Grant title: Type III exported effectors of Chlamydia trachomatis Abstract: The human pathogen Chlamydia trachomatis is a significant concern in the United States due to its prevalence and the combined health and socioeconomic impact of acute and chronic disease. Chlamydiae are obligate intracellular pathogens that possess the ability to modulate host-cell functions while sequestered within a membrane-bound parasitophorous vacuole. Although it is established that a type III secretion system (T3SS) represents one mechanism employed to modulate critical host cell pathways, the overall understanding of this process in Chlamydia remains limited. During the past funding cycle, we made significant progress in understanding the role of T3S effectors designated CT694 and CT695. Importantly, we also leveraged novel genetic manipulation techniques to investigate the impact of T3S in chlamydial pathogenesis. Perhaps our most important contribution was the development of a fluorescence-reported allelic exchange mutagenesis (FRAEM) approach to allow, for the first time, deletion of targeted chlamydial genes. We have used this technique to create null mutants for ct694 and ct695, and these strains are attenuated in both tissue-culture and animal-infection models. We propose to elucidate molecular details by which mutant phenotypes are manifested. A balanced combination of genetic, biochemical, cell-biology, and animal-based studies are proposed that will define developmental defects, identify host targets important for effector function(s), and gauge the overall role of effectors and target proteins in promoting Chlamydia pathogenesis. Completion of this work will extend the efficacy of genetically manipulating a challenging pathogen and lead to an enhanced understanding of Chlamydia-mediated disease.

Public Health Relevance

Chlamydia trachomatis, an agent of sexually transmitted disease, relies on a specialized secretion mechanism to deploy proteins exerting anti-host activities essential to pathogenesis. This proposal contains work designed to use mutagenesis of specific genes to determine their specific contributions to chlamydial disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI065530-13
Application #
9382846
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Hiltke, Thomas J
Project Start
2005-06-01
Project End
2021-10-31
Budget Start
2017-11-01
Budget End
2018-10-31
Support Year
13
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Kentucky
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40526
Mueller, Konrad E; Wolf, Katerina; Fields, Kenneth A (2017) Chlamydia trachomatis Transformation and Allelic Exchange Mutagenesis. Curr Protoc Microbiol 45:11A.3.1-11A.3.15
Ferrell, Joshua C; Fields, Kenneth A (2016) A working model for the type III secretion mechanism in Chlamydia. Microbes Infect 18:84-92
Mueller, Konrad E; Wolf, Katerina; Fields, Kenneth A (2016) Gene Deletion by Fluorescence-Reported Allelic Exchange Mutagenesis in Chlamydia trachomatis. MBio 7:e01817-15
Mueller, Konrad E; Fields, Kenneth A (2015) Application of ?-lactamase reporter fusions as an indicator of effector protein secretion during infections with the obligate intracellular pathogen Chlamydia trachomatis. PLoS One 10:e0135295
Mueller, K E; Plano, G V; Fields, K A (2014) New frontiers in type III secretion biology: the Chlamydia perspective. Infect Immun 82:2-9
McKuen, Mary J; Dahl, Gerhard; Fields, Kenneth A (2013) Assessing a potential role of host Pannexin 1 during Chlamydia trachomatis infection. PLoS One 8:e63732
Fields, K A; McCormack, R; de Armas, L R et al. (2013) Perforin-2 restricts growth of Chlamydia trachomatis in macrophages. Infect Immun 81:3045-54
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
Bullock, Holly D; Hower, Suzanne; Fields, Kenneth A (2012) Domain analyses reveal that Chlamydia trachomatis CT694 protein belongs to the membrane-localized family of type III effector proteins. J Biol Chem 287:28078-86
Chellas-Géry, B; Wolf, K; Tisoncik, J et al. (2011) Biochemical and localization analyses of putative type III secretion translocator proteins CopB and CopB2 of Chlamydia trachomatis reveal significant distinctions. Infect Immun 79:3036-45

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