RsbT and RsbV Contribute to σ^B-Dependent Survival under Environmental, Energy, and Intracellular Stress Conditions inListeria monocytogenes

Abstract

Sigma B (B ) is a stress-responsive alternative sigma factor that has been identified in various gram-positive bacteria. Seven different regulators of sigma B (Rsbs) are located in the sigB operons of both Bacillus subtilis and Listeria monocytogenes. In B. subtilis, these proteins contribute to regulation of B activity by conveying environmental and energy stress signals through two well-established branches of a signal transduction pathway. RsbT contributes to regulation of B activity in response to environmental stresses, while RsbV contributes to B activation under both environmental and energy stresses in B. subtilis. To probe L. monocytogenes Rsb roles in B -mediated responses to various stresses, in-frame deletions were created in rsbT and rsbV. Phenotypic characterization of the L. monocytogenes rsbT and rsbV null mutants revealed that both mutants were similar to the ⌬sigB strain in their abilities to survive under environmental stress conditions (exposure to synthetic gastric fluid, pH 2.5, acidified brain heart infusion broth [BHI], or oxidative stress [13 mM cumene hydroperoxide]). Under energy stress conditions (carbon starvation in defined media, entry into stationary phase, or reduced intracellular ATP), both ⌬rsbT and ⌬rsbV showed survival reductions similar to that of the ⌬sigB strain. These observations suggest that the pathways for Rsb-dependent regulation of B activity differ between L. monocytogenes and B. subtilis. As B also activates transcription of the L. monocytogenes prfAP2 promoter, we evaluated virulence-associated characteristics of ⌬prfAP1rsbT and ⌬prfAP1rsbV double mutants in hemolysis and tissue culture assays. Both double mutants showed identical phenotypes to ⌬prfAP1P2 and ⌬prfAP1sigB double mutants, i.e., reduced hemolysis activity and reduced plaque size in mouse fibroblast cells. These findings indicate that RsbT and RsbV both contribute to B activation in L. monocytogenes during exposure to environmental and energy stresses as well as during tissue culture infection.Listeria monocytogenes, a gram-positive, non-spore-forming rod-shaped bacterium, is recognized as a foodborne pathogen. This organism is capable of surviving in a broad range of ecological niches (e.g., in farm environments and food processing plants) and in a wide range of hosts, including humans and many species of animals. In L. monocytogenes, the alternative sigma factor B ( B ) contributes to survival under stressful environmental conditions, such as exposure to low pH, oxidizing conditions, and starvation (17, 18). Loss of B also reduces L. monocytogenes virulence in a murine model (32, 44).The sigB gene, which encodes B , lies seventh in the sigB operon. This operon also includes seven additional genes, which encode the following regulator of sigma B proteins: RsbR, RsbS, RsbT, RsbU, RsbV, RsbW, and RsbX (4,19,25,44,45). In B. subtilis, activation of B by the Rsb proteins is achieved through a complex phosphorylation/dephosphorylation cascade in response to various cellular stimuli, which have been catego...

Contributions of Listeria monocytogenes σ B and PrfA to expression of virulence and stress response genes during extra- and intracellular growth

“…While these results may seem surprising, considering the well-documented occurrence in the vacuole of the types of stressful conditions that have been shown to activate s B or to affect survival of a DsigB mutant (e.g. low pH, oxidative stress) (Chaturongakul & Boor, 2004;Ferreira et al, 2001;Maul et al, 1995;Sue et al, 2004), virulence gene expression has been shown to differ between L. monocytogenes present in professional and nonprofessional phagocytes (Bubert et al, 1999). Hence, it is possible that the intravacuolar environment of professional phagocytes is a more stressful environment for intracellular bacterial pathogens than that in other cell types, which may Table 2) detects transcripts covering the plcA coding region; probe 2 ('plcA-readthrough') detects plcA readthrough transcription; probe 3 ('prfA-P1') detects P1prfA transcript including plcA readthrough transcripts; probe 4 ('prfA-CDS') detects all prfA coding region transcripts.…”

“…haemolysin production (Freitag & Portnoy, 1994;Nadon et al, 2002), it does not affect L. monocytogenes invasion capabilities in human intestinal epithelial cell lines (Kim et al, 2004(Kim et al, , 2005. In L. monocytogenes, s B also contributes to survival under a variety of lethal environmental stresses (Ferreira et al, 2001), including acidic conditions mimicking mammalian gastric fluid (Chaturongakul & Boor, 2004;Cotter et al, 2001;Wiedmann et al, 1998).…”

Development and optimization of an EGFP-based reporter for measuring the general stress response inListeria monocytogenes

“…13 Indeed there is genetic data from L. monocytogenes 10403S to support this model; rsbV and sigB mutants are found to have essentially the same phenotype, suggesting that RsbV is required for s B activation. 46 This highlights the potential use of the P lmo2230 ::egfp reporter in conducting studies on the regulation of s B . A key question that remains to be answered concerns the nature of the stress sensing mechanism.…”

Refinement of the Listeria monocytogenes σB regulon through quantitative proteomic analysis

“…The contributions of the s B regulon to energy metabolism are also supported by phenotypic data. For example, under energy stress in glucose-limiting media, a L. monocytogenes DsigB mutant was shown to grow more rapidly than the parent strain, while the parent strain displayed greater survival after the carbon source was depleted (Chaturongakul & Boor, 2004). In addition to 30 genes that grouped into the 'energy metabolism' category, the 149 s B -dependent genes and proteins identified here also represented 18 other role categories, including transport and binding proteins (22 genes) and cellular processes (14 genes) (Table S3), illustrating the extensive network of cellular functions regulated by s B .…”