Limits...
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.

Show MeSH

Related in: MedlinePlus

Mutated residues shown on the CtHtrA monomer of the active 24-meric structure. a. The CtHtrA active monomer with black boxes indicating the location of mutated residues. Green: protease domain, orange: PDZ1 domain, blue: PDZ2 domain. b. Close-up of wild-type residues (orange side-chain) aligned with the mutated residue (green side-chain)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Mutated residues shown on the CtHtrA monomer of the active 24-meric structure. a. The CtHtrA active monomer with black boxes indicating the location of mutated residues. Green: protease domain, orange: PDZ1 domain, blue: PDZ2 domain. b. Close-up of wild-type residues (orange side-chain) aligned with the mutated residue (green side-chain)

Mentions: The cthtrA EMS mutations were examined using molecular models of the inactive hexamer and active 24-mer oligomers of CtHtrA, according to 2D and 3D structural motifs known to have a role in CtHtrA protein function [11, 12, 17, 18]. Mutations were identified throughout the entire cthtrA gene, with SNVs identified in the signal peptide, protease domain, PDZ1 domain, and PDZ2 domain (Additional file 1: Table S1). Six mutations that resulted in amino acid substitutions were selected for characterization based on in silico prediction of a structural and/or functional impact: E47K (G – A), R55Q (G – A), A240V (C – T), G268R (G – A), P370L (C – T), and G475E (G – A) (Fig. 1a). The E47K and R55Q mutations were located at the N-terminus of the protease domain, upstream from loop LA, and had the potential to disrupt the trimeric protease domain interface by forming a steric clash with an adjacent protease domain loop (Fig. 1b). A240V represented a minor substitution from alanine to valine, two small and hydrophobic amino acids, however this residue is situated on loop L1 near to the catalytic serine and any conformational shift that may occur as a result of this mutation, however minor, would likely impact CtHtrA proteolytic activity (Fig. 1b). G268R was situated in the protease domain active site in loop L2 and potentially results in a steric clash with loop L2 from an adjacent protease domain, also potentially disrupting proteolysis (Fig. 1b). P370L was located in the PDZ1 domain at the base of the ‘carboxylate binding loop’ and is likely to disrupt the conformational turn of this loop, which may impact the binding of the substrate C-terminus to the PDZ1 domain and the subsequent activation cascade (Fig. 1b). G475E was located in the PDZ2 domain and resulted in a potential steric clash with a nearby PDZ2 domain loop, as well as an adjacent PDZ1 domain in the oligomeric state, thus potentially affecting the oligomerization mechanism (Fig. 1b). These six mutations were then generated in vitro in our CtHtrA recombinant protein expression construct using site-directed mutagenesis for biochemical characterization of the recombinant mutated proteins.Fig. 1


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)

Mutated residues shown on the CtHtrA monomer of the active 24-meric structure. a. The CtHtrA active monomer with black boxes indicating the location of mutated residues. Green: protease domain, orange: PDZ1 domain, blue: PDZ2 domain. b. Close-up of wild-type residues (orange side-chain) aligned with the mutated residue (green side-chain)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Mutated residues shown on the CtHtrA monomer of the active 24-meric structure. a. The CtHtrA active monomer with black boxes indicating the location of mutated residues. Green: protease domain, orange: PDZ1 domain, blue: PDZ2 domain. b. Close-up of wild-type residues (orange side-chain) aligned with the mutated residue (green side-chain)
Mentions: The cthtrA EMS mutations were examined using molecular models of the inactive hexamer and active 24-mer oligomers of CtHtrA, according to 2D and 3D structural motifs known to have a role in CtHtrA protein function [11, 12, 17, 18]. Mutations were identified throughout the entire cthtrA gene, with SNVs identified in the signal peptide, protease domain, PDZ1 domain, and PDZ2 domain (Additional file 1: Table S1). Six mutations that resulted in amino acid substitutions were selected for characterization based on in silico prediction of a structural and/or functional impact: E47K (G – A), R55Q (G – A), A240V (C – T), G268R (G – A), P370L (C – T), and G475E (G – A) (Fig. 1a). The E47K and R55Q mutations were located at the N-terminus of the protease domain, upstream from loop LA, and had the potential to disrupt the trimeric protease domain interface by forming a steric clash with an adjacent protease domain loop (Fig. 1b). A240V represented a minor substitution from alanine to valine, two small and hydrophobic amino acids, however this residue is situated on loop L1 near to the catalytic serine and any conformational shift that may occur as a result of this mutation, however minor, would likely impact CtHtrA proteolytic activity (Fig. 1b). G268R was situated in the protease domain active site in loop L2 and potentially results in a steric clash with loop L2 from an adjacent protease domain, also potentially disrupting proteolysis (Fig. 1b). P370L was located in the PDZ1 domain at the base of the ‘carboxylate binding loop’ and is likely to disrupt the conformational turn of this loop, which may impact the binding of the substrate C-terminus to the PDZ1 domain and the subsequent activation cascade (Fig. 1b). G475E was located in the PDZ2 domain and resulted in a potential steric clash with a nearby PDZ2 domain loop, as well as an adjacent PDZ1 domain in the oligomeric state, thus potentially affecting the oligomerization mechanism (Fig. 1b). These six mutations were then generated in vitro in our CtHtrA recombinant protein expression construct using site-directed mutagenesis for biochemical characterization of the recombinant mutated proteins.Fig. 1

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