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A Chlamydia trachomatis strain with a chemically generated amino acid substitution (P370L) in the cthtrA gene shows reduced elementary body production.

Marsh JW, Wee BA, Tyndall JD, Lott WB, Bastidas RJ, Caldwell HD, Valdivia RH, Kari L, Huston WM - BMC Microbiol. (2015)

Bottom Line: In this study, chemically-induced SNVs in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterized.The strain harboring the SNV with the most marked impact on proteolysis (cthtrA P370L) was detected to have a significant reduction in the production of infectious elementary bodies.This provides genetic evidence that CtHtrA is critical for the C. trachomatis developmental cycle.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), 60 Musk Avenue, Kelvin Grove, QLD, 4059, Australia. james.marsh@qut.edu.au.

ABSTRACT

Background: Chlamydia (C.) trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Genetic approaches to investigate C. trachomatis have been only recently developed due to the organism's intracellular developmental cycle. HtrA is a critical stress response serine protease and chaperone for many bacteria and in C. trachomatis has been previously shown to be important for heat stress and the replicative phase of development using a chemical inhibitor of the CtHtrA activity. In this study, chemically-induced SNVs in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterized.

Methods: SNVs were initially biochemically characterized in vitro using recombinant protein techniques to confirm a functional impact on proteolysis. The C. trachomatis strains containing the SNVs with marked reductions in proteolysis were investigated in cell culture to identify phenotypes that could be linked to CtHtrA function.

Results: The strain harboring the SNV with the most marked impact on proteolysis (cthtrA P370L) was detected to have a significant reduction in the production of infectious elementary bodies.

Conclusions: This provides genetic evidence that CtHtrA is critical for the C. trachomatis developmental cycle.

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Related in: MedlinePlus

Circular representation of the reference L2/434/Bu genome (1.04 Mbp) showing the position of SNVs found on protein coding genes and the annotation of those genes. Each mutant strain is represented by a single ring layer (from inner to outer: cthtrAP3470L, cthtrAG475E, cthtraA240V). Blue labels correspond to synonymous SNVs and black labels indicate non-synonymous SNVs. Figure generated using BRIG [37]
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Fig6: Circular representation of the reference L2/434/Bu genome (1.04 Mbp) showing the position of SNVs found on protein coding genes and the annotation of those genes. Each mutant strain is represented by a single ring layer (from inner to outer: cthtrAP3470L, cthtrAG475E, cthtraA240V). Blue labels correspond to synonymous SNVs and black labels indicate non-synonymous SNVs. Figure generated using BRIG [37]

Mentions: Given that the isolate with the cthtrAP370L mutation displayed the most marked phenotypes and was severely impaired during CtHtrA recombinant protein in vitro protease assays, it was reasoned that this isolate will be the most informative for determining the physiological function of CtHtrA and was selected for further genetic characterization. Of the 19 unique SNVs in the genome of the cthtrAP370L mutant isolate, 11 were non-synonymous, five were synonymous, and three were located in intergenic regions. Notably, there was a mutation in ctl0738, a putative DNA methyltransferase. Of the eleven non-synonymous mutations, three resulted in a change to a similar amino acid (i.e. polar, hydrophobic etc.) and are therefore unlikely to have a functional effect (these were SNVs found in ctl0493, metG, and ctl0220). Consequently, a total of eight SNVs in the following cthtrAP370L isolate were identified as potentially significant, found on the following locus: recB, murC, incA, ydhO, ctl0738 (putative DNA methyltransferase), ctl0791 (putative membrane protein), ctl0885 (conserved hypothetical protein), and cthtrAP370L (Fig. 6, Additional file 1: Table S3).Fig. 6


A Chlamydia trachomatis strain with a chemically generated amino acid substitution (P370L) in the cthtrA gene shows reduced elementary body production.

Marsh JW, Wee BA, Tyndall JD, Lott WB, Bastidas RJ, Caldwell HD, Valdivia RH, Kari L, Huston WM - BMC Microbiol. (2015)

Circular representation of the reference L2/434/Bu genome (1.04 Mbp) showing the position of SNVs found on protein coding genes and the annotation of those genes. Each mutant strain is represented by a single ring layer (from inner to outer: cthtrAP3470L, cthtrAG475E, cthtraA240V). Blue labels correspond to synonymous SNVs and black labels indicate non-synonymous SNVs. Figure generated using BRIG [37]
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4590699&req=5

Fig6: Circular representation of the reference L2/434/Bu genome (1.04 Mbp) showing the position of SNVs found on protein coding genes and the annotation of those genes. Each mutant strain is represented by a single ring layer (from inner to outer: cthtrAP3470L, cthtrAG475E, cthtraA240V). Blue labels correspond to synonymous SNVs and black labels indicate non-synonymous SNVs. Figure generated using BRIG [37]
Mentions: Given that the isolate with the cthtrAP370L mutation displayed the most marked phenotypes and was severely impaired during CtHtrA recombinant protein in vitro protease assays, it was reasoned that this isolate will be the most informative for determining the physiological function of CtHtrA and was selected for further genetic characterization. Of the 19 unique SNVs in the genome of the cthtrAP370L mutant isolate, 11 were non-synonymous, five were synonymous, and three were located in intergenic regions. Notably, there was a mutation in ctl0738, a putative DNA methyltransferase. Of the eleven non-synonymous mutations, three resulted in a change to a similar amino acid (i.e. polar, hydrophobic etc.) and are therefore unlikely to have a functional effect (these were SNVs found in ctl0493, metG, and ctl0220). Consequently, a total of eight SNVs in the following cthtrAP370L isolate were identified as potentially significant, found on the following locus: recB, murC, incA, ydhO, ctl0738 (putative DNA methyltransferase), ctl0791 (putative membrane protein), ctl0885 (conserved hypothetical protein), and cthtrAP370L (Fig. 6, Additional file 1: Table S3).Fig. 6

Bottom Line: In this study, chemically-induced SNVs in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterized.The strain harboring the SNV with the most marked impact on proteolysis (cthtrA P370L) was detected to have a significant reduction in the production of infectious elementary bodies.This provides genetic evidence that CtHtrA is critical for the C. trachomatis developmental cycle.

View Article: PubMed Central - PubMed

Affiliation: Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), 60 Musk Avenue, Kelvin Grove, QLD, 4059, Australia. james.marsh@qut.edu.au.

ABSTRACT

Background: Chlamydia (C.) trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Genetic approaches to investigate C. trachomatis have been only recently developed due to the organism's intracellular developmental cycle. HtrA is a critical stress response serine protease and chaperone for many bacteria and in C. trachomatis has been previously shown to be important for heat stress and the replicative phase of development using a chemical inhibitor of the CtHtrA activity. In this study, chemically-induced SNVs in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterized.

Methods: SNVs were initially biochemically characterized in vitro using recombinant protein techniques to confirm a functional impact on proteolysis. The C. trachomatis strains containing the SNVs with marked reductions in proteolysis were investigated in cell culture to identify phenotypes that could be linked to CtHtrA function.

Results: The strain harboring the SNV with the most marked impact on proteolysis (cthtrA P370L) was detected to have a significant reduction in the production of infectious elementary bodies.

Conclusions: This provides genetic evidence that CtHtrA is critical for the C. trachomatis developmental cycle.

Show MeSH
Related in: MedlinePlus