{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T02:39:17Z","timestamp":1790303957903,"version":"4.1.0"},"reference-count":66,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2021,11,16]],"date-time":"2021-11-16T00:00:00Z","timestamp":1637020800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"crossref","award":["202755"],"award-info":[{"award-number":["202755"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"\n Background:<\/ns3:bold>\n Chlamydia trachomatis<\/ns3:italic>\n is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in\n Chlamydia<\/ns3:italic>\n 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\n C. trachomatis<\/ns3:italic>\n using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the\n Chlamydia<\/ns3:italic>\n genome.\n <\/ns3:p>\n \n Methods:<\/ns3:bold>\n The low transformation efficiency of\n C. trachomatis<\/ns3:italic>\n 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\n tet<\/ns3:italic>\n promoter system with the addition of a riboswitch).\n Chlamydia<\/ns3:italic>\n 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.\n <\/ns3:p>\n \n Results:<\/ns3:bold>\n Here we present the preliminary sequencing results of transposon mutant pools of both\n C. trachomatis<\/ns3:italic>\n biovars, using two plasmid-free representatives: urogenital strain \u00a0\n C. trachomatis<\/ns3:italic>\n SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies\u2019 MinION technology. This enabled \u2018proof of concept\u2019 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.\n <\/ns3:p>\n \n Conclusions:<\/ns3:bold>\n This significant advance provides an efficient method for assaying\n C. trachomatis<\/ns3:italic>\n gene function and will enable the identification of the essential gene set of\n C. trachomatis<\/ns3:italic>\n . 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.\n <\/ns3:p>","DOI":"10.12688\/wellcomeopenres.16068.1","type":"journal-article","created":{"date-parts":[[2021,11,16]],"date-time":"2021-11-16T11:32:48Z","timestamp":1637062368000},"page":"312","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":25,"title":["An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis"],"prefix":"10.12688","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9725-8793","authenticated-orcid":false,"given":"Colette E.","family":"O'Neill","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rachel 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status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.17627.r47094, Isabelle Derr\u00e9, Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA, USA, 15 Dec 2021, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-312\/v1#referee-response-47094","order":0,"name":"referee-response-47094","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"Colette O'Neill<\/b>; \nPosted: 31 Jan 2022<\/i>; We would like to thank Prof Derré for her in-depth critique of our work. We agree that whilst basal transcription and translation of transposase appears to be strongly repressed in this system, there remains a possibility that the additional transposon insertions identified in the output libraries could represent new transposition events due to basal transposase expression, rather than carry-over from the input library. Our next aim will be to optimise the transposon-delivery vector and identifying the cause of these additional mutations will form part of this phase of work. Whilst important to clarify this point, once saturation mutagenesis is proven, individual inserts will have less impact due to the statistical methods used in comparing input and output libraries for the identification of essential genes.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-312\/v1#referee-comment-4892","order":1,"name":"referee-comment-4892","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.17627.r47092, Garry Myers, The iThree Institute, University of Technology Sydney, Sydney, NSW, Australia, 18 Jan 2022, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-312\/v1#referee-response-47092","order":2,"name":"referee-response-47092","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"Colette O'Neill<\/b>; \nPosted: 02 Feb 2022<\/i>; We are grateful to Dr Garry Myers for his detailed analysis of our work. He poses three key questions. In response to these: \n1. How do the obtained transposon insertion sites in these experiments compare to those previously obtained with non-replicating vectors (despite the low numbers)?<\/b> In the study by LaBrie \net al.<\/i>, living mutants were isolated after several passages, so the only comparable dataset from our study was from the \u201coutput library\u201d (i.e. the transposon mutagenesis library passaged in vitro). Only one gene was found to contain a transposon insertion in both ours and LaBrie\u2019s datasets \u2013 namely \nuvrB<\/i>. This might suggest functional redundancy exists for this gene within the chlamydial genome. However, this could also be coincidental and would need to be investigated using the same strains (in our passaging experiment we used SWFP- whereas LaBrie used L2\/434Bu), grown under the same conditions with multiple replicates. We chose not to include this sort of analysis for the reasons given above and more specifically because this study was proof of principle for transposon insertion from a replicating vector. It\u2019s also true to say that once saturation mutagenesis is achieved, these experiments will be made unnecessary as statistical comparison of input vs output libraries will inform on which genes are essential to growth\/infectivity in vitro. \n2. What strategies might be effective to overcome potential DNA topology effects in forthcoming saturation experiments?<\/b> We thank the reviewer for this question. Access to all potential transposon insertion sites (i.e. TA dinucleotides) might be impeded by changes in DNA topology throughout the developmental cycle. Therefore, it follows that the induction time point could affect the distribution of transposon inserts. In these first unoptimized experiments, we chose to induce transposase expression mid-way through the growth cycle, during DNA replication. The thought was that whilst DNA is actively replicating, different regions of the genome will be exposed to the transposase enzyme and therefore given the best opportunity for random transposon mutagenesis to occur. After induction, the media was not exchanged for anhydrotetracycline\/theophylline-free media, meaning that the transposase was continually expressed after 24hpi and thus transposon mutagenesis could continue until the end of the replication cycle. However, induction timing may vary depending on strain (due to strain-specific growth differences \u2013 e.g. L2 has a shorter developmental cycle than SWFP-) and growth conditions; variables such as these will be assessed beyond this proof of principle in future optimisation experiments. \n3. This work is performed in vitro. Given that gene essentiality is likely to be substantially different in in vivo tissue contexts, is there an expectation that this construct and mutagenesis strategy will be effective in animal models?<\/b> The design of these experiments enables transposon mutant libraries to be tested by altering the growth conditions used during passage of mutated chlamydiae. This could include infection of animal models with \u201cinput\u201d transposon mutant libraries to determine which genes are essential for infectivity in vivo \u2013 with those successful at infecting being collected for sequencing at the end of the experiment.  We still need to demonstrate saturation mutagenesis before embarking on in vivo experiments and it may be necessary to optimise the delivery vector since the plasmid has a role in virulence.","URL":"https:\/\/wellcomeopenresearch.org\/articles\/6-312\/v1#referee-comment-4897","order":3,"name":"referee-comment-4897","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by Wellcome [202755, https:\/\/doi.org\/10.35802\/202755<\/a>]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}