Abstract
BACKGROUND: High temperature requirement A (HtrA) is a widely expressed chaperone and serine protease in bacteria. HtrA proteases assemble and hydrolyze misfolded proteins to enhance bacterial survival under stress conditions. Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen that induces listeriosis in humans. In previous studies, it was shown that deletion of htrA in the genome of L. monocytogenes increased the susceptibility to cellular stress and attenuated virulence. However, expression and protease activity of listerial HtrA (LmHtrA) were never analyzed in detail. RESULTS: In this study, we cloned LmHtrA wildtype (LmHtrA(wt)) and generated a proteolytic inactive LmHtrA(SA) mutant. Recombinant LmHtrA(wt) and LmHtrA(SA) were purified and the proteolytic activity was analyzed in casein zymography and in vitro cleavage assays. LmHtrA activity could be efficiently blocked by a small molecule inhibitor targeting bacterial HtrA proteases. The expression of LmHtrA was enhanced in the stationary growth phase of L. monocytogenes and significantly contributed to bacterial survival at high temperatures. CONCLUSIONS: Our data show that LmHtrA is a highly active caseinolytic protease and provide a deeper insight into the function and mechanism, which could lead to medical and biotechnological applications in the future.