Mo, Elaine; (2002) The HtrA Family of Proteins in Salmonella typhimurium and staphylococcus aureus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

High temperature requirement A (HtrA) has been identified in many Gram-negative bacteria to be important for bacterial survival during stress conditions, such as heat-shock and oxidative stress. HtrA has been reported to degrade misfolded proteins in the periplasm. Substrate recognition is proposed to be carried out by C-terminal PDZ domains. In Escherichia coli, a family of HtrA homologues have been identified. HhoA has a similar domain structure to HtrA, with two PDZ domains, whereas HhoB is a smaller protein and has a single PDZ domain. This study reports the analysis of the HtrA family in the Gram-negative bacterium, S. typhimurium, and identifies a family of HtrA-like homologues in the Gram-positive bacterium, S. aureus. Three S. aureus HtrA orthologues were identified and named SAHtrA1, SAHtrA2 and SAHtrA3. SAHtrA3 did not have a PDZ domain, thus, this study examines SAHtrA1 and SAHtrA2, both of which have a single PDZ domain. Northern blot analysis showed that the transcription of sahtrA2 was heat-inducible at 42degree C whereas transcription of sahtrA1 was constitutively expressed. Primer extension experiments predicted that sahtrA1 had promoter sites similar to that seen in the vegetative a factor, In addition, the PDZ domain of SAHtrA2 was constructed as a recombinant protein and shown to bind the S. aureus SsrA-tag, suggesting that this domain was involved in protein:protein interactions. The S. typhimurium HtrA homologues, named STHtrA, STHhoA and STHhoB, exhibited a high level of identity to their E. coli counterparts. Mutants of the htrA gene family were constructed in S. typhimurium. In vitro heat-shock studies suggest that htrA and hhoB are important in bacterial viability at 42 degree C. The hhoB gene also appeared to be important for growth when bacteria were exposed to hydrogen peroxide. Interestingly, under the in vitro stress conditions, mutating the hhoA gene appeared to have little effect on bacterial viability while mutating both the htrA and hhoB genes together severely affected bacterial viability and growth. Bacterial virulence was investigated using a mouse model. These in vivo experiments showed that in common with the in vitro data, htrA and hhoB mutants were attenuated, whereas mutations in hhoA had little affect on bacterial survival. To further characterise the function of the HtrA homologues, recombinant proteins were generated and STHhoA was shown to be proteolytically active.

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