Generation of a toxin/antitoxin-based counterselection marker for Chlamydia trachomatis

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infection in the United States. The high rate of asymptomatic cases and absence of a vaccine often leave infections untreated, increasing the risk of serious complications in women, like pelvic inflammatory disease, ectopic pregnancy, and infertility. The generation of C. trachomatis mutants is crucial for studying C. trachomatis gene function, identifying potential vaccine candidates, and understanding host-pathogen interactions. However, the obligate intracellular nature of the bacteria hinders the development of genetic tools for mutagenesis. Counterselectable markers are effective systems for selecting bacterial mutants; however, such systems have yet to be optimized for use in C. trachomatis. In this study, we created a toxin-antitoxin (TA) system-based counterselection marker. Two TA systems were tested, toxin CcdB and its antitoxin CcdA (CcdAB), and toxin MvpT and its antitoxin MvpA (MvpAT). For each system, the antitoxin was expressed from a constitutive promoter, while the toxin was controlled by an inducible promoter. We first showed that, in Escherichia coli, toxin induction in both TA systems overcame the protective effect of the antitoxin, resulting in growth inhibition. The two systems were subsequently tested in C. trachomatis. While the CcdAB system did not significantly inhibit the growth of C. trachomatis, the MvpAT system did. Altogether, we have developed an MvpAT-based counterselection system for use in C. trachomatis. Implementation of this system will enable more efficient genetic manipulation, facilitating the identification of bacterial virulence factors and advancing translational research toward improved treatment and prevention.