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Genetic transformation of a C. trachomatis ocular isolate with the functional tryptophan synthase operon confers an indole-rescuable phenotype

Genetic transformation of a C. trachomatis ocular isolate with the functional tryptophan synthase operon confers an indole-rescuable phenotype
Genetic transformation of a C. trachomatis ocular isolate with the functional tryptophan synthase operon confers an indole-rescuable phenotype
Chlamydia trachomatis is the leading cause of preventable blindness and the most common bacterial sexually transmitted infection. Different strains are associated with ocular or urogenital infections, and a proposed mechanism that may explain this tissue tropism is the active tryptophan biosynthesis pathway encoded by the genomic trpRBA operon in urogenital strains. Here we describe genetic complementation studies that are essential to confirm the role of tryptophan synthase in the context of an ocular C. trachomatis genomic background. Ocular strain A2497 was transformed with the (urogenital) pSW2::GFP shuttle vector showing that there is no strain tropism barrier to this plasmid vector; moreover, transformation had no detrimental effect on the growth kinetics of A2497, which is important given the low transformation efficiency of C. trachomatis. A derivative of the pSW2::GFP vector was used to deliver the active tryptophan biosynthesis genes from a urogenital strain of C. trachomatis (Soton D1) to A2497 with the aim of complementing the truncated trpA gene common to most ocular strains. After confirmation of intact TrpA protein expression in the transformed A2497, the resulting transformants were cultivated in tryptophan-depleted medium with and without indole or tryptophan, showing that complementation of the truncated trpA gene by the intact and functional urogenital trpRBA operon was sufficient to bestow an indole rescuable phenotype upon A2497. This study proves that pSW2::GFP derived vectors do not conform to the cross-strain transformation barrier reported for other chlamydia shuttle vectors, suggesting these as a universal vector for transformation of all C. trachomatis strains. This vector promiscuity enabled us to test the indole rescue hypothesis by transforming ocular strain A2497 with the functional urogenital trpRBA operon, which complemented the non-functional tryptophan synthase. These data confirm that the trpRBA operon is necessary and sufficient for chlamydia to survive in tryptophan-limited environments such as the female urogenital tract.
Chlamydia trachomatis, Trachoma, Interferon Gamma, Genetic complementation, Transformation, Tropism, tryptophan, Plasmid
O'Neill, Colette
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Skilton, Rachel
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Pearson, Sarah
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Filardo, Simone
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Andersson, Patiyan
1d7813cd-a206-449b-b5e1-d6976a530797
Clarke, Ian
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O'Neill, Colette
3de0c221-6578-4a1a-96bd-2a3fba2b6193
Skilton, Rachel
b02d4f32-609c-4074-b616-ec819b018dbe
Pearson, Sarah
0965edba-1d1f-4080-85a0-41b53d3cb553
Filardo, Simone
9215c7e9-889d-418f-b519-4d62e700d72f
Andersson, Patiyan
1d7813cd-a206-449b-b5e1-d6976a530797
Clarke, Ian
ff6c9324-3547-4039-bb2c-10c0b3327a8b

O'Neill, Colette, Skilton, Rachel, Pearson, Sarah, Filardo, Simone, Andersson, Patiyan and Clarke, Ian (2018) Genetic transformation of a C. trachomatis ocular isolate with the functional tryptophan synthase operon confers an indole-rescuable phenotype. Frontiers in Cellular and Infection Microbiology, 8, [434]. (doi:10.3389/fcimb.2018.00434).

Record type: Article

Abstract

Chlamydia trachomatis is the leading cause of preventable blindness and the most common bacterial sexually transmitted infection. Different strains are associated with ocular or urogenital infections, and a proposed mechanism that may explain this tissue tropism is the active tryptophan biosynthesis pathway encoded by the genomic trpRBA operon in urogenital strains. Here we describe genetic complementation studies that are essential to confirm the role of tryptophan synthase in the context of an ocular C. trachomatis genomic background. Ocular strain A2497 was transformed with the (urogenital) pSW2::GFP shuttle vector showing that there is no strain tropism barrier to this plasmid vector; moreover, transformation had no detrimental effect on the growth kinetics of A2497, which is important given the low transformation efficiency of C. trachomatis. A derivative of the pSW2::GFP vector was used to deliver the active tryptophan biosynthesis genes from a urogenital strain of C. trachomatis (Soton D1) to A2497 with the aim of complementing the truncated trpA gene common to most ocular strains. After confirmation of intact TrpA protein expression in the transformed A2497, the resulting transformants were cultivated in tryptophan-depleted medium with and without indole or tryptophan, showing that complementation of the truncated trpA gene by the intact and functional urogenital trpRBA operon was sufficient to bestow an indole rescuable phenotype upon A2497. This study proves that pSW2::GFP derived vectors do not conform to the cross-strain transformation barrier reported for other chlamydia shuttle vectors, suggesting these as a universal vector for transformation of all C. trachomatis strains. This vector promiscuity enabled us to test the indole rescue hypothesis by transforming ocular strain A2497 with the functional urogenital trpRBA operon, which complemented the non-functional tryptophan synthase. These data confirm that the trpRBA operon is necessary and sufficient for chlamydia to survive in tryptophan-limited environments such as the female urogenital tract.

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ONeill et al 2018 - Version of Record
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e-pub ahead of print date: 14 December 2018
Keywords: Chlamydia trachomatis, Trachoma, Interferon Gamma, Genetic complementation, Transformation, Tropism, tryptophan, Plasmid

Identifiers

Local EPrints ID: 426960
URI: http://eprints.soton.ac.uk/id/eprint/426960
PURE UUID: 89ae9945-8c64-4b19-ad96-3c644a3c3cd4
ORCID for Ian Clarke: ORCID iD orcid.org/0000-0002-4938-1620

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Date deposited: 19 Dec 2018 17:30
Last modified: 26 Nov 2021 02:32

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Contributors

Author: Colette O'Neill
Author: Rachel Skilton
Author: Sarah Pearson
Author: Simone Filardo
Author: Patiyan Andersson
Author: Ian Clarke ORCID iD

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