPATHICITY OF CPAF € Paschen et al. dying cells, identifying this cell death as nonapoptotic or necrotic. All the observed features recapitulated the changes seen in cells infected by C. trachomatis . Infections with Chlamydia commonly induce strong in ammatory responses. These reactions may thus be the result of the CPAF-mediated release of not only bacterial but also pro-in ammatory cellular molecules. We were prompted to test for the possibility of activation by induced proximity by similarities between the known characteristics of CPAF and the well-established process of activation Discussion This study shows that C. trachomatis CPAF can be activated by oligomerization to induce degradation of host cell proteins. On the one hand, CPAF caused the degradation of pro-apoptotic BH3-only proteins and is therefore a mediator of chlamydial anti-apoptotic activity. On the other hand, CPAF induced cell death in the absence of hallmarks of apoptosis. Activation of the apoptotic pathway was observed only in a small minority of Figure 5. CPAF expression leads to changes in cellular morphology. (A) Changes in cell morphology during CPAF expression. CPAF K6 cells were incubated either with 6 ng/ml AHT, CM, or both for 16 h. Cells were analyzed by light microscopy (left). Arrows indicate smaller ve sicular fragments. Black bar, 10 µ m. The right panel shows an enlarged section of either a control sample or a sample treated with AHT and CM. White bar, 3 µ m. (B) Inhibition of CPAF oligomerization by novobiocin. CPAF expression was induced in CPAF K6 cells by adding AHT, or cells were left untreated. 3 0 min before addition of CM, the cells were incubated with indicated amounts of NB. Cells were analyzed by light microscopy. Bar, 10 µ m.

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JCB € VOLUME 182 € NUMBER 1 € 2008 124or suspected (CPAF) to be autocatalysis, and both form, during physiological activation, complexes of the two subunits derived from intramolecular cleavage. Our analysis shows that they of initiator caspases, especially caspase-9. Both CPAF and caspase-9 are synthesized as zymogens that have low proteolytic activity. Both are activated by what has been shown (caspase-9) Figure 6. CPAF expression causes nonapoptotic cell death. (A) CPAF reduces cell viability. CPAF K6 or T-REx-293 cells were treated with the indicated combinations of TET, CM, and the caspase inhibitor zVAD-fmk (zVAD). As a positive control, cells were treated with TNF(TNF) and cycloheximide (CHX). After indicated time points, cell viabilities were measured by MTT assay. Relative cell viability was calculated (untreated cel ls were set to 100%). Data are normalized means/SEM of three independent experiments. (B) Analysis of nuclear morphology after CPAF expression by Hoechst stai ning. CPAF K6 cells were treated with 10 ng/ml AHT, CM, or zVAD-fmk as indicated. As a positive control of apoptosis, cells were treated with TNF/C HX (as described in A). After 16 h, cells were stained with the Hoechst 33342 dye and analyzed by ” uorescence microscopy. Bar, 15 µ m. (C) Caspase-3 activation during CPAFexpression. CPAF K6 cells were treated as described in B and analyzed by ” ow cytometry using an antibody speci“ c for active caspase-3. (D) Analysis of caspase-3 activation by Western blotting. CPAF K6 cells were treated as described in B, and cell extracts were analyzed by W estern blotting using an antibody speci“ c for active caspase-3. Arrowheads indicate speci“ c cleavage products of activated caspase-3. Detection of actin served as loading control. (E) Cleavage of PARP by infection with C. trachomatis or expression of active CPAF. (Left) CPAF K6 cells were infected with C. trachomatis for the indicated periods of time. (Right) CPAF K6 cells were treated as described in B. Cell extracts were analyzed by Western blottin g using a PARP antibody. The arrowheads indicate cleavage products resulting from chlamydial infection or CPAF expression. The asterisk indicates a spec i“ c PARP cleavage product due to caspase activation by TNF/CHX.

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125 CYTOPATHICITY OF CPAF € Paschen et al. mammals, and has been worked out in great detail ( Bao and Shi, 2007 ). Our results suggest that this principle of protease activation is even much older in evolutionary terms, as it is already found in bacteria. CPAF is ef ciently translocated from the bacteria-harboring vacuole into the cytosol, although the mechanism of this translocation is unclear. Our data indicate that oligomerization of CPAF is an essential step in its maturation. This is supported by the observation that CPAF secreted from the vacuole into the host cytosol during infection with C. trachomatis was also found in a complex of 200 kD, which probably corresponds to 3 … 4 subunits (unpublished data). When and how CPAF oligomerizes is unclear. One possibility is that a bacterial chaperone binds and translocates CPAF into the host cytosol, during which process the spatial requirements for CPAF activation might be met. It is also conceivable that CPAF may transiently associate with the inclusion membrane at a suf ciently high local concentration to cause its own activation. Initiator caspases are activated by clustering induced by speci c adaptors. In the best characterized example, caspase-9 is clustered after heptamerization of its adaptor, Apaf-1 ( Bao and Shi, 2007 ). The existence of a speci c bacterial adaptor for CPAF is therefore also possible. Limited homology between CPAF and Tsp has been observed. Our results show that the Tsp-like active site in C. trachomatis CPAF is required for its activity. Chlamydia has a protein, CT441 in C. trachomatis , that shows higher similarity to known bacterial Tsp and that has recently been characterized ( Lad et al., 2007 ). The role of Tsp in bacteria is not well understood, but these enzymes appear to be involved with proteolytic modi cations and degradation of various bacterial proteins ( Paetzel and Dalbey, 1997 ). The conservation of the active site in CPAF might be an indication of evolutionary origin, but the low overall homology suggests that it has assumed other functions. The translocation of CPAF into the host cytosol also indicates that its physiological targets may not be bacterial but host cell proteins. It is interesting to note that CPAF both has anti-apoptotic activity and induces nonapoptotic cell death, reproducing two salient features of chlamydial infection. Although the relevance of cell death modulation for chlamydial pathogenesis is not known, it is likely that apoptosis inhibition may contribute to the ability of chlamydiae to complete their developmental cycle and perhaps to maintain persistent human infections. During viral infection, the host cell defense often includes the induction of apoptosis through the mitochondrial, BH3-only protein controlled pathway ( Everett and McFadden, 2002 ). Although Chlamydia differs from viruses in important aspects, it shares their dependency on cellular integrity for its replication. The cell  s response to chlamydial infection may include the attempt to undergo apoptosis through activation of BH3-only proteins. These proteins are general sensors of cell stress and can be activated to induce apoptosis in many different situations ( Strasser, 2005 ). It is therefore even conceivable that they are activated in response to CPAF activity. CPAF might thus induce apoptosis but at the same time counter it by causing the degradation of BH3-only proteins. In this scenario, as BH3-only proteins are mostly degraded, the net result of the activity of CPAF would be the observed nonapoptotic cell death. CPAF-induced cell death could therefore be a masked form of apoptosis that serves as a cellular defense reaction. differ in that a cleavage-defective mutant of CPAF is inactive, whereas auto-cleavage defective caspase-9 still can cleave its substrates ( Stennicke et al., 1999 ). A mutant carrying a point mutation in the CPAF processing site was defective in both processing and activity. These results strongly suggest that CPAF is activated by an autocatalytic process. Although we cannot exclude the possibility that a cellular protease is involved in gyrBCPAF processing, this seems unlikely. The induced proximity model has been initially proposed to explain the activation of initiator caspases by adaptor-induced clustering during apoptosis. This mode of protease activation is conserved between the nematode Caenorhabditis elegans and Figure 7. CPAF expression leads to changes in cellular ultrastructure. CPAF K6 cells were treated with 5 ng/ml AHT and CM for 20 h (middle and bottom), or left untreated (control; top). Cells were harvested, pelleted, and analyzed by electron microscopy. The bottom panel shows a 10-fold magni“ cation. Scale bars are indicated. M, mitochondria.

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JCB € VOLUME 182 € NUMBER 1 € 2008 126cells were infected at a MOI = 3. After 2 h, 10% FCS were added. At indicated time points, cells were harvested and lysed by incubation with RIPA buffer (1% Triton X-100, 0.5% SDS, 0.5% deoxycholate,1 mM EDTA, 150 mM NaCl, and 50 mM Tris, pH 8.0), supplemented with a protease inhibitor cocktail (Roche). Transient transfection of T-REx-293 cells and induction of protein expression Transient transfections of T-REx-293 cells were performed using FuGene HD (Roche), following the manufacturer  s instructions. CPAF expression was induced by addition of 0.5 ng/ µ l anhydrotetracycline (AHT; IBA) for 13 … 14 h unless otherwise indicated. In some experiments 4 µ g/ml tetracycline (TET) was used. For oligomerization experiments 1 µ M coumermycin (CM; SigmaAldrich) was added 7 h before harvesting to the cells. When indicated, novobiocin (NB; Sigma-Aldrich) was added 30 min before CM addition. Immunoblotting Cells were harvested and lysed in Triton X-100 buffer (1% Triton X-100, 1 mM EDTA, 150 mM NaCl, 50 mM Tris, pH 8.0, and protease inhibitor cocktail). Cell extracts were separated using SDS-PAGE and proteins were transferred onto nitrocellulose membranes. Equivalent amounts of protein were loaded and equal loading was con“ rmed by detection of -actin or tubulin using speci“ c antibodies (Sigma-Aldrich). Membranes were probed with anti-Bim, anti-cyclin B1, anti-FLAG, anti-myc, anti-PARP, anti-Puma (all from Cell Signaling Technology), anti-Bak, anti-Bcl-2 (both from BD Biosciences), anti-RFX 5, anti-vimentin, anti-CK8 (all three from Acris), or anti-caspase 3 (Abcam) antibodies. Proteins were visualized using peroxidase-conjugated secondary antibodies and a chemoluminescence detection system (GE Healthcare). Size exclusion chromatography Cells were lysed in 1% Triton X-100 and 1 mM EDTA in PBS, and lysates were cleared by centrifugation. NB was used at a concentration of 20 µ M and was added 30 min before chromatography. All extracts were separated on a Superose 200 gel “ ltration column (GE Healthcare). Molecular sizes were calculated by plotting the log of the molecular weight of standard marker proteins (Sigma-Aldrich) against their elution volume. Cell-free cleavage assay CPAF K6 cells expressing oligomerized CPAF were lysed in NP-40 buffer (1% NP-40, 150 mM NaCl, 1 mM EDTA, and 20 mM MOPS, pH 7.4), and equivalent amounts of the extract were mixed either with PBS as a control or with NP-40 extracts of T-REx-293 cells, transiently transfected with the CK8myc construct. Extracts were incubated at 37 ° C for 1 h. CPAF K6 cell extracts were preincubated with the proteasome inhibitors clasto-lactacystin -lactone (LC; Sigma-Aldrich) or MG-132 (EMD) at 37 ° C for 20 min before substrate addition. MTT assay Cell viability was tested by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; Sigma-Aldrich) assay. MTT was added to cells at a concentration of 0.5 mg/ µ l and incubated at 37 ° C for 1 h. Generated formazane crystals were dissolved in DMSO, and the OD at a wavelength of 570 nm was measured. As a positive control, cells treated with TNF (R & D Systems)/cycloheximide (0.5 ng/ml each) for 16 h were used. The caspase inhibitor zVAD-fmk was used at a concentration of 25 µ M. Analysis of cell morphology and detection of apoptosis Cell morphology was analyzed in culture media by light microscopy at RT (CKX41 inverted microscope, 40x/0.55 lens, U-CMAD3 video adaptor, F-View II Camera, Cell-F Soft Imaging Solution; all from Olympus). For detection of apoptosis, cells were stained with 1 µ g/ µ l Hoechst 33342 dye (Roche) and incubated for 30 min at 37 ° C. Cells were harvested, washed with PBS, resuspended in PBS, and embedded in mounting ” uid (Labsystems Oy). Nuclei were examined using an Epi” uorescence microscope at RT (DMRBE microscope, 40x/0.70 lens, both from Leica; AxioCam MRc camera with AxioVision software; Carl Zeiss, Inc.). Adobe Photoshop and Microsoft PhotoEditor were used to adjust image size and resolution, and to enhance contrast of the whole image for better visibility in some pictures. Flow cytometry Caspase-3 activation was detected by ” ow cytometry analysis. CPAF K6 cells were harvested, “ xed in 2% neutral-buffered paraformaldehyde, and permeabilized with 0.5% saponin (Sigma-Aldrich). Active caspase-3 was detected with an anti-active caspase-3 antibody (Abcam) and FITC-conjugated The molecular mechanism for the cell death … inducing activity of CPAF is still unknown. The appearance of lamellar structures in the cell might suggest changes to organelles and intracellular membranes, such as lysosomes and the ER, and their membranes. The ultrastructural changes to these organelles that have been described during chlamydial infection ( Todd and Storz, 1975 ; Todd et al., 1976 ) are thus likely connected to CPAF activity, and it could be the release of, for instance, lysosomal peptidases, that causes the damage and eventual death of the cell. All sequenced chlamydial strains have a gene coding for CPAF. The genome of an endosymbiont of free-living amoeba, candidatus protochlamydia amoebophila (UWE25), has recently been sequenced, and even this distantly related bacterium carries a recognizable CPAF homologue ( Horn et al., 2004 ; Collingro et al., 2005 ). This suggests that CPAF serves a function that is similar for the different species and perhaps even the very different requirements of infection of human cells and amoeba. When rst discovered, CPAF  s activity to degrade transcription factors required for MHC-expression was noted. Although such a mechanism might contribute to immune evasion by Chlamydia , it appears more likely that this is not the evolutionarily selected function of CPAF, especially not in amoeba lacking MHC. Cytoskeletal structures like the intermediate lament components CK8 and vimentin are good candidates as essential targets of CPAF activity. The host cell cytosol has to accommodate the rapidly growing inclusion, and the disruption of intermediate laments might facilitate expansion of the inclusion and perhaps eventually the release of the inclusion by lysis or extrusion ( Hybiske and Stephens, 2007 ). Whether CPAF-induced cell death is bene cial for the host or for the bacteria is therefore uncertain at this stage. Either way, our results strongly suggest that CPAF is an important factor in chlamydial pathogenicity, and cellular alterations and responses induced by CPAF might be involved in causing protracted infections. Materials and methods Cloning of expression vectors A fragment encoding for amino acid residues 2 … 221 of gyrB of E. coli was ampli“ ed by PCR and cloned into a pcDNA4/TO/ myc -His vector (Invitrogen). Consecutively, two additional gyrB fragments, separated by linker sequences of 16 amino acid residues, were inserted 5 of the “ rst copy. A sequence coding for a FLAG tag was added 3 of the gyrB fragments (gyrB construct). The coding sequence of CPAF of C. trachomatis (amino acid residues 18 … 601) was inserted behind the FLAG-gyrB construct (gyrB-CPAF). CPAF mutants were generated by point mutations using a Stratagene XL Mutagenesis kit. The open reading frame of human cytokeratin 8 (CK8; ATCC #61515) was ampli“ ed by PCR, thereby adding a myc-tag coding sequence to the 3 end and cloned into a pENTR/SD/D-TOPO vector (Invitrogen). Subsequently, the CK8-myc fragment was shuf” ed into a pcDNA6.2/ V5-DEST (CK8-myc construct) by Gateway LR reaction, according to the manufacturer  s instructions (Invitrogen). Cell lines and cell culture The human embryonic kidney cell line, T-REx-293, and T-REx-HeLa cells, which stably express the tetracycline repressor (Invitrogen), were grown and maintained in humidi“ ed air containing 5% CO 2 at 37 ° C in DMEM supplemented with 10% fetal calf serum (tetracycline negative; PAA Laboratories), 50 µ g/ µ l penicillin/streptomycin, and 5 µ g/ µ l blasticidin. T-REx-293 clones stably expressing gyrB-CPAF were generated by electroporation with the construct and antibiotic selection. Chlamydial infections of T-REx-293 cells The C. trachomatis strain L2 was obtained from ATCC. Before infection, the culture medium was replaced with DMEM without FCS and antibiotics, and

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127 CYTOPATHICITY OF CPAF € Paschen et al. Horn , M. , A. Collingro , S. Schmitz-Esser , C.L. Beier , U. Purkhold , B. Fartmann , P. Brandt , G.J. Nyakatura , M. Droege , D. Frishman , et al . 2004 . Illuminating the evolutionary history of chlamydiae. Science . 304 : 728 … 730 . Hybiske , K. , and R.S. Stephens . 2007 . Mechanisms of host cell exit by the intracellular bacterium Chlamydia . Proc. Natl. Acad. Sci. USA . 104 : 11430 … 11435 . Kawana , K. , A.J. Quayle , M. Ficarra , J.A. Ibana , L. Shen , Y. Kawana , H. Yang , L. Marrero , S. Yavagal , S.J. Greene , et al . 2007 . CD1d degradation in Chlamydia trachomatis -infected epithelial cells is the result of both cellular and chlamydial proteasomal activity. J. Biol. Chem. 282 : 7368 … 7375 . Lad , S.P. , G. Yang , D.A. Scott , G. Wang , P. Nair , J. Mathison , V.S. Reddy , and E. Li . 2007 . Chlamydial CT441 is a PDZ domain-containing tailspeci c protease that interferes with the NF-kappaB pathway of immune response. J. 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Role of Bcl-2 family members in caspase-independent apoptosis during Chlamydia infection. Infect. Immun. 70 : 55 … 61 . Peters , J. , D.P. Wilson , G. Myers , P. Timms , and P.M. Bavoil . 2007 . Type III secretion a la Chlamydia . Trends Microbiol. 15 : 241 … 251 . Pirbhai , M. , F. Dong , Y. Zhong , K.Z. Pan , and G. Zhong . 2006 . The secreted protease factor CPAF is responsible for degrading pro-apoptotic BH3only proteins in Chlamydia trachomatis-infected cells. J. Biol. Chem. 281 : 31495 … 31501 . Pop , C. , J. Timmer , S. Sperandio , and G.S. Salvesen . 2006 . The apoptosome activates caspase-9 by dimerization. Mol. Cell . 22 : 269 … 275 . Salvesen , G.S. , and V.M. Dixit . 1999 . Caspase activation: the induced-proximity model. Proc. Natl. Acad. Sci. USA . 96 : 10964 … 10967 . Shaw , A.C. , B.B. Vandahl , M.R. Larsen , P. Roepstorff , K. Gevaert , J. Vandekerckhove , G. Christiansen , and S. Birkelund . 2002 . Characterization of a secreted Chlamydia protease. Cell. 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Identi cation of a chlamydial protease-like activity factor responsible for the degradation of host transcription factors. J. Exp. Med. 193 : 935 … 942 . goat anti … rabbit secondary antibody (Dianova). Flow cytometric analysis was performed with a FACSCalibur (Becton Dickinson). Electron microscopy Cells were harvested and “ xed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4; Electron Microscopy Sciences) and embedded in epoxy resin (epon 812; Electron Microscopy Sciences). Ultrathin sections were examined with an EM 10 CR transmission electron microscope (Carl Zeiss, Inc.). For image acquisition, a MegaView III camera system (Olympus) was used. Online supplemental material Fig. S1 shows the morphological changes in T-REx-293 and T-REx-HeLa cells, respectively, due to transient transfection of CPAF. Fig. S2 compares the changes in cellular morphology between infection with C. trachomatis and the expression of CPAF in CPAF K6 cells. 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