The use of molecular genetic tools to generate and complement mutations in pathogenic bacteria is central to our understanding of how microorganisms cause disease. Unfortunately, many clinically important pathogens, such as Chlamydia trachomatis -the causative agent of blinding trachoma and many sexually transmitted diseases- are not amenable to such analysis. Here we propose to establish a comprehensive system for forward and reverse genetic analysis in Chlamydia based on the use of chemical mutagens, genetic recombination and "deep" DNA sequencing technologies. Our specific goals are to 1) identify factors important in C. trachomatis pathogenesis by implementing forward genetic screens, including a genetic analysis of Type II secretion and 2) develop a method for systematic reverse genetic analysis in C. trachomatis. By the completion of these experiments we will have established methods to perform routine forward and genetic screens - the lynchpin of microbial pathogenesis research. The tools and reagents generated through the proposed work will constitute an important resource for the Chlamydia research community.
Chlamydia trachomatis is the leading cause of infectious blindness (trachoma) and many sexually transmitted infections. Despite their clinical and public health importance, these obligate intracellular bacterial pathogens remain understudied because of their intractability to molecular genetic manipulation. Here we describe methods to perform genetic analysis in these pathogens, which will significantly accelerate our understanding of virulence gene function and the potential development of attenuated strains for vaccine applications.
|Young, Hayley E; Zhao, Jinshi; Barker, Jeffrey R et al. (2016) Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis. MBio 7:e00090|
|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|
|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|
|EngstrÃ¶m, Patrik; Krishnan, K Syam; Ngyuen, Bidong D et al. (2014) A 2-pyridone-amide inhibitor targets the glucose metabolism pathway of Chlamydia trachomatis. MBio 6:e02304-14|
|Snavely, Emily A; Kokes, Marcela; Dunn, Joe Dan et al. (2014) Reassessing the role of the secreted protease CPAF in Chlamydia trachomatis infection through genetic approaches. Pathog Dis 71:336-51|
|Furuse, Yuki; Finethy, Ryan; Saka, Hector A et al. (2014) Search for microRNAs expressed by intracellular bacterial pathogens in infected mammalian cells. PLoS One 9:e106434|
|EngstrÃ¶m, 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|
|Nguyen, Bidong D; Valdivia, Raphael H (2012) Virulence determinants in the obligate intracellular pathogen Chlamydia trachomatis revealed by forward genetic approaches. Proc Natl Acad Sci U S A 109:1263-8|