The proteolytic activity of Listeria monocytogenes HtrA.

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 (LmHtrAwt) and generated a proteolytic inactive LmHtrASA mutant. Recombinant LmHtrAwt and LmHtrASA 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.

HtrA-mediated E-cadherin cleavage is limited to DegP and DegQ homologs expressed by gram-negative pathogens.

BACKGROUND: The serine proteases HtrA/DegP secreted by the human gastrointestinal pathogens Helicobacter pylori (H. pylori) and Campylobacter jejuni (C. jejuni) cleave the mammalian cell adhesion protein E-cadherin to open intercellular adhesions. A wide range of bacteria also expresses the HtrA/DegP homologs DegQ and/or DegS, which significantly differ in structure and function. METHODS: E-cadherin shedding was investigated in infection experiments with the Gram-negative pathogens H. pylori, enteropathogenic Escherichia coli (EPEC), Salmonella enterica subsp. Enterica (S. Typhimurium), Yersinia enterocolitica (Y. enterocolitica), and Proteus mirabilis (P. mirabilis), which express different combinations of HtrAs. Annotated wild-type htrA/degP, degQ and degS genes were cloned and proteolytically inactive mutants were generated by a serine-to-alanine exchange in the active center. All HtrA variants were overexpressed and purified to compare their proteolytic activities in casein zymography and in vitro E-cadherin cleavage experiments. RESULTS: Infection of epithelial cells resulted in a strong E-cadherin ectodomain shedding as reflected by the loss of full length E-cadherin in whole cell lysates and formation of the soluble 90 kDa extracellular domain of E-cadherin (NTF) in the supernatants of infected cells. Importantly, comparing the caseinolytic and E-cadherin cleavage activities of HtrA/DegP, DegQ and DegS proteins revealed that DegP and DegQ homologs from H. pylori, S. Typhimurium, Y. enterocolitica, EPEC and P. mirabilis, but not activated DegS, cleaved E-cadherin as a substrate in vitro. CONCLUSIONS: These data indicate that E-cadherin cleavage is confined to HtrA/DegP and DegQ proteins representing an important prevalent step in bacterial pathogenesis.

Cloning, Purification and Characterization of the Collagenase ColA Expressed by Bacillus cereus ATCC 14579.

Bacterial collagenases differ considerably in their structure and functions. The collagenases ColH and ColG from Clostridium histolyticum and ColA expressed by Clostridium perfringens are well-characterized collagenases that cleave triple-helical collagen, which were therefore termed as ´true´ collagenases. ColA from Bacillus cereus (B. cereus) has been added to the collection of true collagenases. However, the molecular characteristics of B. cereus ColA are less understood. In this study, we identified ColA as a secreted true collagenase from B. cereus ATCC 14579, which is transcriptionally controlled by the regulon phospholipase C regulator (PlcR). B. cereus ATCC 14579 ColA was cloned to express recombinant wildtype ColA (ColAwt) and mutated to a proteolytically inactive (ColAE501A) version. Recombinant ColAwt was tested for gelatinolytic and collagenolytic activities and ColAE501A was used for the production of a polyclonal anti-ColA antibody. Comparison of ColAwt activity with homologous proteases in additional strains of B. cereus sensu lato (B. cereus s.l.) and related clostridial collagenases revealed that B. cereus ATCC 14579 ColA is a highly active peptidolytic and collagenolytic protease. These findings could lead to a deeper insight into the function and mechanism of bacterial collagenases which are used in medical and biotechnological applications.

Proteolytic Activities Expressed by Gastrointestinal Pathogens Bacillus cereus, Listeria monocytogenes and Enterococcus faecium in Different Growth Phases.

AIMS: Bacterial proteases are implicated in protein quality control, biofilm formation or might have a direct function in pathogenesis by processing virulence factors or cleaving host factors. In recent years, knowledge of proteases expressed by Gram-negative pathogens remarkably increased. However, investigation of proteases from Gram-positive bacteria is rather rare, but required for the analysis of pathogenesis-relevant proteases. In this study, we extracted and detected proteases from the gastrointestinal pathogens Bacillus cereus, Listeria monocytogenes, and Enterococcus faecium in different growth phases. METHODOLOGY: Bacteria were grown to logarithmic or stationary phases, harvested and extracted by sonication and French press. For the detection of active proteases, zymography analyses were performed using casein and gelatin as substrates to monitor caseinolytic and gelatinolytic activities. RESULTS: We observed different active proteases with different intensities in bacteria grown to logarithmic or stationary phases. Strong activities as gelatinases were detected in B. cereus and distinct caseinolytic proteases exhibiting molecular weights of > 170 kDa, 70 kDa and 45 kDa were shown in L. monocytogenes and E. faecium, respectively. Interestingly, detected proteases were differentially regulated in bacteria grown to logarithmic or stationary phases. CONCLUSION: In summary, the data clearly indicated proteases that are differentially regulated in the Gram-positive pathogens B. cereus, L. monocytogenes, and E. faecium, which might contribute to bacterial pathogenesis.