Date of Award

12-1-2015

Degree Name

Master of Science

Department

Molecular Biology Microbiology and Biochemistry

First Advisor

Fisher, Derek

Abstract

TITLE: DEVELOPING GENETIC TOOLS TO EXPLORE PATHOGENESIS OF CHLAMYDIA TRACHOMATIS MAJOR PROFESSOR: Dr. Derek Fisher Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for sexually transmitted infections (STIs) and the ocular disease, trachoma, in humans. While antibiotic treatment is currently effective, antibiotic resistance has been seen in C. suis and the C. suis tet(C) resistance marker can be transferred to C. trachomatis. In addition, approximately two-thirds of C. trachomatis STIs are asymptomatic in women leading to untreated infections that can result in pelvic inflammatory disease, infertility, and ectopic pregnancy. The identification of new drug targets and vaccine development for Chlamydia spp., in general, has largely been hindered until more recently by a lack of genetic tools. While breakthroughs in DNA transformation, shuttle-vector design, and targeted gene insertion approaches opened the door for chlamydial genetics, the field remained limited by the number of drug resistance markers available for mutant selection. Furthermore, the most widely used marker, bla, may not be used with C. trachomatis genital serovars D-K. Because of these limitations, we sought to develop alternative resistance markers. Using a mobile group II intron (TargeTronTM), we tested whether the aadA, cat, and arr-2 markers could be used for mutant selection in C. trachomatis L2 using the incA and rsbV1 genes as intron targets. The aadA and cat markers, but not arr-2, proved effective for mutant selection. Using a combination of markers, we demonstrated that multiple chromosomal mutations within the same strain could be constructed using TargeTron, that intron insertion is stable in an animal infection model, and that an intron marked-strain could be complemented using an Escherichia coli-C. trachomatis shuttle vector. Recombinant strains were verified through PCR, DNA sequencing, and western blot analysis. Collectively, our results increase the chlamydial molecular “tool kit” and will allow researchers to address fundamental questions regarding genotypic contributions to disease using approaches that fulfill Molecular Koch’s postulates.

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