scholarly journals RsbT and RsbV Contribute to σB-Dependent Survival under Environmental, Energy, and Intracellular Stress Conditions in Listeria monocytogenes

2004 ◽  
Vol 70 (9) ◽  
pp. 5349-5356 ◽  
Author(s):  
Soraya Chaturongakul ◽  
Kathryn J. Boor
Keyword(s):  
Tissue Culture ◽  
Listeria Monocytogenes ◽  
Cumene Hydroperoxide ◽  
Signal Transduction Pathway ◽  
Brain Heart Infusion ◽  
Mouse Fibroblast ◽  
Stress Conditions ◽  
Phenotypic Characterization ◽  
Energy Stress ◽  
Sigma B

ABSTRACT Sigma B (σB) 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 σB activity by conveying environmental and energy stress signals through two well-established branches of a signal transduction pathway. RsbT contributes to regulation of σB activity in response to environmental stresses, while RsbV contributes to σB activation under both environmental and energy stresses in B. subtilis. To probe L. monocytogenes Rsb roles in σB-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 ΔsigB 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 ΔrsbT and ΔrsbV showed survival reductions similar to that of the ΔsigB strain. These observations suggest that the pathways for Rsb-dependent regulation of σB activity differ between L. monocytogenes and B. subtilis. As σB also activates transcription of the L. monocytogenes prfAP2 promoter, we evaluated virulence-associated characteristics of ΔprfAP1rsbT and ΔprfAP1rsbV double mutants in hemolysis and tissue culture assays. Both double mutants showed identical phenotypes to ΔprfAP1P2 and ΔprfAP1sigB 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 σB activation in L. monocytogenes during exposure to environmental and energy stresses as well as during tissue culture infection.

scholarly journals σB Activation under Environmental and Energy Stress Conditions in Listeria monocytogenes

2006 ◽  
Vol 72 (8) ◽  
pp. 5197-5203 ◽  
Author(s):  
Soraya Chaturongakul ◽  
Kathryn J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Cumene Hydroperoxide ◽  
Stress Conditions ◽  
Wild Type ◽  
Regulation Of Expression ◽  
Transcript Levels ◽  
Energy Stress ◽  
Expression Studies ◽  
Genes Encoding ◽  
Gene Expression Studies

ABSTRACT To measure σB activation in Listeria monocytogenes under environmental or energy stress conditions, quantitative reverse transcriptase PCR (TaqMan) was used to determine the levels of transcripts for the σB-dependent opuCA and clpC genes in strains having null mutations in genes encoding regulator of sigma B proteins (rsbT and rsbV) and sigma B (sigB) and in the L. monocytogenes wild-type 10403S strain under different stress conditions. The ΔsigB, ΔrsbT, and ΔrsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for hly were also determined. RsbT, RsbV, and σB were all required for opuCA expression during growth under carbon-limiting conditions or following exposure to pH 4.5, salt, ethanol, or the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of clpC was RsbT, RsbV, and σB dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or σB dependent in the presence of either CCCP or salt. opuCA transcript levels did not increase in the presence of rapidly lethal stresses (i.e., pH 2.5 or 13 mM cumene hydroperoxide) despite the enhanced survival of the wild type compared with the survival of the mutant strains under these conditions. These findings highlight the importance of complementing phenotypic characterizations with gene expression studies to identify direct and indirect effects of null mutations in regulatory genes, such as sigB. Overall, our data show that while σB activation occurs through a single pathway under both environmental and energy stress conditions, regulation of expression of some stress response and virulence genes in the σB regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators.

scholarly journals Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance

2008 ◽  
Vol 74 (22) ◽  
pp. 6848-6858 ◽  
Author(s):  
F. Abram ◽  
E. Starr ◽  
K. A. G. Karatzas ◽  
K. Matlawska-Wasowska ◽  
A. Boyd ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Stress Tolerance ◽  
Intact Cell ◽  
Microscopic Analysis ◽  
Phenotypic Characterization ◽  
Carbon Utilization ◽  
Two Dimensional Gel Electrophoresis ◽  
Sigma B ◽  
The Impact

ABSTRACT Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

scholarly journals Biofilm Formation among Clinical and Food Isolates ofListeria monocytogenes

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Joana Barbosa ◽  
Sandra Borges ◽  
Ruth Camilo ◽  
Rui Magalhães ◽  
Vânia Ferreira ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Listeria Monocytogenes ◽  
Osmotic Stress ◽  
Environmental Conditions ◽  
Biofilm Formation ◽  
Stress Conditions ◽  
Acidic Stress ◽  
Biofilm Production ◽  
Oflisteria Monocytogenes ◽  
Clinical Cases

Objective. A total of 725Listeria monocytogenesisolates, 607 from various foods and 118 from clinical cases of listeriosis, were investigated concerning their ability to form biofilms, at 4°C during 5 days and at 37°C during 24 h.Methods. Biofilm production was carried out on polystyrene tissue culture plates. FiveL. monocytogenesisolates were tested for biofilm formation after being exposed to acidic and osmotic stress conditions.Results. Significant differences (P<0.01) between clinical and food isolates were observed. At 37°C for 24 h, most food isolates were classified as weak or moderate biofilm formers whereas all the clinical isolates were biofilm producers, although the majority were weak. At 4°C during 5 days, 65 and 59% isolates, from food and clinical cases, respectively, were classified as weak. After both sublethal stresses, at 37°C just one of the five isolates tested was shown to be more sensitive to subsequent acidic exposure. However, at 4°C both stresses did not confer either sensitivity or resistance.Conclusions. Significant differences between isolates origin, temperature, and sublethal acidic stress were observed concerning the ability to form biofilms. Strain, origin, and environmental conditions can determine the level of biofilm production byL. monocytogenesisolates.

scholarly journals Role of hemolysin for the intracellular growth of Listeria monocytogenes.

1988 ◽  
Vol 167 (4) ◽  
pp. 1459-1471 ◽  
Author(s):  
D A Portnoy ◽  
P S Jacks ◽  
D J Hinrichs
Keyword(s):  
Tissue Culture ◽  
Listeria Monocytogenes ◽  
Cell Line ◽  
Mouse Fibroblast ◽  
Epithelial Cell Line ◽  
Mouse Bone Marrow ◽  
Intracellular Growth ◽  
Embryonic Mouse ◽  
Culture Cells ◽  
Tissue Culture Cells

Listeria monocytogenes insertion mutants defective in hemolysin production were generated using the conjugative transposons Tn916 and Tn1545. All of the nonhemolytic mutants (hly-) lacked a secreted 58-kD polypeptide, presumedly hemolysin, and were avirulent in a mouse model. An intracellular multiplication assay was established in monolayers of mouse bone marrow-derived macrophages, the J774 macrophage-like cell line, the CL.7 embryonic mouse fibroblast cell line, and the Henle 407 human epithelial cell line. The hly+ strain grew intracellularly in all of the tissue culture cells with a doubling time of approximately 60 min. In contrast, the hly- mutants failed to grow in the murine-derived tissue culture cells, but retained the ability to grow in the human tissue culture cells examined. Hemolytic-positive revertants were selected after passage of the hly- mutants through monolayers of J774 cells. In each case, the hemolytic revertants possessed the 58-kD polypeptide, were capable of intracellular growth in tissue culture monolayers and were virulent for mice.

Growth Kinetics and Cell Morphology of Listeria monocytogenes Scott A as Affected by Temperature, NaCl, and EDTA†,‡

2003 ◽  
Vol 66 (7) ◽  
pp. 1208-1215 ◽  
Author(s):  
LAURA L. ZAIKA ◽  
JOSEPH S. FANELLI
Keyword(s):  
Listeria Monocytogenes ◽  
Growth Kinetics ◽  
Morphological Characteristics ◽  
Brain Heart Infusion ◽  
Growth Curves ◽  
Stress Conditions ◽  
Plate Count ◽  
Lag Phase ◽  
Kinetics Parameters ◽  
Phase Duration

Growth kinetics and morphological characteristics of Listeria monocytogenes Scott A grown under stress conditions induced by increasing levels of NaCl and EDTA were studied as a function of temperature. L. monocytogenes Scott A was inoculated into brain heart infusion broth (pH 6) at 19, 28, 37, and 42°C. Test cultures contained NaCl (at concentrations of 4.5, 6.0, and 7.5%) or EDTA (at concentrations of 0.1, 0.2, and 0.3 mM); control cultures contained 0.5% NaCl. Growth curves were fitted from plate count data by the Gompertz equation, and growth kinetics parameters were derived. Stationary-phase cells were examined by scanning and transmission electron microscopy. Generation times (GTs) and lag phase duration times (LPDs) increased as additive levels were increased. The bacterium grew at all NaCl levels. At 37 and 42°C, growth was slow in media containing 7.5% NaCl, and no growth occurred in media containing 0.3 mM EDTA. Temperature was a major factor in certain stress conditions that led to cell elongation and loss of flagella. Cells in control media at 28°C grew as short rods (0.5 by 1.0 to 2.0 μm), while at 42°C most cells were 4 to 10 times as long. Higher levels of NaCl at higher temperatures resulted in longer and thicker cells. At 28°C, 0.1 mM EDTA had little effect on growth kinetics and morphology; however, 0.3 mM EDTA caused a sixfold increase in GT and LPD and loss of flagellae, with most cells being two to six times as long as normal. Cell length did not correlate with growth kinetics. The results of this study suggest that the effect of altered morphological characteristics of L. monocytogenes cells grown under stress on the virulence and subsequent survival of these cells should be investigated.

scholarly journals PSIX-18 Laurate and its glycerol monoester, monolaurin, as potential additives to control Listeria monocytogenes and tetracycline resistant Enterococcus faecalis in air-exposed silage

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 414-415
Author(s):  
Yamicela Castillo-Castillo ◽  
Marina Ontiveros ◽  
Eric J Scholljegerdes ◽  
Robin Anderson ◽  
Claudio Arzola-Alvarez ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Enterococcus Faecalis ◽  
Brain Heart Infusion ◽  
Bactericidal Effect ◽  
Complete Elimination ◽  
Gram Positive Bacteria ◽  
Viable Cells ◽  
Colony Forming Units ◽  
Feed Value ◽  
Risk Infection

Abstract Silages can harbor pathogenic and antimicrobial resistant microbes which risk infection of food-producing animals. Livestock producers need effective yet environmentally friendly interventions to preserve the feed value of these fermented materials. Medium chain fatty acids such as laurate and its glycerol monoester, monolaurin, are potent inhibitors of many Gram-positive bacteria and when tested at 5 mg/mL in anaerobic cultures (n = 3/treatment) inoculated with 105 colony forming units (CFU) of Listeria monocytogenes and grown at 37oC in ½ strength Brain Heart infusion broth achieved near complete elimination of viable cells after 6 h compared to a 2.2 ± 0.1 log10 CFU/mL increase observed in controls. Culture of a tetracycline-resistant Enterococcus faecalis with 5 mg laurate/mL likewise achieved near complete elimination of viable cells (5 log10 CFU/mL) by 6 h incubation. The bactericidal effect of 5 mg monolaurin was less against E. faecalis, achieving a decrease of 1.8 ± 0.2 log10 CFU/mL and not decreased further after 24 h. When tested against air-exposed silage, pH 7.53 (4 g), mixed with 4 mL water, 5 mg laurate or monolaurin decreased viability of experimentally-inoculated L. monocytogenes (105 CFU/g silage) more (P &lt; 0.05) than untreated controls after 24 h aerobic incubation (22oC), with viable counts being decreased 6.3 ± 0.1, 5.9 ± 0.8 and 4.5 ± 0.1 log10 CFU/g, respectively. In contrast, viable recovery of the experimentally-inoculated (105 CFU/g) tetracycline-resistant E. faecalis was reduced more (P &lt; 0.05) than controls (decreased 0.7 ± 0.1 log10 CFU/g) after 6 h incubation when similarly tested with laurate and monolaurin (1.7 ± 0.5 and 3.0 ± 0.9 log10 CFU/g, respectively) but counts after 24 h were similar, decreasing on average 2.0 ± 0.5 log10 CFU/g). Results indicate laurate and monolaurin may be useful in killing L. monocytogenes and tetracycline-resistant E. faecalis during silage feed-out.

scholarly journals Identification of Listeria monocytogenes Genes Contributing to Intracellular Replication by Expression Profiling and Mutant Screening

2006 ◽  
Vol 188 (2) ◽  
pp. 556-568 ◽  
Author(s):  
Biju Joseph ◽  
Karin Przybilla ◽  
Claudia Stühler ◽  
Kristina Schauer ◽  
Jörg Slaghuis ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Proteins ◽  
Carbon Sources ◽  
Brain Heart Infusion ◽  
Nitrogen Sources ◽  
Pentose Phosphate ◽  
Intracellular Growth ◽  
Cell Infection ◽  
Phosphotransferase System ◽  
Isoleucine Leucine

ABSTRACT A successful transition of Listeria monocytogenes from the extracellular to the intracellular environment requires a precise adaptation response to conditions encountered in the host milieu. Although many key steps in the intracellular lifestyle of this gram-positive pathogen are well characterized, our knowledge about the factors required for cytosolic proliferation is still rather limited. We used DNA microarray and real-time reverse transcriptase PCR analyses to investigate the transcriptional profile of intracellular L. monocytogenes following epithelial cell infection. Approximately 19% of the genes were differentially expressed by at least 1.6-fold relative to their level of transcription when grown in brain heart infusion medium, including genes encoding transporter proteins essential for the uptake of carbon and nitrogen sources, factors involved in anabolic pathways, stress proteins, transcriptional regulators, and proteins of unknown function. To validate the biological relevance of the intracellular gene expression profile, a random mutant library of L. monocytogenes was constructed by insertion-duplication mutagenesis and screened for intracellular-growth-deficient strains. By interfacing the results of both approaches, we provide evidence that L. monocytogenes can use alternative carbon sources like phosphorylated glucose and glycerol and nitrogen sources like ethanolamine during replication in epithelial cells and that the pentose phosphate cycle, but not glycolysis, is the predominant pathway of sugar metabolism in the host environment. Additionally, we show that the synthesis of arginine, isoleucine, leucine, and valine, as well as a species-specific phosphoenolpyruvate-dependent phosphotransferase system, play a major role in the intracellular growth of L. monocytogenes.

scholarly journals Sigma B Contributes to PrfA-Mediated Virulence in Listeria monocytogenes

2002 ◽  
Vol 70 (7) ◽  
pp. 3948-3952 ◽  
Author(s):  
Celine A. Nadon ◽  
Barbara M. Bowen ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Sigma Factor ◽  
Deletion Mutation ◽  
Alternative Sigma Factor ◽  
Regulatory Factor ◽  
Virulence Phenotype ◽  
Sigma B

ABSTRACT 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 σB-dependent promoter and that a sigB deletion mutation affects the virulence phenotype of L. monocytogenes. Thus, the alternative sigma factor σB contributes to virulence in L. monocytogenes.

scholarly journals Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Duarte N. Guerreiro ◽  
Jialun Wu ◽  
Charlotte Dessaux ◽  
Ana H. Oliveira ◽  
Teresa Tiensuu ◽  
...  
Keyword(s):  
Heat Stress ◽  
Listeria Monocytogenes ◽  
Laboratory Culture ◽  
Stress Conditions ◽  
Mild Stress ◽  
Whole Genome ◽  
Content Type ◽  
Acid Sensitivity ◽  
Stop Codons ◽  
Mild Heat

ABSTRACT In Listeria monocytogenes, the full details of how stress signals are integrated into the σB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for σB activation, independently of the transposon location. Reduced σB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of σB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting σB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype. IMPORTANCE In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating σB activity. Together, these findings suggest that the occurrence of mutations that attenuate σB activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.

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