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The proteolytic activity of Listeria monocytogenes HtrA.

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 (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.

SUBMITTER: Abfalter CM

PROVIDER: S-EPMC6857308 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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The proteolytic activity of Listeria monocytogenes HtrA.

Abfalter Carmen M CM Bernegger Sabine S Jarzab Miroslaw M Posselt Gernot G Ponnuraj Karthe K Wessler Silja S

BMC microbiology 20191114 1

<h4>Background</h4>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. ...[more]

PMID: 31726993

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Project description:Listeria monocytogenes is able to efficiently utilize glycerol as a carbon source. In a defined minimal medium, the growth rate (during balanced growth) in the presence of glycerol is similar to that in the presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed high-level transcriptional upregulation of the genes known to be involved in glycerol uptake and metabolism (glpFK and glpD) in the presence of glycerol (compared to that in the presence of glucose and/or cellobiose). Levels of expression of the genes encoding a second putative glycerol uptake facilitator (GlpF(2)) and a second putative glycerol kinase (GlpK(2)) were less enhanced under these conditions. GlpK(1) but not GlpK(2) was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while the loss of GlpK(1) affected replication in Caco-2 cells less than did the loss of GlpK(2) and GlpD. Additional genes whose transcription levels were higher in the presence of glycerol than in the presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression control. Transcriptional downregulation in the presence of glycerol (compared to those in the presence glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N metabolism, and the biosynthesis of branched-chain amino acids. The highest level of transcriptional upregulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions, a low level of HPr-Ser-P and a high level of HPr-His-P were present in the cells, suggesting that all enzyme IIA (EIIA) (or EIIB) components of the phosphotransferase system (PTS) permeases expressed will be phosphorylated. These and other data suggest that the phosphorylation state of PTS permeases correlates with PrfA activity.

Project description:Listeria monocytogenes is able to efficiently utilize glycerol as carbon source. In a defined minimal medium the growth rate is similar (during balanced growth) in presence of glycerol as in presence of glucose or cellobiose. Comparative transcriptome analyses of L. monocytogenes showed in the presence of glycerol (compared to glucose and/or cellobiose) high transcriptional upregulation of the known genes involved in glycerol uptake and metabolism (glpFK, glpD). Expression of the genes encoding a second putative glycerol uptake facilitator (GlpF-2) and a second putative glycerol kinase (GlpK-2) was less enhanced under these conditions. GlpK-1 but not GlpK-2 was essential for glycerol catabolism in L. monocytogenes under extracellular conditions, while loss of GlpK-1 affected replication in Caco-2 cells less than loss of GlpK-2 and GlpD. Additional genes whose transcription was higher in presence of glycerol than in presence of glucose and cellobiose included those for two dihydroxyacetone (Dha) kinases and many genes that are under carbon catabolite repression (CCR) control. Transcriptional down-regulation in the presence of glycerol (compared to glucose and cellobiose) was observed for several genes and operons that are positively regulated by glucose, including genes involved in glycolysis, N-metabolism and biosynthesis of branched chain amino acids. The highest transcriptional up-regulation was observed for all PrfA-dependent genes during early and late logarithmic growth in glycerol. Under these conditions a low level of HPr-Ser-P and a high level of HPr-His-P was present in the cells, suggesting that all EIIA (B) components of the PTS permeases expressed will be phosphorylated. These and other data reported suggest that the phosphorylation state of PTS permeases correlates with PrfA activity. Keywords: Response of Listeria monocytogenes to different carbon sources A total of four independently isolated RNA samples from each condition at each growth phase were used for the analysis. RNA from two isolations were pooled and hybridized onto two microarray slides with dye swap. Another two microarray slides were hybridized using the same principle. In total, we used four RNAs and four microarray slides to generate 16 replicate expression values for each combination except for the comparison between glucose and cellobiose, phase B where data generated from three microarray slides were used for further analysis

Dataset Information

The proteolytic activity of Listeria monocytogenes HtrA.

Publications

The proteolytic activity of Listeria monocytogenes HtrA.

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