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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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Related in: MedlinePlus

Cell culture analysis of growth and heat shock phenotypes for the cthtrAP370L and ctl0738 mutants. a. One-step growth curve for wild-type C. trachomatis L2 and mutants over an 88 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. b. Viable infectious yield of wild-type C. trachomatis L2 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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Fig7: Cell culture analysis of growth and heat shock phenotypes for the cthtrAP370L and ctl0738 mutants. a. One-step growth curve for wild-type C. trachomatis L2 and mutants over an 88 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. b. Viable infectious yield of wild-type C. trachomatis L2 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: The lateral gene transfer experiments did not enable the generation of an isogenic cthtrAP370L mutant isolate. The SNV that could be contributing to the phenotype observed in the cthtrAP370L strain was the mutation in the DNA methyltransferase, ctl0738. This mutation was able to be transferred to the genome of C. trachomatis L2 spectinomycin-resistant strain (CtL2spc) in the absence of the eight other EMS mutations that were considered potentially significant on the ctHtrAP370L isolate genome. Therefore, further analysis of the isogenic strain containing the DNA methyltransferase (ctl0738) was conducted to examine the role of this mutation for the phenotypes observed in the cthtrAP370L strain. Growth curve and heat shock experiments were conducted using the wild type strains (CtL2wt and CtL2spc), the original cthtrAP370L mutant strain, and the ctl0738 strain. The cthtrAP370L strain has the same severely impacted reduced infectious progeny yield (8–25-fold reduction in infectious progeny; Fig. 7a) and the ctl0738 strain showed infectious progeny production similar to the wild type. Notably, by including additional time-points at the beginning of the replicative phase (16–22 h post infection), it was observed that both the cthtrAP370L and ctl0738 strains displayed a delayed start to the log growth phase implying slowed RB to EB conversion. Somewhat unexpectedly, the heat stress phenotype was similar in the cthtrAP370L and ctl0738 strains with a significant reduction in infectious progeny (CtL2wt: 4.5 × 107 EBs, cthtrAP370L: 1.2 × 106 EBs; ctl0738: 1.4 × 106 EBs; p < 0.0001), corresponding with a 30–40-fold reduction in infectious progeny after heat stress relative to CtL2wt (Fig. 7b).Fig. 7


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)

Cell culture analysis of growth and heat shock phenotypes for the cthtrAP370L and ctl0738 mutants. a. One-step growth curve for wild-type C. trachomatis L2 and mutants over an 88 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. b. Viable infectious yield of wild-type C. trachomatis L2 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

Fig7: Cell culture analysis of growth and heat shock phenotypes for the cthtrAP370L and ctl0738 mutants. a. One-step growth curve for wild-type C. trachomatis L2 and mutants over an 88 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. b. Viable infectious yield of wild-type C. trachomatis L2 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: The lateral gene transfer experiments did not enable the generation of an isogenic cthtrAP370L mutant isolate. The SNV that could be contributing to the phenotype observed in the cthtrAP370L strain was the mutation in the DNA methyltransferase, ctl0738. This mutation was able to be transferred to the genome of C. trachomatis L2 spectinomycin-resistant strain (CtL2spc) in the absence of the eight other EMS mutations that were considered potentially significant on the ctHtrAP370L isolate genome. Therefore, further analysis of the isogenic strain containing the DNA methyltransferase (ctl0738) was conducted to examine the role of this mutation for the phenotypes observed in the cthtrAP370L strain. Growth curve and heat shock experiments were conducted using the wild type strains (CtL2wt and CtL2spc), the original cthtrAP370L mutant strain, and the ctl0738 strain. The cthtrAP370L strain has the same severely impacted reduced infectious progeny yield (8–25-fold reduction in infectious progeny; Fig. 7a) and the ctl0738 strain showed infectious progeny production similar to the wild type. Notably, by including additional time-points at the beginning of the replicative phase (16–22 h post infection), it was observed that both the cthtrAP370L and ctl0738 strains displayed a delayed start to the log growth phase implying slowed RB to EB conversion. Somewhat unexpectedly, the heat stress phenotype was similar in the cthtrAP370L and ctl0738 strains with a significant reduction in infectious progeny (CtL2wt: 4.5 × 107 EBs, cthtrAP370L: 1.2 × 106 EBs; ctl0738: 1.4 × 106 EBs; p < 0.0001), corresponding with a 30–40-fold reduction in infectious progeny after heat stress relative to CtL2wt (Fig. 7b).Fig. 7

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