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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.SNVs were initially biochemically characterized in vitro using recombinant protein techniques to confirm a functional impact on proteolysis.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.

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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Growth and heat shock response phenotypes for C. trachomatis mutants containing cthtrA SNVs. a. One-step growth curve for CtL2wt and mutants over a 48 h time course. McCoy B cells were infected at an MOI of 0.3 and collected over the time course for determination of inclusion-forming units (IFUs). b. Infectious yield of CtL2wt and mutants at 44 h post infection after 4 h heat shock (42 °C, 5 % CO2; 20–24 h post infection). **** indicates p < 0.0001. Error bars represent standard error of the mean (n = 27)
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Fig3: Growth and heat shock response phenotypes for C. trachomatis mutants containing cthtrA SNVs. a. One-step growth curve for CtL2wt and mutants over a 48 h time course. McCoy B cells were infected at an MOI of 0.3 and collected over the time course for determination of inclusion-forming units (IFUs). b. Infectious yield of CtL2wt and mutants at 44 h post infection after 4 h heat shock (42 °C, 5 % CO2; 20–24 h post infection). **** indicates p < 0.0001. Error bars represent standard error of the mean (n = 27)

Mentions: Previous work by our group suggested that CtHtrA has a crucial role in the replicative phase of C. trachomatis development, as the JO146 CtHtrA inhibitor was lethal when added to the cultures at mid-replication [15]. Accordingly, growth rates were assessed for Chlamydia strains containing the cthtrA variants cthtrAA240V, cthtrAP370L, cthtrAG475E and wild-type C. trachomatis serovar L2 (referred to as for this study CtL2wt) (Additional file 1: Table S2), by determining the time at which infectious EBs were first detected, with additional times points during the growth cycle. The presence of infectious EBs was detected between 20 and 48 h post-infection, with each mutant strain producing fewer EBs than the wild-type (Fig. 3a). The cthtrAP370L mutant produced significantly fewer infectious EBs compared to the wild type and other mutants, displaying a ~20–40-fold reduction (~95 % reduction) in infectious EB progeny (for example at 32 h post infection, 2.26 × 107 EBs were detected for CtL2wt while 5.45 × 105 EBs were detected for cthtrAP370L, corresponding to a 41.4-fold reduction in infectious EB production at this time point).Fig. 3


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)

Growth and heat shock response phenotypes for C. trachomatis mutants containing cthtrA SNVs. a. One-step growth curve for CtL2wt and mutants over a 48 h time course. McCoy B cells were infected at an MOI of 0.3 and collected over the time course for determination of inclusion-forming units (IFUs). b. Infectious yield of CtL2wt and mutants at 44 h post infection after 4 h heat shock (42 °C, 5 % CO2; 20–24 h post infection). **** indicates p < 0.0001. Error bars represent standard error of the mean (n = 27)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Growth and heat shock response phenotypes for C. trachomatis mutants containing cthtrA SNVs. a. One-step growth curve for CtL2wt and mutants over a 48 h time course. McCoy B cells were infected at an MOI of 0.3 and collected over the time course for determination of inclusion-forming units (IFUs). b. Infectious yield of CtL2wt and mutants at 44 h post infection after 4 h heat shock (42 °C, 5 % CO2; 20–24 h post infection). **** indicates p < 0.0001. Error bars represent standard error of the mean (n = 27)
Mentions: Previous work by our group suggested that CtHtrA has a crucial role in the replicative phase of C. trachomatis development, as the JO146 CtHtrA inhibitor was lethal when added to the cultures at mid-replication [15]. Accordingly, growth rates were assessed for Chlamydia strains containing the cthtrA variants cthtrAA240V, cthtrAP370L, cthtrAG475E and wild-type C. trachomatis serovar L2 (referred to as for this study CtL2wt) (Additional file 1: Table S2), by determining the time at which infectious EBs were first detected, with additional times points during the growth cycle. The presence of infectious EBs was detected between 20 and 48 h post-infection, with each mutant strain producing fewer EBs than the wild-type (Fig. 3a). The cthtrAP370L mutant produced significantly fewer infectious EBs compared to the wild type and other mutants, displaying a ~20–40-fold reduction (~95 % reduction) in infectious EB progeny (for example at 32 h post infection, 2.26 × 107 EBs were detected for CtL2wt while 5.45 × 105 EBs were detected for cthtrAP370L, corresponding to a 41.4-fold reduction in infectious EB production at this time point).Fig. 3

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.SNVs were initially biochemically characterized in vitro using recombinant protein techniques to confirm a functional impact on proteolysis.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.

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