Poster Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

Chemically generated chromosomal mutants suggest a critical role for C. trachomatis HtrA in heat shock and the production of infectious progeny (#123)

James W. Marsh 1 , Garry Myers 2 , Wilhelmina Huston 1
  1. Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), Brisbane, QLD, Australia
  2. i3 Institute, University of Technology Sydney (UTS), Sydney, NSW, Australia

Chlamydia (C.) trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness. Genetic approaches to investigate Chlamydia have only recently been developed, largely due to the organism’s complex intracellular developmental cycle. Thus, much remains to be uncovered about the virulence pathways of this unique organism. HtrA is a critical stress response serine protease and chaperone for many bacteria, while in C. trachomatis, studies using a (Ct)HtrA-specific inhibitor have shown that this protein is important for the replicative phase of development and during penicillin-induced persistence. In this study, chemically-induced single nucleotide variants (SNV) in the cthtrA gene that resulted in amino acid substitutions (A240V, G475E, and P370L) were identified and characterised. Initially, SNVs were biochemically investigated in vitro using recombinant protein techniques to confirm a functional impact on CtHtrA activity. The SNVs resulting in marked reductions in proteolysis were then investigated in cell culture to identify specific phenotypes that could be linked to CtHtrA function. The P370L SNV displayed the most marked impact on in vitro proteolysis, and the C. trachomatis strain harbouring this SNV was detected to have a significant reduction in the production of infectious elementary bodies, an increased susceptibility to heat shock, and a reduced inclusion size. This data provides the first genetic evidence for CtHtrA’s role during the C. trachomatis developmental cycle, as well as during heat stress conditions, and represents the first step towards a genotype-phenotype characterisation of CtHtrA function in C. trachomatis.