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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

Confocal microscopy images of CtL2wt and mutants at 24 h and 44 h post infection. The Chlamydia isolates (CtL2wt and mutant) are shown to the left of the panels and the time point is shown above the panels. The second and fourth images for each isolate are enlarged representations of single inclusions. The image colours are, green: LPS (FITC anti-chlamydial LPS); red: host cells (Evans blue). The scale bars (bottom right) indicate 50 μm and 25 μm for the enlarged images
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Fig4: Confocal microscopy images of CtL2wt and mutants at 24 h and 44 h post infection. The Chlamydia isolates (CtL2wt and mutant) are shown to the left of the panels and the time point is shown above the panels. The second and fourth images for each isolate are enlarged representations of single inclusions. The image colours are, green: LPS (FITC anti-chlamydial LPS); red: host cells (Evans blue). The scale bars (bottom right) indicate 50 μm and 25 μm for the enlarged images

Mentions: The impact of the mutations on the chlamydial inclusion morphology was examined using immunocytochemistry and confocal laser scanning microscopy. Cultures were examined at 24 h post-infection (log phase) and 40 h post-infection (stationary phase; Fig. 4). At 24 h post-infection, the inclusion size of each mutant appeared smaller compared to the wild-type, while in the cthtrAP370L strain there appeared to be fewer chlamydial cells within the inclusions. At 40 h post-infection, the inclusion sizes appeared to be more comparable to the wild-type, if slightly smaller. The inclusion sizes were measured to allow statistical comparison of the mutants against the wild-type, confirming that the mutant inclusion sizes were appreciably smaller compared to the wild-type at 24 h post-infection (Fig. 5). The difference was less pronounced at 40 h post-infection, where the cthtrAG475E inclusion sizes were not significantly different compared to the wild-type. Alternatively, in the cthtrAA240V and, to a greater extent, cthtrAP370L strains, inclusion sizes were significantly smaller than the wild-type (p < 0.0001; Fig. 5).Fig. 4


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)

Confocal microscopy images of CtL2wt and mutants at 24 h and 44 h post infection. The Chlamydia isolates (CtL2wt and mutant) are shown to the left of the panels and the time point is shown above the panels. The second and fourth images for each isolate are enlarged representations of single inclusions. The image colours are, green: LPS (FITC anti-chlamydial LPS); red: host cells (Evans blue). The scale bars (bottom right) indicate 50 μm and 25 μm for the enlarged images
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Confocal microscopy images of CtL2wt and mutants at 24 h and 44 h post infection. The Chlamydia isolates (CtL2wt and mutant) are shown to the left of the panels and the time point is shown above the panels. The second and fourth images for each isolate are enlarged representations of single inclusions. The image colours are, green: LPS (FITC anti-chlamydial LPS); red: host cells (Evans blue). The scale bars (bottom right) indicate 50 μm and 25 μm for the enlarged images
Mentions: The impact of the mutations on the chlamydial inclusion morphology was examined using immunocytochemistry and confocal laser scanning microscopy. Cultures were examined at 24 h post-infection (log phase) and 40 h post-infection (stationary phase; Fig. 4). At 24 h post-infection, the inclusion size of each mutant appeared smaller compared to the wild-type, while in the cthtrAP370L strain there appeared to be fewer chlamydial cells within the inclusions. At 40 h post-infection, the inclusion sizes appeared to be more comparable to the wild-type, if slightly smaller. The inclusion sizes were measured to allow statistical comparison of the mutants against the wild-type, confirming that the mutant inclusion sizes were appreciably smaller compared to the wild-type at 24 h post-infection (Fig. 5). The difference was less pronounced at 40 h post-infection, where the cthtrAG475E inclusion sizes were not significantly different compared to the wild-type. Alternatively, in the cthtrAA240V and, to a greater extent, cthtrAP370L strains, inclusion sizes were significantly smaller than the wild-type (p < 0.0001; Fig. 5).Fig. 4

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