The obligate intracellular bacterial pathogen Chlamydia trachomatis infects the ocular and genital epithelium to cause diseases of significant clinical importance. C. trachomatis modulates host cellular functions by translocating "effector" proteins across host and inclusion membranes. The lack of tools to perform classical molecular genetic analysis in Chlamydia, has hampered progress in identifying and characterizing effector proteins. However, given the small size of the its genome, expression-based functional approaches can be brought to genomic scale and significantly accelerate research in Chlamydia pathogenesis. Here, we propose to develop functional genomic tools to identify and characterize effector proteins and the dynamics of effector protein export during chlamydial infection. In our first aim, we propose to generate comprehensive yeast and mammalian expression libraries to systematically survey chlamydial proteins for functions related to their pathogenicity and apply immunological-based screens to identify novel secreted chlamydial proteins. These findings will be merged with proteomic data of Chlamydia elementary bodies to identify and characterize bacterial factors mediating entry and biogenesis of the nascent pathogenic vacuole. In our second aim, we extend the application of functional tools to characterize the chlamydial Type III secretion (TTS) system, the main portal for the delivery of effector proteins. Through two-hybrid technologies and biochemical approaches we propose to construct a protein-protein interaction map of this ancestral TTS apparatus to identify non-conserved components of the core translocon, needle complex and putative regulatory subunits. In particular, we will focus our studies on Chlamydia-specific proteins that interact with the basal structure of the TSS system, including a novel TTS chaperone we have identified and its cognate cargo.

Public Health Relevance

The extent to which the widely disseminated pathogen bacteria Chlamydia trachomatis subverts immune responses is determined by virulence factors they inject into host cells ("effector" proteins). The proposed study will develop genomic tools to identify and characterize these effector proteins. In this manner we will significantly further our understanding of chlamydial pathogenesis and generate new targets for vaccine design and therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI081694-02
Application #
7990426
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Hiltke, Thomas J
Project Start
2009-12-01
Project End
2014-11-30
Budget Start
2010-12-01
Budget End
2011-11-30
Support Year
2
Fiscal Year
2011
Total Cost
$433,703
Indirect Cost
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Bastidas, Robert J; Valdivia, Raphael H (2016) Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen. Microbiol Mol Biol Rev 80:411-27
Kokes, Marcela; Valdivia, Raphael H (2015) Differential Translocation of Host Cellular Materials into the Chlamydia trachomatis Inclusion Lumen during Chemical Fixation. PLoS One 10:e0139153
Mirrashidi, Kathleen M; Elwell, Cherilyn A; Verschueren, Erik et al. (2015) Global Mapping of the Inc-Human Interactome Reveals that Retromer Restricts Chlamydia Infection. Cell Host Microbe 18:109-21
Kokes, Marcela; Dunn, Joe Dan; Granek, Joshua A et al. (2015) Integrating chemical mutagenesis and whole-genome sequencing as a platform for forward and reverse genetic analysis of Chlamydia. Cell Host Microbe 17:716-25
Chen, Yi-Shan; Bastidas, Robert J; Saka, Hector A et al. (2014) The Chlamydia trachomatis type III secretion chaperone Slc1 engages multiple early effectors, including TepP, a tyrosine-phosphorylated protein required for the recruitment of CrkI-II to nascent inclusions and innate immune signaling. PLoS Pathog 10:e1003954
Haldar, Arun K; Saka, Hector A; Piro, Anthony S et al. (2013) IRG and GBP host resistance factors target aberrant, "non-self" vacuoles characterized by the missing of "self" IRGM proteins. PLoS Pathog 9:e1003414
Barker, Jeffrey R; Koestler, Benjamin J; Carpenter, Victoria K et al. (2013) STING-dependent recognition of cyclic di-AMP mediates type I interferon responses during Chlamydia trachomatis infection. MBio 4:e00018-13
Bastidas, Robert J; Elwell, Cherilyn A; Engel, Joanne N et al. (2013) Chlamydial intracellular survival strategies. Cold Spring Harb Perspect Med 3:a010256
Engström, Patrik; Nguyen, Bidong D; Normark, Johan et al. (2013) Mutations in hemG mediate resistance to salicylidene acylhydrazides, demonstrating a novel link between protoporphyrinogen oxidase (HemG) and Chlamydia trachomatis infectivity. J Bacteriol 195:4221-30
Valdivia, Raphael H (2012) Thinking outside the box: new strategies for antichlamydial control. Future Microbiol 7:427-9

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