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

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

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.

2398-502X
O'Neill, Colette E
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Skilton, Rachel J.
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Forster, Jade
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Cleary, David W.
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Pearson, Sarah A.
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Lampe, David J.
eb2a4b89-c302-42cd-b92c-d32c1a5fda7e
Thomson, Nicholas R.
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Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b
O'Neill, Colette E
3de0c221-6578-4a1a-96bd-2a3fba2b6193
Skilton, Rachel J.
b02d4f32-609c-4074-b616-ec819b018dbe
Forster, Jade
239097a0-1f53-470f-8d7c-efc786e18a46
Cleary, David W.
f4079c6d-d54b-4108-b346-b0069035bec0
Pearson, Sarah A.
0965edba-1d1f-4080-85a0-41b53d3cb553
Lampe, David J.
eb2a4b89-c302-42cd-b92c-d32c1a5fda7e
Thomson, Nicholas R.
5497a110-069d-4156-bccb-c77db572b1c2
Clarke, Ian N.
ff6c9324-3547-4039-bb2c-10c0b3327a8b

O'Neill, Colette E, Skilton, Rachel J., Forster, Jade, Cleary, David W., Pearson, Sarah A., Lampe, David J., Thomson, Nicholas R. and Clarke, Ian N. (2021) An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis. Wellcome Open Research, 6, [312]. (doi:10.12688/wellcomeopenres.16068.1).

Record type: Article

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.

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e-pub ahead of print date: 16 November 2021

Identifiers

Local EPrints ID: 457230
URI: http://eprints.soton.ac.uk/id/eprint/457230
ISSN: 2398-502X
PURE UUID: b114f85f-a436-4fec-b1d0-5539a39f1b81
ORCID for David W. Cleary: ORCID iD orcid.org/0000-0003-4533-0700
ORCID for Ian N. Clarke: ORCID iD orcid.org/0000-0002-4938-1620

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Date deposited: 26 May 2022 16:54
Last modified: 17 Mar 2024 03:35

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Contributors

Author: Colette E O'Neill
Author: Rachel J. Skilton
Author: Jade Forster
Author: David W. Cleary ORCID iD
Author: Sarah A. Pearson
Author: David J. Lampe
Author: Nicholas R. Thomson
Author: Ian N. Clarke ORCID iD

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