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Downregulation of motility during stress conditions requires stressosome input in Listeria monocytogenes

ABSTRACT: Motility in Listeria monocytogenes is negatively regulated within the mammalian host in response to temperature through a complex interplay between positively acting and negatively acting regulators. Motility genes are expressed under saprophytic conditions where the temperature is 30ºC or less. Motility is costly on cellular resources due to the large molecular structures that need to be synthesized, and the proton motive force required to produce flagellar rotation. Under stressful environmental conditions there is a trade off between motility and energy conservation and bacteria typically repress motility when adverse conditions are encountered. Here we investigated the impact of the SigB-mediated general stress response on the regulation of motility in L. monocytogenes and sought to elucidate the regulatory steps involved. We show that an rsbX mutation that compromises the ability to inactivate the stressosome, the sensory hub at the top of the SigB activation pathway, results in motility repression. Escaping from this repressed state occurred through the acquisition of mutations that decreased SigB activity. Flagellar expression was abolished in these mutants and a transcriptomic analysis revealed that the entire flagellar operon is strongly repressed. The motility anti-repressor gene gmaR was also downregulated in an ΔrsbX background. These effects can be reversed by providing functional copies of rsbX or gmaR in trans. Deletion of an internal SigB dependent promoter that produces an antisense RNA for the large motility operon (including gmaR) restores the ability to produce flagellin in the ΔrsbX background. A similar phenotype is observed by deleting MogR, which acts as a negative regulator of flagella motility. Stressosome mutations that negatively affect SigB activity result in derepressed motility whereas those that increase SigB activity result in a decreased motility phenotype similar to the ΔrsbX strain. Overall, the data indicate that stress sensing via the stressosome negatively impacts motility in L. monocytogenes and shows that the general stress response can take priority over motility when L. monocytogenes encounters harsh environmental conditions.

ORGANISM(S): Listeria monocytogenes

PROVIDER: GSE310143 | GEO | 2025/11/24

REPOSITORIES: GEO

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Project description:Background: The Bacillus cereus Sigma B (SigB) dependent general stress response is activated via the two-component RsbKY system, which involves a phosphate transfer from RsbK to RsbY. It has been hypothesized that the Hpr-like phosphocarrier protein (Bc1009) encoded by bc1009 in the SigB gene cluster may play a role in this transfer, thereby acting as a regulator of SigB activation. Alternatively, Bc1009 may be involved in the activation of a subset of SigB regulon members. Results: We first investigated the potential role of bc1009 to act as a SigB regulator but ruled out this possibility as the deletion of bc1009 did not affect the expression of sigB and other SigB gene cluster members. The SigB-dependent functions of Bc1009 were further examined in B. cereus ATCC14579 via comparative proteome profiling (backed up by transcriptomics) of wt, Δbc1009 and ΔsigB deletion mutants under heat stress at 42°C. This revealed 284 proteins displaying SigB-dependent alterations in protein expression levels in heat-stressed cells, including a subgroup of 138 proteins for which alterations were also Bc1009-dependent. Next to proteins with roles in stress defense, newly identified SigB and Bc1009-dependent proteins have roles in cell motility, signal transduction, transcription, cell wall biogenesis, and amino acid transport and metabolism. Analysis of lethal stress survival at 50°C after pre-adaptation at 42°C showed intermediate survival efficacy of Δbc1009 cells, highest survival of wt, and lowest survival of ΔsigB cells, respectively. Additional comparative proteome analysis of non-stressed wt and mutant cells at 30°C revealed 96 proteins with SigB and Bc1009-dependent differences in levels, 51 were also identified under heat stress, and 45 showed significant differential expression at 30°C, including proteins with roles in carbohydrate/ion transport and metabolism. Overlapping functions at 30°C and 42°C included proteins involved in motility, and ΔsigB and Δbc1009 cells showed reduced motility compared to wt cells in swimming assays at both temperatures. Conclusion: our results extend the B. cereus SigB regulon to >300 members, with a novel role of SigB-dependent Bc1009 in the activation of a subregulon of >180 members, conceivably via phosphotransfer-based interactions with other transcriptional regulatory networks.

Project description:Listeria monocytogenes is well known to have the ability to survive and grow under a variety of stress conditions. The ability to survive and grow under osmotic stress conditions in particular appears to be important for both growth in certain foods and food associated environments as well as for host infection. To characterize the contributions of transcriptional regulators important for stress response and virulence (i.e., Sigma B - M-OM-^CB and PrfA), we initially analyzed three L. monocytogenes parent strains and isogenic mutants ( delta sigB, delta prfA, and delta sigB delta prfA), representing different serotypes and lineages, for their ability to grow in BHI with 11% NaCl (1.9M) at 25M-BM-0C. No significant differences were observed in terms of growth between the parent strains and their respective mutants lacking prfA (i.e. delta prfA, and delta sigB delta prfA mutant strains). While for all strains, the delta sigB mutant showed a prolonged lag phase as compared to the parent strains, maximum growth rates were only reduced for the delta sigB mutant of lineage I and IV strains. Interestingly, for the serotype 1/2b strain, the delta sigB mutant reached a higher maximum cell density than the parent strain or the delta prfA mutant. Caco-2 intestinal epithelial cells invasion assays and hemolytic activity assays showed a significant role for M-OM-^CB in the former and for PrfA in the latter. To initially explore the mechanism that may contribute to the extended lag phase in the delta sigB mutant, microarray was performed to compare transcript levels between the lineage I, serotype 1/2b, parent strain and its isogenic delta sigB mutant in lag phase at 25M-BM-0C in the presence of 11% NaCl. Microarray data showed significant lower and higher transcript levels for 135 and 173 genes, respectively, in the parent strain as compared to the delta sigB strains. Overall, 51 of the 173 M-OM-^CB up-regulated genes had previously been found among the 249 M-OM-^CB-dependent genes identified in a microarray study conducted in stationary phase cells, indicating that up to 122 genes may be transcribed in a M-OM-^CB-dependent manner during lag phase under salt stress. Notable genes that showed higher transcript levels in the parent strain include inlD (encoding an internalin protein that may be involved in virulence), sigH (encoding an alternative M-OM-^C factor), glpK (encoding a glycerol kinase) and resD (encoding a two-component response regulator ResD); while, genes that showed lower transcript levels in the parent strain include dnaK (encoding a dihydroxyacetone kinase), groES (encoding a class I heat-shock protein GroES), grpE (encoding a co-chaperone GrpE) and fri (encoding a non-heme iron-binding ferritin). These data showed that although PrfA does not contribute to growth under osmotic stress at 25M-BM-0C, M-OM-^CB does contribute to survival of L. monocytogenes under high salt conditions. Moreover, the M-OM-^CB-dependent transcriptome of L. monocytogenes lag phase cells under salt stress was characterized and includes previously identified as well as novel M-OM-^CB-dependent genes, including a number of stress response and virulence-associated genes. Independent RNA isolations were performed for one wildtype (lineage I) and M-NM-^TsigB strains from cells grown to lag phase. Three biological replicates were used in competitive whole-genome microarray experiments. For each set of hybridizations, RNA from a L. monocytogenes wildtype strain was hybridized with RNA from its isogenic M-NM-^TsigB null mutant.

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Downregulation of motility during stress conditions requires stressosome input in Listeria monocytogenes

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