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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

In vitro biochemical activity of wild type C. trachomatis L2 and recombinant mutants. a. Cleavage of full-length β-casein by wild type CtHtrA and recombinant mutants over a 60 min time course. The corresponding SDS-PAGE gels are provided in Additional file 2: Figure S1. b. Rate of proteolysis of the βcas1 peptide substrate with and without allosteric activation (Act1 peptide) by recombinant proteins. Numbers above the bars represent the fold-change in proteolytic activity following the addition of the Act1 activator. Rate of proteolysis is measured as μM MCA min−1 μg CtHtrA−1. Error bars represent standard error of the mean (n = 6). c. Measurement of the proteolytic activity of wild type CtHtrA and mutants in the presence of pNA1-4 peptide substrates. Rate of proteolysis is measured as pNA 405 nm min−1. Error bars represent standard error of the mean (n = 6). d. Wild type CtHtrA and the mutants oligomerise to 24-mer in the presence of full-length β-casein. The figure shows glutaraldehyde crosslinked protein samples prepared by oligomerisation assays following separation on 3–8 % TrisAcetate gels prior to silver staining. The size of the 24-meric oligomer is indicated to the right of the gel and the molecular weights are indicated on the left (High Molecular Weight Marker, Invitrogen)
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Fig2: In vitro biochemical activity of wild type C. trachomatis L2 and recombinant mutants. a. Cleavage of full-length β-casein by wild type CtHtrA and recombinant mutants over a 60 min time course. The corresponding SDS-PAGE gels are provided in Additional file 2: Figure S1. b. Rate of proteolysis of the βcas1 peptide substrate with and without allosteric activation (Act1 peptide) by recombinant proteins. Numbers above the bars represent the fold-change in proteolytic activity following the addition of the Act1 activator. Rate of proteolysis is measured as μM MCA min−1 μg CtHtrA−1. Error bars represent standard error of the mean (n = 6). c. Measurement of the proteolytic activity of wild type CtHtrA and mutants in the presence of pNA1-4 peptide substrates. Rate of proteolysis is measured as pNA 405 nm min−1. Error bars represent standard error of the mean (n = 6). d. Wild type CtHtrA and the mutants oligomerise to 24-mer in the presence of full-length β-casein. The figure shows glutaraldehyde crosslinked protein samples prepared by oligomerisation assays following separation on 3–8 % TrisAcetate gels prior to silver staining. The size of the 24-meric oligomer is indicated to the right of the gel and the molecular weights are indicated on the left (High Molecular Weight Marker, Invitrogen)

Mentions: The proteolysis and oligomerization activities of these recombinant proteins were compared to the wild-type recombinant protein (CtHtrA). All recombinant proteins were able to cleave full-length β-casein at 37 °C, with proteins CtHtrAE47K, CtHtrAG268R, and CtHtrAR55Q displaying similar proteolytic activity to the wild-type (Fig. 2a). The rate of proteolysis was slower for the CtHtrAA240V, and CtHtrAG475E proteins compared to the wild type, while the proteolysis activity of the CtHtrAP370L was substantially reduced (Fig. 2a; Additional file 2: Figure S1). Similarly, in the presence of a peptide substrate, the CtHtrAA240V and CtHtrAG475E proteins demonstrated a reduction in proteolytic rate, respectively, compared to the wild-type, while no proteolytic activity could be detected for the CtHtrAP370L protein (Fig. 2b). The proteolytic rate of the CtHtrAG268R and CtHtrAR55Q proteins were not substantially different from the wild-type. We have previously shown that the addition of a second peptide based on the 12 C-terminal residues of β-casein (Act1) can activate or increase the proteolytic activity of CtHtrA [11, 12]. Thus, the activation of proteolysis by the recombinant proteins was investigated and a 1.4–2.3-fold increase in the rate of proteolysis was observed for CtHtrAE47K, CtHtrAG268R, CtHtrAR55Q, CtHtrAA240V and CtHtrAG475E. Alternatively, CtHtrAP370L displayed no detectable proteolytic activity against this peptide substrate (Fig. 2b), which may be due to this mutation causing a conformational change to the ‘PDZ1 activation cleft’, impacting the correct binding of Act1. Substrate specificity of the proteins was also examined using different para-Nitroanilide (pNA)-labeled substrates that differ in length and sequence (P1 – P4). No differences in substrate specificity were observed for any of the proteins compared to the wild type, suggesting that the mutations do not appreciably change the conformation of the catalytic domain (at least using these substrates; Fig. 2c). The CtHtrAP370L protein again displayed no proteolytic activity. CtHtrA has been previously shown to oligomerize from a resting hexamer to an activated 12–/24-mer in the presence of both peptide and protein substrates [11, 12]. To investigate whether any of the recombinant mutated protein disrupted or impaired the activation of oligomerization, each were incubated with full-length β-casein at 37 °C and cross-linked with glutaraldehyde. In the presence of substrate, each mutant formed particles that are consistent with a higher order oligomer of CtHtrA (such as a 24-mer), with no evidence of the hexameric or trimeric forms that are observed when the oligomerization mechanism has been disrupted [12, 31]. While this method cannot detect differences in oligomeric structure or the exact number of monomers present, oligomeric activation to some form of oligomer appears to be unperturbed for each of the mutants (Fig. 2d) [11]. These data demonstrated that the CtHtrAA240V, CtHtrAG475E, and CtHtrAP370L mutations resulted in an appreciable disruption to the in vitro proteolytic activity of CtHtrA and therefore isolates with these three mutations were selected from the libraries for further in vitro analysis of their phenotypic impact on chlamydial growth.Fig. 2


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)

In vitro biochemical activity of wild type C. trachomatis L2 and recombinant mutants. a. Cleavage of full-length β-casein by wild type CtHtrA and recombinant mutants over a 60 min time course. The corresponding SDS-PAGE gels are provided in Additional file 2: Figure S1. b. Rate of proteolysis of the βcas1 peptide substrate with and without allosteric activation (Act1 peptide) by recombinant proteins. Numbers above the bars represent the fold-change in proteolytic activity following the addition of the Act1 activator. Rate of proteolysis is measured as μM MCA min−1 μg CtHtrA−1. Error bars represent standard error of the mean (n = 6). c. Measurement of the proteolytic activity of wild type CtHtrA and mutants in the presence of pNA1-4 peptide substrates. Rate of proteolysis is measured as pNA 405 nm min−1. Error bars represent standard error of the mean (n = 6). d. Wild type CtHtrA and the mutants oligomerise to 24-mer in the presence of full-length β-casein. The figure shows glutaraldehyde crosslinked protein samples prepared by oligomerisation assays following separation on 3–8 % TrisAcetate gels prior to silver staining. The size of the 24-meric oligomer is indicated to the right of the gel and the molecular weights are indicated on the left (High Molecular Weight Marker, Invitrogen)
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Related In: Results  -  Collection

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Fig2: In vitro biochemical activity of wild type C. trachomatis L2 and recombinant mutants. a. Cleavage of full-length β-casein by wild type CtHtrA and recombinant mutants over a 60 min time course. The corresponding SDS-PAGE gels are provided in Additional file 2: Figure S1. b. Rate of proteolysis of the βcas1 peptide substrate with and without allosteric activation (Act1 peptide) by recombinant proteins. Numbers above the bars represent the fold-change in proteolytic activity following the addition of the Act1 activator. Rate of proteolysis is measured as μM MCA min−1 μg CtHtrA−1. Error bars represent standard error of the mean (n = 6). c. Measurement of the proteolytic activity of wild type CtHtrA and mutants in the presence of pNA1-4 peptide substrates. Rate of proteolysis is measured as pNA 405 nm min−1. Error bars represent standard error of the mean (n = 6). d. Wild type CtHtrA and the mutants oligomerise to 24-mer in the presence of full-length β-casein. The figure shows glutaraldehyde crosslinked protein samples prepared by oligomerisation assays following separation on 3–8 % TrisAcetate gels prior to silver staining. The size of the 24-meric oligomer is indicated to the right of the gel and the molecular weights are indicated on the left (High Molecular Weight Marker, Invitrogen)
Mentions: The proteolysis and oligomerization activities of these recombinant proteins were compared to the wild-type recombinant protein (CtHtrA). All recombinant proteins were able to cleave full-length β-casein at 37 °C, with proteins CtHtrAE47K, CtHtrAG268R, and CtHtrAR55Q displaying similar proteolytic activity to the wild-type (Fig. 2a). The rate of proteolysis was slower for the CtHtrAA240V, and CtHtrAG475E proteins compared to the wild type, while the proteolysis activity of the CtHtrAP370L was substantially reduced (Fig. 2a; Additional file 2: Figure S1). Similarly, in the presence of a peptide substrate, the CtHtrAA240V and CtHtrAG475E proteins demonstrated a reduction in proteolytic rate, respectively, compared to the wild-type, while no proteolytic activity could be detected for the CtHtrAP370L protein (Fig. 2b). The proteolytic rate of the CtHtrAG268R and CtHtrAR55Q proteins were not substantially different from the wild-type. We have previously shown that the addition of a second peptide based on the 12 C-terminal residues of β-casein (Act1) can activate or increase the proteolytic activity of CtHtrA [11, 12]. Thus, the activation of proteolysis by the recombinant proteins was investigated and a 1.4–2.3-fold increase in the rate of proteolysis was observed for CtHtrAE47K, CtHtrAG268R, CtHtrAR55Q, CtHtrAA240V and CtHtrAG475E. Alternatively, CtHtrAP370L displayed no detectable proteolytic activity against this peptide substrate (Fig. 2b), which may be due to this mutation causing a conformational change to the ‘PDZ1 activation cleft’, impacting the correct binding of Act1. Substrate specificity of the proteins was also examined using different para-Nitroanilide (pNA)-labeled substrates that differ in length and sequence (P1 – P4). No differences in substrate specificity were observed for any of the proteins compared to the wild type, suggesting that the mutations do not appreciably change the conformation of the catalytic domain (at least using these substrates; Fig. 2c). The CtHtrAP370L protein again displayed no proteolytic activity. CtHtrA has been previously shown to oligomerize from a resting hexamer to an activated 12–/24-mer in the presence of both peptide and protein substrates [11, 12]. To investigate whether any of the recombinant mutated protein disrupted or impaired the activation of oligomerization, each were incubated with full-length β-casein at 37 °C and cross-linked with glutaraldehyde. In the presence of substrate, each mutant formed particles that are consistent with a higher order oligomer of CtHtrA (such as a 24-mer), with no evidence of the hexameric or trimeric forms that are observed when the oligomerization mechanism has been disrupted [12, 31]. While this method cannot detect differences in oligomeric structure or the exact number of monomers present, oligomeric activation to some form of oligomer appears to be unperturbed for each of the mutants (Fig. 2d) [11]. These data demonstrated that the CtHtrAA240V, CtHtrAG475E, and CtHtrAP370L mutations resulted in an appreciable disruption to the in vitro proteolytic activity of CtHtrA and therefore isolates with these three mutations were selected from the libraries for further in vitro analysis of their phenotypic impact on chlamydial growth.Fig. 2

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