An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis

Abstract

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies’ MinION technology. This enabled ‘proof of concept’ for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

In vivo and In vitro Characterization of the ClpC AAA+ ATPase of Chlamydia trachomatis

“…Collectively, these data suggest that the inability of ClpC (by its absence or impairment) to target specific substrate(s) for degradation by ClpP or for possible chaperone functions impairs growth and/or differentiation in Chlamydia . Further, this indicates that ClpC is likely essential to Chlamydia and is consistent with failure to obtain a clpC ::intron mutant (D. Fisher, unpublished observation) and loss of a clpC ::Tn mutant from a transposon mutant pool during serial passaging [65]. Overall, both the overexpression and knockdown data suggest that the activity of ClpC must be tightly regulated to ensure proper developmental cycle progression.…”

“…Fisher, unpublished observation) and loss of a clpC::Tn mutant from a transposon mutant pool during serial passaging [65]. Overall, both the overexpression and knockdown data suggest that the activity of ClpC must be tightly regulated to ensure proper developmental cycle progression.…”

Bringing genetics to heretofore intractable obligate intracellular bacterial pathogens: Chlamydia and beyond

“…While the low transformation efficiencies in Chlamydia have been considered a major obstacle for establishing such approach, the recent development of inducible transposon mutagenesis in C . trachomatis suggests that it can be bypassed [ 79 ].…”

“…Finally, a better understanding of Chlamydia ’s unique developmental biology will also require a further refinement of our capabilities to study the function of essential genes, such as by developing more tightly controlled inducible expression systems to improve conditional gene expression and disruption approaches. Again, most recent developments in the field, such as the description of riboswitches for translational control of gene expression in Chlamydia [ 79 , 80 ], provide a highly encouraging perspective.…”

“…Transposon-insertion sequencing holds great promise in identifying essential genes and genes providing such context-specific fitness benefits. While the low transformation efficiencies in Chlamydia have been considered a major obstacle for establishing such approach, the recent development of inducible transposon mutagenesis in C. trachomatis suggests that it can be bypassed [79].…”