Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in theChlamydia trachomatisdevelopmental cycle

Abstract

AbstractChlamydia trachomatisis an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infections. Changes in DNA topology in this pathogen have been linked to its pathogenicity-associated developmental cycle. Here, evidence is provided that the balanced activity of DNA topoisomerases (Topos) contributes toChlamydiadevelopmental processes. Utilizing catalytically inactivated Cas12 (dCas12) based-clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomaltopAtranscription inC. trachomatiswithout detected toxicity of dCas12. Repression oftopAimpaired the growth ofC. trachomatismostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes ofC. trachomatiswas downregulated while early genes maintained their expression. Importantly, the growth defect associated withtopAknockdown was rescued by overexpressingtopAat an appropriate degree and time, directly linking the growth patterns to the levels oftopAexpression. Interestingly,topAknockdown had pleiotropic effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency.C. trachomatiswithtopAknocked down displayed hypersensitivity to moxifloxacin that targets DNA gyrase in comparison with the wild type. These data underscore the requirement of integrated topoisomerase actions to support the essential development and transcriptional processes ofC. trachomatis.ImportanceWe used genetic and chemical tools to demonstrate the relationship of topoisomerase activities and their obligatory role for the chlamydial developmental cycle. Successfully targeting the essential genetopAwith a CRISPRi approach, using dCas12, inC. trachomatisindicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanism(s) by which well-balanced topoisomerase activities enableC. trachomatisto adapt to unfavorable growth conditions imposed by antibiotics.