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.
Highlights
Chlamydia trachomatis is a common human pathogen that infects epithelial cells of mucosal sites where it causes distinct clinical manifestations including blinding trachoma and the sexually transmitted infection chlamydia
Transformation of C. trachomatis with the pSW2-RiboAC9 delivery vector was successful, but transformants failed to expand under spectinomycin selection in cell culture Previous attempts to introduce the transposon and transposase to C. trachomatis on a single replicating vector were unsuccessful; we hypothesised that basal expression from the tet promoter was causing premature transposition, resulting in immediate vector loss and killing of early transformants[30]
To reduce this basal expression, we investigated the use of translational regulation by incorporating a riboswitch between the promoter and start codon of the C9 transposase
Summary
Chlamydia trachomatis is a common human pathogen that infects epithelial cells of mucosal sites where it causes distinct clinical manifestations including blinding trachoma and the sexually transmitted infection chlamydia. We chose to first revert the Q358R mutation, changing the amino acid back from arginine (hyperactive variant) to glutamine (wild-type); in E. coli, this reduces the activity of the transposase enzyme to less than half that of the C9 mutant[22] The vector with this mutation (pSW2-RiboA-C9Q) was successfully transformed into C. trachomatis SWFP- on the first attempt, and this time the transformant survived serial passage and expanded in cell culture (Figure 2b). 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. This enabled ‘proof of concept’ for the methods as an initial low-throughput screen of mutant libraries; the step is to employ high throughput sequencing to assess saturation mutagenesis
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