Role for HtrA in Stress Induction and Virulence Potential in Listeria monocytogenes

Abstract

In silico analysis of the Listeria monocytogenes genome revealed lmo0292, a gene predicted to encode a HtrA-like serine protease. A stable insertion mutant was constructed, revealing a requirement for htrA in the listerial response to heat, acid, and penicillin stress. Transcriptional analysis revealed that htrA is not induced in response to heat shock but is induced in response to low pH and penicillin G stress. Furthermore, htrA expression was shown to be dependent upon the LisRK two-component sensor-kinase, a system known to respond to changes in integrity of the cell envelope. In addition, we demonstrated that a second in-frame start codon, upstream of that previously annotated for L. monocytogenes htrA, incorporating a putative signal sequence appears to influence virulence potential. Finally, a significant virulence defect was observed for the htrA mutant, indicating that this gene is required for full virulence in mice. Our findings suggest that L. monocytogenes lmo0292 encodes an HtrA-like serine protease that is not part of the classical heat shock response but is involved in stress responses and virulence.L. monocytogenes is a food-borne pathogen with an ability to sense and appropriately respond to hostile changes in its environment, an adaptive response, which is pivotal to mounting a successful infection. To overcome stresses encountered in food and during infection, L. monocytogenes has evolved elaborate systems for sensing and responding to a variety of adverse environments (2,9,15,28).The publication of the complete chromosomal sequence of L. monocytogenes EGDe (11) facilitated significant advancements in the identification and characterization of loci which potentially play important roles in listerial growth and survival in foods and during infection, one such locus is lmo0292 (encoding an HtrA-like homologue). Initially characterized in Escherichia coli, HtrA is one of several proteins, collectively known as heat shock proteins, whose expression is essential for survival of bacteria at high temperatures (17). In addition, htrA has been shown to be essential for the pathogenicity of several gram-negative and gram-positive bacteria, namely, Salmonella enterica serovar Typhimurium (13), Klebsiella pneumoniae (3), Streptococcus pyogenes (14), and Streptococcus pneumoniae (12), as well as the antibiotic stress response in Lactococcus lactis (7) and Staphylococcus aureus (30).Three HtrA homologues-HtrA, YvtA, and YycK-are encoded in Bacillus subtilis (22,23). In silico analysis revealed that the immediate genomic organization of the region encoding the HtrA-like serine protease in L. monocytogenes corresponds to the B. subtilis six-gene operon (yycF-yycK). The B. subtilis yycF gene and its ortholog in S. aureus encode a response regulator that is essential for cell growth (6, 21). Fukuchi et al. (8) showed that the two-component system encoded by yycF and yycG is essential and has the potential to modulate expression of the cell division operon ftsAZ in B. subtilis. Attempts to disrupt the correspo...

Genome-wide transcriptional profiling of the cell envelope stress response and the role of LisRK and CesRK in Listeria monocytogenes

“…Interestingly, the genes encoding the LisRK two-component system were found to be induced 4.17-fold (lisR) and 3.93-fold (lisK), respectively (Supplementary Table S2). In addition, four genes previously shown to be affected by LisRK (htrA, lmo2210, lmo2229 and lmo2487) were highly upregulated in response to cefuroxime exposure (Supplementary Table S2) (Cotter et al, 2002;Gottschalk et al, 2008;Stack et al, 2005). Our transcriptional analysis also showed that the CesRK-regulated genes lmo0443, lmo1416, lmo2420 and lmo2812 were highly upregulated by cefuroxime exposure (Supplementary Table S2) (Gottschalk et al, 2008;Kallipolitis et al, 2003).…”

“…Thus, the cell envelope stress response of L. monocytogenes appears to be activated at multiple stages of the infectious process. In accordance with this, LisRK, CesRK and VirRS have been shown to contribute to pathogenesis in mice, and several of the genes shown to be highly upregulated in response to cefuroxime exposure play important roles in virulence, including dacA, lmo2714, htrA and the dlt-operon (Abachin et al, 2002;Camejo et al, 2009;Stack et al, 2005;Wilson et al, 2006;Woodward et al, 2010).…”

“…Furthermore, the expression of the htrA gene (lmo0292) encoding the serine protease HtrA, was induced 18-fold by cefuroxime exposure, corresponding to the third most upregulated gene in this study (Supplementary Table S2). HtrA is important for growth under stress conditions and contributes to virulence in L. monocytogenes (Stack et al, 2005;Wilson et al, 2006;Wonderling et al, 2004).…”

“…Furthermore, a lisK deletion mutant displays a growth-phase-dependent response to acidic conditions and is less tolerant to osmotic stress but more resistant to the lantibiotic nisin (Cotter et al, 1999(Cotter et al, , 2002. LisRK has been shown to be a positive regulator of five genes, including htrA (lmo0292), encoding the serine protease HtrA, lmo1021, encoding the HK of the TCS LiaSR, and lmo2229, encoding the penicillin-binding protein PBPA2 (Cotter et al, 2002;Gottschalk et al, 2008;Stack et al, 2005). Transcription of the LisR-regulated gene lmo2210, encoding a protein of unknown function, is induced by subinhibitory concentrations of ethanol and the cephalosporin cefuroxime (Gottschalk et al, 2008).…”

The heat-shock response of Listeria monocytogenes comprises genes involved in heat shock, cell division, cell wall synthesis, and the SOS response

“…In L. monocytogenes, nine orthologues of these genes showed induction, including htrA. Other research has shown that HtrA is important for survival during exposure to environmental and cellular stresses (Stack et al, 2005;Wilson et al, 2006).…”