Targeted repression of topA by CRISPRi reveals a critical function for balanced DNA topoisomerase I activity in the Chlamydia trachomatis developmental cycle

Abstract

ABSTRACT Chlamydia trachomatis is an obligate intracellular bacterium that is responsible for the most prevalent bacterial sexually transmitted infection. 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 contributes to controlling Chlamydia developmental processes. Utilizing catalytically inactivated Cas12 (dCas12)-based clustered regularly interspaced short palindromic repeats interference (CRISPRi) technology, we demonstrate targeted knockdown of chromosomal topA transcription in C. trachomatis without detected toxicity of dCas12. Repression of topA impaired the developmental cycle of C. trachomatis mostly through disruption of its differentiation from a replicative form to an infectious form. Consistent with this, expression of late developmental genes of C. trachomatis was downregulated, while early genes maintained their expression. Importantly, the developmental defect associated with topA knockdown was rescued by overexpressing topA at an appropriate degree and time, directly linking the growth patterns to the levels of topA expression. Interestingly, topA knockdown had effects on DNA gyrase expression, indicating a potential compensatory mechanism for survival to offset TopA deficiency. C. trachomatis with topA knocked 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 developmental and transcriptional processes of C. 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 gene topA with a CRISPRi approach, using dCas12, in C. trachomatis indicates that this method will facilitate the characterization of the essential genome. These findings have an important impact on our understanding of the mechanisms by which well-balanced topoisomerase functions in adaptation of C. trachomatis to unfavorable growth conditions imposed by antibiotics.

Keywords

• Chlamydia trachomatis
• DNA topoisomerase
• topA
• CRISPRi
• dCas12
• bacterial developmental cycle