Friday, May 9, 2025
5/9/2025
Saturating transposon mutagenesis for Chlamydia - ABSTRACT Chlamydia species are important pathogens that represent a paradigm for understanding successful obligate intracellular parasitism. C. trachomatis is a prevalent human pathogen exerting a tremendous negative impact on reproductive fitness, particularly in females. A complete understanding is lacking regarding how these bacteria create and maintain an intracellular niche, avoid/subvert host defense mechanisms, and cause disease. This information is particularly important in the absence of an efficacious, preventative vaccine. The ability to genetically manipulate an organism is essential for the definitive investigation of relevant biology. Forward genetic approaches provide one of the most powerful strategies available to accomplish untargeted elucidation of genes contributing to a particular phenotype. For obligate intracellular bacteria, the full power of forward genetics has been generally confounded by factors such as low transformation efficiency, complex developmental cycles, and a requirement for cultivation under selective conditions. The utility of transposon-mediated random mutagenesis in supporting forward genetics is well established in a multitude of genetically tractable systems. Although initial progress has been made in Chlamydia, significant barriers remain that preclude leveraging the full benefit of transposon mutagenesis. We have engineered a platform that promises to overcome these barriers. We will build on initial progress to i) create a robust, reproducible, and user-friendly transposon system, and ii) provide genome- scale evidence for which genes are essential for chlamydial survival in HeLa cells. Both goals will result in reagents and techniques capable of supporting down-stream studies interrogating chlamydial biology.
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