Date of Award
Master of Science
Molecular Biology Microbiology and Biochemistry
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects both humans and domestically important animals. Research in the field has until more recently been hindered by a lack of genetic tools. We have modified Sigma's TargeTronTM Gene Knockout System, which utilizes a mobile group II intron for site-specific insertion and gene inactivation, for use in C. trachomatis. As proof of principle, we used the system to inactivate incA, creating mutant strains DFCT3 and DFCT4 (independent clones both carrying incA::GII[bla]). IncA is a chlamydial inclusion membrane protein involved in homotypic fusion of inclusions when cells are infected with more than one bacterium. Genotypic and phenotypic analysis was performed to ensure successful intron insertion into incA and loss of IncA function. Further characterization of the incA::GII(bla) mutant examined its pathogenicity relative to the wild type strain and indicated that the mutant was attenuated for growth in a mouse infection model, but not in a cell culture infection model. Complementation of the incA mutant confirmed that the phenotype differences between the wild type strain and the mutant were due to inactivation of incA. As incA mutants arise spontaneously during human infections, future work will focus on the role of IncA in pathogenesis using the mutant strains derived from this study.
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