Developing and implementing genetic tools designed to understand host takeover by Chlamydia trachomatis.

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Montana State University - Bozeman, College of Letters & Science


Chlamydia are gram negative obligate intracellular parasites that are responsible for millions of new infections in humans and animals every year. C. trachomatis is the number one cause of bacterial sexually transmitted infections in the United States, the number one cause of infectious blindness worldwide. Since 2001, there has been a steady increase in the number of new cases of C. trachomatis infections each year. Despite the prevalence and medical importance of C. trachomatis, we still know relatively little about the lifecycle of this parasite and the host factors that are essential for the lifecycle of C. trachomatis. To address this critical gap in our knowledge, my thesis work aimed to develop and implement genetic tools to understand host takeover by C. trachomatis. In this thesis I present results suggesting that I have transformed C. trachomatis with a plasmid carrying the Cas9 gene from Campylobacter jejuni. Additional experiments are necessary to determine if the CjCas9 is expressed, nuclease active, and functional for programable editing in C. trachomatis. In addition to my work aimed at developing a CRISPR-Cas9-based genetic engineering system in C. trachomatis, I also participated in a genome wide knockout screen aimed at identifying human genes necessary for completion of the C. trachomatis lifecycle. The CRISPR-Cas9 genome wide knockout screen identified 103 genes as critical factors for C. trachomatis. To validate results for the screen I have been involved in creating clonal cell lines with deletions in three of the genes that form the Adaptor Protein (AP) Complex (i.e., AP3S2, AP1B2 and AP1G2). The genes have been deleted and future experiments are aimed at measuring the impact of these genes on the C. trachomatis lifecycle.




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