The Periplasmic Tail-Specific Protease, Tsp, Is Essential for Secondary Differentiation in Chlamydia trachomatis.

The obligate intracellular human pathogen Chlamydia trachomatis (Ctr) undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms: the elementary body (EB) and the reticulate body (RB). The EB is the smaller, infectious, nondividing form which initiates infection of a susceptible host cell, whereas the RB is the larger, non-infectious form which replicates within a membrane-bound vesicle called an inclusion. The mechanism(s) which drives differentiation between these developmental forms is poorly understood. Bulk protein turnover is likely required for chlamydial differentiation given the significant differences in the protein repertoires and functions of the EB and RB. We hypothesize that periplasmic protein turnover is also critical for the reorganization of an RB into an EB, referred to as secondary differentiation. Ct441 is a periplasmic protease ortholog of tail-specific proteases (i.e., Tsp, Prc) and is expressed in Ctr during secondary differentiation. We investigated the effect of altering Tsp expression on developmental cycle progression. Through assessment of bacterial morphology and infectious progeny production, we found that both overexpression and CRISPR interference/dCas9 (CRISPRi)-mediated knockdown of Tsp negatively impacted chlamydial development through different mechanisms. We also confirmed that catalytic activity is required for the negative effect of overexpression and confirmed the effect of the mutation in in vitro assays. Electron microscopic assessments during knockdown experiments revealed a defect in EB morphology, directly linking Tsp function to secondary differentiation. These data implicate Ct441/Tsp as a critical factor in secondary differentiation. IMPORTANCE The human pathogen Chlamydia trachomatis is the leading cause of preventable infectious blindness and bacterial sexually transmitted infections worldwide. This pathogen has a unique developmental cycle that alternates between distinct forms. However, the key processes of chlamydial development remain obscure. Uncovering the mechanisms of differentiation between its metabolically and functionally distinct developmental forms may foster the discovery of novel Chlamydia-specific therapeutics and limit development of resistant bacterial populations derived from the clinical use of broad-spectrum antibiotics. In this study, we investigate chlamydial tail-specific protease (Tsp) and its function in chlamydial growth and development. Our work implicates Tsp as essential to chlamydial developmental cycle progression and indicates that Tsp is a potential drug target for Chlamydia infections.

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

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.

Targeted repression of DNA topoisomerase I by CRISPRi reveals a critical function for it in the Chlamydia trachomatis developmental cycle.

Chlamydia trachomatis is 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 to 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 growth 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 growth 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 pleiotropic 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 development and transcriptional processes of C. trachomatis.

Tag-Dependent Substrate Selection of ClpX Underlies Secondary Differentiation of Chlamydia trachomatis.

Despite having a highly reduced genome, Chlamydia trachomatis undergoes a complex developmental cycle in which the bacteria differentiate between the following two functionally and morphologically distinct forms: the infectious, nonreplicative elementary body (EB) and the noninfectious, replicative reticulate body (RB). The transitions between EBs and RBs are not mediated by division events that redistribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation likely require bulk protein turnover. One system for targeted protein degradation is the trans-translation system for ribosomal rescue, where polypeptides stalled during translation are marked with an SsrA tag encoded by a hybrid tRNA-mRNA, tmRNA. ClpX recognizes the SsrA tag, leading to ClpXP-mediated degradation. We hypothesize that ClpX functions in chlamydial differentiation through targeted protein degradation. We found that mutation of a key residue (R230A) within the specific motif in ClpX associated with the recognition of SsrA-tagged substrates resulted in abrogated secondary differentiation while not reducing chlamydial replication or developmental cycle progression as measured by transcripts. Furthermore, inhibition of trans-translation through chemical and targeted genetic approaches also impeded chlamydial development. Knockdown of tmRNA and subsequent complementation with an allele mutated in the SsrA tag closely phenocopied the overexpression of ClpXR230A, thus suggesting that ClpX recognition of SsrA-tagged substrates plays a critical function in secondary differentiation. Taken together, these data provide mechanistic insight into the requirements for transitions between chlamydial developmental forms. IMPORTANCE Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections and preventable infectious blindness. This unique organism undergoes developmental transitions between infectious, nondividing forms and noninfectious, dividing forms. Therefore, the chlamydial developmental cycle is an attractive target for Chlamydia-specific antibiotics, which would minimize effects of broad-spectrum antibiotics on the spread of antibiotic resistance in other organisms. However, the lack of knowledge about chlamydial development on a molecular level impedes the identification of specific, druggable targets. This work describes a mechanism through which both the fundamental processes of trans-translation and proteomic turnover by ClpXP contribute to chlamydial differentiation, a critical facet of chlamydial growth and survival. Given the almost universal presence of trans-translation and ClpX in eubacteria, this mechanism may be conserved in developmental cycles of other bacterial species. Additionally, this study expands the fields of trans-translation and Clp proteases by emphasizing the functional diversity of these systems throughout bacterial evolution.