Mechanisms of Chlamydia Manipulation of Host Cell Biology Revealed Through Genetic Approaches by Marcela Kokes Department of Molecular Genetics and Microbiology Program in Cell and Molecular Biology Duke University Date:_______________________ Approved: ___________________________ Raphael H. Valdivia, Supervisor ___________________________ Daniel J. Lew ___________________________ Jörn Coers ___________________________ Patrick C. Seed Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University 2015 i v ABSTRACT Mechanisms of Chlamydia Manipulation of Host Cell Biology Revealed Through Genetic Approaches by Marcela Kokes Department of Molecular Genetics and Microbiology Program in Cell and Molecular Biology Duke University Date:_______________________ Approved: ___________________________ Raphael H. Valdivia, Supervisor ___________________________ Daniel J. Lew ___________________________ Jörn Coers ___________________________ Patrick C. Seed An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Molecular Genetics and Microbiology in the Graduate School of Duke University 2015 Copyright by Marcela Kokes 2015 i v Abstract Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen and is the leading cause of preventable blindness worldwide. Chlamydia is particularly intriguing from the perspective of cell biology because it is an obligate intracellular pathogen that manipulates host cellular pathways to ensure its proliferation and survival. This is achieved through a significant remodeling of the host cell’s internal architecture from within a membrane-bound vacuole, termed the inclusion. However, given a previous lack of tools to perform genetic analysis, the mechanisms by which Chlamydia induces host cellular changes remained unclear. Here I present genetic and molecular mechanisms of chlamydial manipulation of the host cytoskeleton and organelles. Using a forward genetics screen, InaC was identified as a necessary factor for the assembly of an F-actin structure surrounding the inclusion. InaC associated with the vacuolar membrane where it recruited Golgi-specific ARF-family GTPases. Actin dynamics and ARF GTPases regulate Golgi morphology and positioning within cells, and InaC acted to redistribute the Golgi to surround the Chlamydia inclusion. These findings suggest that Chlamydia places InaC at the inclusion-cytosolic interface to recruit host ARF GTPases and F-actin to form a platform for rearranging intracellular organelles around the inclusion. The inclusion is also surrounded by the intermediate filament vimentin and the chlamydial protease CPAF cleaves vimentin in vitro. CPAF-dependent remodeling of vimentin occurred selectively in late stages of the infection. In living cells, this cleavage occurred only after a loss of inclusion membrane integrity, suggesting that CPAF cleaves intermediate filaments specifically during chlamydial exit of host cells. In summary, I have implemented recent forward and reverse genetic approaches in Chlamydia to iv reveal how it employs effector proteins to manipulate the internal organization of cells in novel ways. v Contents Abstract .......................................................................................................................... iv List of Tables .................................................................................................................. xi List of Figures ................................................................................................................ xii Acknowledgements ...................................................................................................... xiv 1. Cell Biology of the Chlamydia Inclusion ...................................................................... 1 1.1 Introduction ......................................................................................................... 1 1.2 Chlamydia heavily interacts with host trafficking machinery ................................ 4 1.2.1 The nascent inclusion is rapidly segregated from classical early endocytic pathways ................................................................................................................ 4 1.2.2 Inclusion membrane proteins mediate inclusion interactions .......................... 5 1.2.3 The nascent inclusion migrates to the host centrosome ................................. 9 1.2.4 The inclusion membrane recruits critical mediators of membrane traffic .......10 1.2.5 Chlamydia acquires host lipids ......................................................................13 1.2.6 Chlamydia acquires host lipids partly via Golgi-dependent vesicular trafficking mechanisms ..........................................................................................................14 1.2.7 Chlamydia acquires host lipids via non-vesicular pathways...........................16 1.3 The inclusion interacts with and redistributes host organelles ............................19 1.3.1 Chlamydia induces Golgi fragmentation ........................................................19 1.3.2 Chlamydia interactions with multivesicular bodies .........................................21 1.3.3 Chlamydia targets lipid droplets and peroxisomes ........................................22 1.3.4 Recycling endosomes and lysosomes closely appose the inclusion ..............24 1.3.5 Chlamydia species interact with mitochondria ...............................................25 1.4 Manipulation of the host cytoskeleton during mid to late stages of infection .......26 vi 1.4.1 F-actin and intermediate filaments surround the inclusion .............................26 1.4.2 Septins are enriched in proximity to the inclusion ..........................................28 1.4.3 Microtubules near the inclusion are post-translational modified .....................29 1.4.4 Chlamydia can exit host cells by actin-dependent extrusion ..........................30 1.5 Conclusions and future perspectives .................................................................32 2. F-actin assembly at the C. trachomatis inclusion .......................................................34 2.1 Introduction ........................................................................................................34 2.2 Genetic approach to investigate F-actin assembly .............................................35 2.2.1 Forward genetic screen for bacterial effectors involved in F-actin rearrangements at the C. trachomatis inclusion .....................................................35 2.2.2 F-actin assembly around the inclusion is enhanced by a S314F mutation in the uncharacterized Chlamydia predicted ORF CTL0496 ......................................37 2.2.2.1 Primary sequence analysis of CTL0496 .................................................38 2.2.3 The inclusion membrane protein InaC is necessary for F-actin assembly at the inclusion periphery .................................................................................................38 2.2.3.1 Expression of full-length InaC in M407 rescues F-actin assembly ..........40 2.2.3.2 Primary sequence analysis of InaC ........................................................42 2.3 Mechanisms of F-actin assembly at the inclusion ..............................................43 2.3.1 Filamins and α-actinin co-assemble with F-actin at inclusions .......................43 2.3.2 InaC recruits ARF and 14-3-3 proteins to the inclusion .................................47 2.3.2.1 InaC selectively recruits a GTP-locked ARF1 variant to the inclusion .....51 2.3.2.2 Different aspects of InaC mediate interactions with ARF and 14-3-3 proteins ..............................................................................................................53 2.4 Functions of F-actin assembly at the inclusion ...................................................56 2.4.1 Golgi redistribution around the inclusion requires InaC and F-actin ...............56 vii 2.4.2 InaC-mediated Golgi redistribution is dispensable for Chlamydia sphingolipid acquisition and growth ...........................................................................................58 2.4.2.1 Mutant 338 is defective in the generation of infectious progeny but forms wild-type-sized inclusions ...................................................................................61 2.4.3 InaC may affect the distribution of various organelles ...................................63 2.4.3.1 InaC affects recycling endosome accumulation at the inclusion .............66 2.4.3.2 InaC alters STING translocation kinetics ................................................66 2.4.4 InaC is dispensable for maintenance of gross inclusion integrity and morphology ............................................................................................................69 2.4.5 InaC does not globally affect IFN transcriptional responses during C. trachomatis infection ..............................................................................................70 2.5 Discussion .........................................................................................................75 2.5.1 Insights into mechanisms of F-actin assembly at the inclusion ......................75 2.5.2 Impact of F-actin assembly at the C. trachomatis inclusion