The DegU Orphan Response Regulator Contributes to Heat Stress Resistance in Listeria monocytogenes.
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ABSTRACT:
SUBMITTER: Cheng C
PROVIDER: S-EPMC8711649 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Listeria monocytogenes is a pathogenic bacterium that produces flagella, the locomotory organelles, in a temperature-dependent manner. At 37 °C inside humans, L. monocytogenes employs MogR to repress the expression of flagellar proteins, thereby preventing the production of flagella. However, in the low-temperature environment outside of the host, the antirepressor GmaR inactivates MogR, allowing flagellar formation. Additionally, DegU is necessary for flagellar expression at low temperatures. DegU transcriptionally activates the expression of GmaR and flagellar proteins by binding the operator DNA in the fliN-gmaR promoter as a response regulator of a two-component regulatory system. To determine the DegU-mediated regulation mechanism, we performed structural and biochemical analyses on the recognition of operator DNA by DegU. The DegU-DNA interaction is primarily mediated by a C-terminal DNA-binding domain (DBD) and can be fortified by an N-terminal receiver domain (RD). The DegU DBD adopts a tetrahelical helix-turn-helix structure and assembles into a dimer. The DegU DBD dimer recognizes the operator DNA using a positive patch. Unexpectedly, unlike typical response regulators, DegU interacts with operator DNA in both unphosphorylated and phosphorylated states with similar binding affinities. Therefore, we conclude that DegU is a noncanonical response regulator that is constitutively active irrespective of phosphorylation.
| S-EPMC9263151 | biostudies-literature
Project description:Listeria monocytogenes is one of the most heat-resistant non-spore-forming food-borne pathogens and poses a notable risk to food safety, particularly when mild heat treatments are used in food processing and preparation. While general heat stress properties and response mechanisms of L. monocytogenes have been described, accessory mechanisms providing particular L. monocytogenes strains with the advantage of enhanced heat resistance are unknown. Here, we report plasmid-mediated heat resistance of L. monocytogenes for the first time. This resistance is mediated by the ATP-dependent protease ClpL. We tested the survival of two wild-type L. monocytogenes strains-both of serotype 1/2c, sequence type ST9, and high sequence identity-at high temperatures and compared their genome composition in order to identify genetic mechanisms involved in their heat survival phenotype. L. monocytogenes AT3E was more heat resistant (0.0 CFU/ml log10 reduction) than strain AL4E (1.4 CFU/ml log10 reduction) after heating at 55°C for 40 min. A prominent difference in the genome compositions of the two strains was a 58-kb plasmid (pLM58) harbored by the heat-resistant AT3E strain, suggesting plasmid-mediated heat resistance. Indeed, plasmid curing resulted in significantly decreased heat resistance (1.1 CFU/ml log10 reduction) at 55°C. pLM58 harbored a 2,115-bp open reading frame annotated as an ATP-dependent protease (ClpL)-encoding clpL gene. Introducing the clpL gene into a natively heat-sensitive L. monocytogenes strain (1.2 CFU/ml log10 reduction) significantly increased the heat resistance of the recipient strain (0.4 CFU/ml log10 reduction) at 55°C. Plasmid-borne ClpL is thus a potential predictor of elevated heat resistance in L. monocytogenes. IMPORTANCEListeria monocytogenes is a dangerous food pathogen causing the severe illness listeriosis that has a high mortality rate in immunocompromised individuals. Although destroyed by pasteurization, L. monocytogenes is among the most heat-resistant non-spore-forming bacteria. This poses a risk to food safety, as listeriosis is commonly associated with ready-to-eat foods that are consumed without thorough heating. However, L. monocytogenes strains differ in their ability to survive high temperatures, and comprehensive understanding of the genetic mechanisms underlying these differences is still limited. Whole-genome-sequence analysis and phenotypic characterization allowed us to identify a novel plasmid, designated pLM58, and a plasmid-borne ATP-dependent protease (ClpL), which mediated heat resistance in L. monocytogenes. As the first report on plasmid-mediated heat resistance in L. monocytogenes, our study sheds light on the accessory genetic mechanisms rendering certain L. monocytogenes strains particularly capable of surviving high temperatures-with plasmid-borne ClpL being a potential predictor of elevated heat resistance.
| S-EPMC5663981 | biostudies-literature
Project description:While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.
| S-EPMC193959 | biostudies-other
Project description:To determine the contribution of sigma B (sigma(B)) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50 degrees C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45 degrees C, 5% ethanol, or pH 4.5). The DeltasigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the DeltasigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the DeltasigB strain. These results suggest the existence in L. monocytogenes of both a sigma(B)-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. sigma(B) contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The DeltasigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the DeltasigB strain lost viability more rapidly than the parent strain. sigma(B) contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that sigma(B) plays a role in protecting L. monocytogenes against environmental adversities.
| S-EPMC93189 | biostudies-literature
Project description:The formation of Listeria monocytogenes biofilms contributes to persistent contamination in food processing facilities. A microarray comparison of L. monocytogenes between the transcriptome of the strong biofilm forming strain (Bfm(s)) Scott A and the weak biofilm forming (Bfm(w)) strain F2365 was conducted to identify genes potentially involved in biofilm formation. Among 951 genes with significant difference in expression between the two strains, a GntR-family response regulator encoding gene (LMOf2365_0414), designated lbrA, was found to be highly expressed in Scott A relative to F2365. A Scott A lbrA-deletion mutant, designated AW3, formed biofilm to a much lesser extent as compared to the parent strain by a rapid attachment assay and scanning electron microscopy. Complementation with lbrA from Scott A restored the Bfm(s) phenotype in the AW3 derivative. A second microarray assessment using the lbrA deletion mutant AW3 and the wild type Scott A revealed a total of 304 genes with expression significantly different between the two strains, indicating the potential regulatory role of LbrA in L. monocytogenes. A cloned copy of Scott A lbrA was unable to confer enhanced biofilm forming potential in F2365, suggesting that additional factors contributed to weak biofilm formation by F2365.
| S-EPMC3720924 | biostudies-literature
Project description:Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.
| S-EPMC3457240 | biostudies-literature
Project description:Listeria monocytogenes is a foodborne pathogen that can cause severe disease (listeriosis) in susceptible individuals. It is ubiquitous in the environment and often exhibits resistance to heavy metals. One of the determinants that enables Listeria to tolerate exposure to cadmium is the cadAC efflux system, with CadA being a P-type ATPase. Three different cadA genes (designated cadA1 to cadA3) were previously characterized in L. monocytogenes A novel putative cadmium resistance gene (cadA4) was recently identified through whole-genome sequencing, but experimental confirmation for its involvement in cadmium resistance is lacking. In this study, we characterized cadA4 in L. monocytogenes strain F8027, a cadmium-resistant strain of serotype 4b. By screening a mariner-based transposon library of this strain, we identified a mutant with reduced tolerance to cadmium and that harbored a single transposon insertion in cadA4 The tolerance to cadmium was restored by genetic complementation with the cadmium resistance cassette (cadA4C), and enhanced cadmium tolerance was conferred to two unrelated cadmium-sensitive strains via heterologous complementation with cadA4C Cadmium exposure induced cadA4 expression, even at noninhibitory levels. Virulence assessments in the Galleria mellonella model suggested that a functional cadA4 suppressed virulence, potentially promoting commensal colonization of the insect larvae. Biofilm assays suggested that cadA4 inactivation reduced biofilm formation. These data not only confirm cadA4 as a novel cadmium resistance determinant in L. monocytogenes but also provide evidence for roles in virulence and biofilm formation.IMPORTANCEListeria monocytogenes is an intracellular foodborne pathogen causing the disease listeriosis, which is responsible for numerous hospitalizations and deaths every year. Among the adaptations that enable the survival of Listeria in the environment are the abilities to persist in biofilms, grow in the cold, and tolerate toxic compounds, such as heavy metals. Here, we characterized a novel determinant that was recently identified on a larger mobile genetic island through whole-genome sequencing. This gene (cadA4) was found to be responsible for cadmium detoxification and to be a divergent member of the Cad family of cadmium efflux pumps. Virulence assessments in a Galleria mellonella model suggested that cadA4 may suppress virulence. Additionally, cadA4 may be involved in the ability of Listeria to form biofilms. Beyond the role in cadmium detoxification, the involvement of cadA4 in other cellular functions potentially explains its retention and wide distribution in L. monocytogenes.
| S-EPMC5311389 | biostudies-literature
Project description:The sessile biofilms of Vibrio parahaemolyticus and Listeria monocytogenes have increasingly become a critical threat in seafood safety. This study aimed to evaluate the effects of modified atmospheres on the formation ability of V. parahaemolyticus and L. monocytogenes biofilms. The stress responses of bacterial biofilm formation to modified atmospheres including anaerobiosis (20% carbon dioxide, 80% nitrogen), micro-aerobiosis (20% oxygen, 80% nitrogen), and aerobiosis (60% oxygen, 40% nitrogen) were illuminated by determining the live cells, chemical composition analysis, textural parameter changes, expression of regulatory genes, etc. Results showed that the biofilm formation ability of V. parahaemolyticus was efficiently decreased, supported by the fact that the modified atmospheres significantly reduced the key chemical composition [extracellular DNA (eDNA) and extracellular proteins] of the extracellular polymeric substance (EPS) and negatively altered the textural parameters (biovolume, thickness, and bio-roughness) of biofilms during the physiological conversion from anaerobiosis to aerobiosis, while the modified atmosphere treatment increased the key chemical composition of EPS and the textural parameters of L. monocytogenes biofilms from anaerobiosis to aerobiosis. Meanwhile, the expression of biofilm formation genes (luxS, aphA, mshA, oxyR, and opaR), EPS production genes (cpsA, cpsC, and cpsR), and virulence genes (vopS, vopD1, vcrD1, vopP2?, and vcrD2?) of V. parahaemolyticus was downregulated. For the L. monocytogenes cells, the expression of biofilm formation genes (flgA, flgU, and degU), EPS production genes (Imo2554, Imo2504, inlA, rmlB), and virulence genes (vopS, vopD1, vcrD1, vopP2?, and vcrD2?) was upregulated during the physiological conversion. All these results indicated that the modified atmospheres possessed significantly different regulation on the biofilm formation of Gram-negative V. parahaemolyticus and Gram-positive L. monocytogenes, which will provide a novel insight to unlock the efficient control of Gram-negative and Gram-positive bacteria in modified-atmosphere packaged food.
| S-EPMC7044124 | biostudies-literature
Project description:Transcription of the Listeria monocytogenes positive regulatory factor A protein (PrfA) is initiated from either of two promoters immediately upstream of prfA (prfAp(1) and prfAp(2)) or from the upstream plcA promoter. We demonstrate that prfAp(2) is a functional sigma(B)-dependent promoter and that a sigB deletion mutation affects the virulence phenotype of L. monocytogenes. Thus, the alternative sigma factor sigma(B) contributes to virulence in L. monocytogenes.
| S-EPMC128067 | biostudies-literature
Project description:Listeria monocytogenes is a food-borne facultative intracellular pathogen. It is widespread in the environment and has several distinct life-styles. The key transcriptional activator PrfA positively regulates L. monocytogenes virulence genes to mediate the transition from extracellular, flagellum-propelled cell to intracellular pathogen. Here we report the first evidence that PrfA also has a significant positive impact on extracellular biofilm formation. Mutants lacking prfA were defective in surface-adhered biofilm formation. The DeltaprfA mutant exhibited wild-type flagellar motility, and its biofilm defect occurred after initial surface adhesion. We also observed that mutations that led to the constitutive expression of PrfA-dependent virulence genes had a minimal impact on biofilm formation. Furthermore, biofilm development was enhanced in a mutant encoding a PrfA protein variant unable to fully transition from the extracellular form to the virulent, intracellular activity conformation. These results indicate that PrfA positively regulates biofilm formation and suggest that PrfA has a global role in modulating the life-style of L. monocytogenes. The requirement of PrfA for optimal biofilm formation may provide selective pressure to maintain this critical virulence regulator when L. monocytogenes is outside host cells in the environment.
| S-EPMC2916369 | biostudies-literature