scholarly journals Phenotypic and Transcriptomic Analyses Demonstrate Interactions between the Transcriptional Regulators CtsR and Sigma B in Listeria monocytogenes

2007 ◽  
Vol 73 (24) ◽  
pp. 7967-7980 ◽  
Author(s):  
Yuewei Hu ◽  
Sarita Raengpradub ◽  
Ute Schwab ◽  
Chris Loss ◽  
Renato H. Orsi ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Response ◽  
Heat Resistance ◽  
Parent Strain ◽  
Inducible Promoter ◽  
Heat Sensitivity ◽  
Intestinal Epithelial ◽  
Class Iii ◽  
Stress Response Genes ◽  
Genes Encoding

ABSTRACT Listeria monocytogenes σB positively regulates the transcription of class II stress response genes; CtsR negatively regulates class III stress response genes. To identify interactions between these two stress response systems, we constructed L. monocytogenes ΔctsR and ΔctsR ΔsigB strains, as well as a ΔctsR strain expressing ctsR in trans under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter. These strains, along with a parent and a ΔsigB strain, were assayed for motility, heat resistance, and invasion of human intestinal epithelial cells, as well as by whole-genome transcriptomic and quantitative real-time PCR analyses. Both ΔctsR and ΔctsR ΔsigB strains had significantly higher thermotolerances than the parent strain; however, full heat sensitivity was restored to the ΔctsR strain when ctsR was expressed in trans. Although log-phase ΔctsR was not reduced in its ability to infect human intestinal cells, the ΔctsR ΔsigB strain showed significantly lower invasion efficiency than either the parent strain or the ΔsigB strain, indicating that interactions between CtsR and σB contribute to invasiveness. Statistical analyses also confirmed interactions between the ctsR and the sigB null mutations in both heat resistance and invasion phenotypes. Microarray transcriptomic analyses and promoter searches identified (i) 42 CtsR-repressed genes, (ii) 22 genes with lower transcript levels in the ΔctsR strain, and (iii) at least 40 genes coregulated by both CtsR and σB, including genes encoding proteins with confirmed or plausible roles in virulence and stress response. Our data demonstrate that interactions between CtsR and σB play an important role in L. monocytogenes stress resistance and virulence.

scholarly journals Transcriptomic and Phenotypic Analyses Suggest a Network between the Transcriptional Regulators HrcA and σB in Listeria monocytogenes

2007 ◽  
Vol 73 (24) ◽  
pp. 7981-7991 ◽  
Author(s):  
Yuewei Hu ◽  
Haley F. Oliver ◽  
Sarita Raengpradub ◽  
M. Elizabeth Palmer ◽  
Renato H. Orsi ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Response ◽  
Heat Resistance ◽  
Acid Resistance ◽  
Transcriptional Regulators ◽  
Intestinal Epithelial ◽  
Quantitative Real Time Pcr ◽  
Stress Response Genes ◽  
Human Intestinal Epithelial Cells ◽  
Number Of Genes

ABSTRACT Listeria monocytogenes HrcA and CtsR negatively regulate class I and III stress response genes, respectively, while σB positively regulates the transcription of class II stress response genes. To define the HrcA regulon and identify interactions between HrcA, CtsR, and σB, we characterized newly generated L. monocytogenes ΔhrcA, ΔctsR ΔhrcA, and ΔhrcA ΔsigB strains, along with previously described ΔsigB, ΔctsR, and ΔctsR ΔsigB strains, using phenotypic assays (i.e., heat resistance, acid resistance, and invasion of human intestinal epithelial cells) and performed whole-genome transcriptome analysis of the ΔhrcA strain. The hrcA and sigB deletions had significant effects on heat resistance. While the hrcA deletion had no significant effect on acid resistance or invasion efficiency in Caco-2 cells, a linear regression model revealed a significant (P = 0.0493) effect of interactions between the hrcA deletion and the ctsR deletion on invasiveness. Microarray-based transcriptome analyses and promoter searches identified (i) 25 HrcA-repressed genes, including two operons (the groESL and dnaK operons, both confirmed as HrcA regulated by quantitative real-time PCR) and one gene directly repressed by HrcA, and (ii) 36 genes that showed lower transcript levels in the ΔhrcA strain and thus appear to be indirectly upregulated by HrcA. A number of genes were found to be coregulated by either HrcA and CtsR (2 genes), HrcA and σB (31 genes), or all three regulators (5 genes, e.g., gadCB). Combined with previous evidence that σB appears to directly regulate hrcA transcription, our data suggest that HrcA and σB, as well as CtsR, form a regulatory network that contributes to the transcription of a number of L. monocytogenes genes.

scholarly journals Listeria monocytogenes Uses Listeria Adhesion Protein (LAP) To Promote Bacterial Transepithelial Translocation and Induces Expression of LAP Receptor Hsp60

2010 ◽  
Vol 78 (12) ◽  
pp. 5062-5073 ◽  
Author(s):  
Kristin M. Burkholder ◽  
Arun K. Bhunia
Keyword(s):  
Plasma Membrane ◽  
Listeria Monocytogenes ◽  
Stress Response ◽  
Cell Model ◽  
Infection Process ◽  
Intestinal Epithelial ◽  
Necrosis Factor Alpha ◽  
Adhesion Protein ◽  
Hsp60 Expression

ABSTRACT Listeria monocytogenes interaction with the intestinal epithelium is a key step in the infection process. We demonstrated that Listeria adhesion protein (LAP) promotes adhesion to intestinal epithelial cells and facilitates extraintestinal dissemination in vivo. The LAP receptor is a stress response protein, Hsp60, but the precise role for the LAP-Hsp60 interaction during Listeria infection is unknown. Here we investigated the influence of physiological stressors and Listeria infection on host Hsp60 expression and LAP-mediated bacterial adhesion, invasion, and transepithelial translocation in an enterocyte-like Caco-2 cell model. Stressors such as heat (41°C), tumor necrosis factor alpha (TNF-α) (100 U), and L. monocytogenes infection (104 to 106 CFU/ml) significantly (P < 0.05) increased plasma membrane and intracellular Hsp60 levels in Caco-2 cells and consequently enhanced LAP-mediated L. monocytogenes adhesion but not invasion of Caco-2 cells. In transepithelial translocation experiments, the wild type (WT) exhibited 2.7-fold more translocation through Caco-2 monolayers than a lap mutant, suggesting that LAP is involved in transepithelial translocation, potentially via a paracellular route. Short hairpin RNA (shRNA) suppression of Hsp60 in Caco-2 cells reduced WT adhesion and translocation 4.5- and 3-fold, respectively, while adhesion remained unchanged for the lap mutant. Conversely, overexpression of Hsp60 in Caco-2 cells enhanced WT adhesion and transepithelial translocation, but not those of the lap mutant. Furthermore, initial infection with a low dosage (106 CFU/ml) of L. monocytogenes increased plasma membrane and intracellular expression of Hsp60 significantly, which rendered Caco-2 cells more susceptible to subsequent LAP-mediated adhesion and translocation. These data provide insight into the role of LAP as a virulence factor during intestinal epithelial infection and pose new questions regarding the dynamics between the host stress response and pathogen infection.

scholarly journals Listeria monocytogenes σB Regulates Stress Response and Virulence Functions

2003 ◽  
Vol 185 (19) ◽  
pp. 5722-5734 ◽  
Author(s):  
Mark J. Kazmierczak ◽  
Sharon C. Mithoe ◽  
Kathryn J. Boor ◽  
Martin Wiedmann
Keyword(s):  
Gene Expression ◽  
Listeria Monocytogenes ◽  
Stress Response ◽  
Consensus Sequence ◽  
Virulence Gene ◽  
Wild Type ◽  
Promoter Regions ◽  
Virulence Gene Expression ◽  
Stress Response Genes ◽  
In The Wild

ABSTRACT While the stress-responsive alternative sigma factor σB has been identified in different species of Bacillus, Listeria, and Staphylococcus, theσ B regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify σB-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidateσ 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 σB-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and ΔsigB 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 significantσ 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 σB-dependent expression, 54 were preceded by a sequence resembling the σB promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the σB-dependent nature of a subset of eight selected promoter regions. Notably, theσ 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, σB also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest thatσ B contributes to L. monocytogenes gene expression during infection.

σ B-dependent gene induction and expression in Listeria monocytogenes during osmotic and acid stress conditions simulating the intestinal environment

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3843-3855 ◽  
Author(s):  
David Sue ◽  
Daniel Fink ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Response ◽  
Virulence Genes ◽  
Acid Stress ◽  
Exponential Phase ◽  
Wild Type ◽  
Transcript Accumulation ◽  
Gastrointestinal Infections ◽  
Stress Response Genes ◽  
Foodborne Infections

Listeria monocytogenes must overcome a variety of stress conditions in the host digestive tract to cause foodborne infections. The alternative sigma factor σ B, encoded by sigB, is responsible for regulating transcription of several L. monocytogenes virulence and stress-response genes, including genes that contribute to establishment of gastrointestinal infections. A quantitative RT-PCR assay was used to measure mRNA transcript accumulation for the virulence genes inlA and bsh, the stress-response genes opuCA and lmo0669 (encoding a carnitine transporter and an oxidoreductase, respectively) and the housekeeping gene rpoB. Assays were conducted on mid-exponential phase L. monocytogenes cells exposed to conditions reflecting osmotic (0·3 M NaCl) or acid (pH 4·5) conditions typical for the human intestinal lumen. In exponential-phase cells, as well as under osmotic and acid stress, inlA, opuCA and bsh showed significantly lower absolute expression levels in a L. monocytogenes ΔsigB null mutant compared to wild-type. A statistical model that normalized target gene expression relative to rpoB showed that accumulation of inlA, opuCA and bsh transcripts was significantly increased in the wild-type strain within 5 min of acid and osmotic stress exposure; lmo0669 transcript accumulation increased significantly only after acid exposure. It was concluded that σ B is essential for rapid induction of the tested stress-response and virulence genes under conditions typically encountered during gastrointestinal passage. As inlA, bsh and opuCA are critical for gastrointestinal infections in animal models, the data also suggest that σ B contributes to the ability of L. monocytogenes to cause foodborne infections.

scholarly journals Comparative Analysis of the σB-Dependent Stress Responses in Listeria monocytogenes and Listeria innocua Strains Exposed to Selected Stress Conditions

2007 ◽  
Vol 74 (1) ◽  
pp. 158-171 ◽  
Author(s):  
Sarita Raengpradub ◽  
Martin Wiedmann ◽  
Kathryn J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Response ◽  
Stationary Phase ◽  
Stress Responses ◽  
Bacterial Species ◽  
Acid Stress ◽  
Listeria Innocua ◽  
Transcript Levels ◽  
Genes Encoding ◽  
Flagellar Proteins

ABSTRACT The alternative sigma factor σB contributes to transcription of stress response and virulence genes in diverse gram-positive bacterial species. The composition and functions of the Listeria monocytogenes and Listeria innocua σB regulons were hypothesized to differ due to virulence differences between these closely related species. Transcript levels in stationary-phase cells and in cells exposed to salt stress were characterized by microarray analyses for both species. In L. monocytogenes, 168 genes were positively regulated by σB; 145 of these genes were preceded by a putative σB consensus promoter. In L. innocua, 64 genes were positively regulated by σB. σB contributed to acid stress survival in log-phase cells for both species but to survival in stationary-phase cells only for L. monocytogenes. In summary, (i) the L. monocytogenes σB regulon includes >140 genes that are both directly and positively regulated by σB, including genes encoding proteins with importance in stress response, virulence, transcriptional regulation, carbohydrate metabolism, and transport; (ii) a number of L. monocytogenes genes encoding flagellar proteins show higher transcript levels in the ΔsigB mutant, and both L. monocytogenes and L. innocua ΔsigB null mutants have increased motility compared to the respective isogenic parent strains, suggesting that σB affects motility and chemotaxis; and (iii) although L. monocytogenes and L. innocua differ in σB-dependent acid stress resistance and have species-specific σB-dependent genes, the L. monocytogenes and L. innocua σB regulons show considerable conservation, with a common set of at least 49 genes that are σB dependent in both species.

scholarly journals Validation of Predicted Virulence Factors in Listeria monocytogenes Identified Using Comparative Genomics

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 508 ◽  
Author(s):  
Hossam Abdelhamed ◽  
Mark Lawrence ◽  
Reshma Ramachandran ◽  
Attila Karsi
Keyword(s):  
Listeria Monocytogenes ◽  
Virulence Factors ◽  
Parent Strain ◽  
Intestinal Epithelial ◽  
Zoonotic Infection ◽  
Phosphotransferase System ◽  
Genome Sequences ◽  
Pathogenic Potential ◽  
Mutant Strains ◽  
Class 1

Listeria monocytogenes is an intracellular facultative pathogen that causes listeriosis, a foodborne zoonotic infection. There are differences in the pathogenic potential of L. monocytogenes subtypes and strains. Comparison of the genome sequences among L. monocytogenes pathogenic strains EGD-e and F2365 with nonpathogenic L. innocua CLIP1182 and L. monocytogenes strain HCC23 revealed a set of proteins that were present in pathogenic strains and had no orthologs among the nonpathogenic strains. Among the candidate virulence factors are five proteins: putrescine carbamoyltransferase; InlH/InlC2 family class 1 internalin; phosphotransferase system (PTS) fructose transporter subunit EIIC; putative transketolase; and transcription antiterminator BglG family. To determine if these proteins have a role in adherence and invasion of intestinal epithelial Caco-2 cells and/or contribute to virulence, five mutant strains were constructed. F2365ΔinlC2, F2365Δeiic, and F2365Δtkt exhibited a significant (p < 0.05) reduction in adhesion to Caco-2 cells compared to parent F2365 strain. The invasion of F2365ΔaguB, F2365ΔinlC2, and F2365ΔbglG decreased significantly (p < 0.05) compared with the parent strain. Bacterial loads in mouse liver and spleen infected by F2365 was significantly (p < 0.05) higher than it was for F2365ΔaguB, F2365ΔinlC2, F2365Δeiic, F2365Δtkt, and F2365ΔbglG strains. This study demonstrates that aguB, inlC2, eiic, tkt, and bglG play a role in L. monocytogenes pathogenicity.

Characterization of new stress response genes in Listeria monocytogenes

2018 ◽  
Vol 120 ◽  
pp. S151
Author(s):  
Sofia Boavida ◽  
Ricardo dos Santos ◽  
Ana Quendera ◽  
Cecilia Arraiano ◽  
José Andrade
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Response ◽  
Stress Response Genes

scholarly journals AITRL, an evolutionarily conserved plant specific transcription repressor regulates ABA response in Arabidopsis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yanxing Ma ◽  
Hainan Tian ◽  
Rao Lin ◽  
Wei Wang ◽  
Na Zhang ◽  
...  
Keyword(s):  
Stress Response ◽  
Protein Phosphatase ◽  
Aba Signaling ◽  
Response Gene ◽  
Stress Response Genes ◽  
Arabidopsis Protein ◽  
Evolutionarily Conserved ◽  
Increased Sensitivity ◽  
Aba Response ◽  
Transcription Repressors

AbstractExpression of stress response genes can be regulated by abscisic acid (ABA) dependent and ABA independent pathways. Osmotic stresses promote ABA accumulation, therefore inducing the expression of stress response genes via ABA signaling. Whereas cold and heat stresses induce the expression of stress response genes via ABA independent pathway. ABA induced transcription repressors (AITRs) are a family of novel transcription factors that play a role in ABA signaling, and Drought response gene (DRG) has previously been shown to play a role in regulating plant response to drought and freezing stresses. We report here the identification of DRG as a novel transcription factor and a regulator of ABA response in Arabidopsis. We found that the expression of DRG was induced by ABA treatment. Homologs searching identified AITR5 as the most closely related Arabidopsis protein to DRG, and homologs of DRG, including the AITR-like (AITRL) proteins in bryophytes and gymnosperms, are specifically presented in embryophytes. Therefore we renamed DRG as AITRL. Protoplast transfection assays show that AITRL functioned as a transcription repressor. In seed germination and seedling greening assays, the aitrl mutants showed an increased sensitivity to ABA. By using qRT-PCR, we show that ABA responses of some ABA signaling component genes including some PYR1-likes (PYLs), PROTEIN PHOSPHATASE 2Cs (PP2Cs) and SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2s (SnRK2s) were reduced in the aitrl mutants. Taken together, our results suggest that AITRLs are a family of novel transcription repressors evolutionally conserved in embryophytes, and AITRL regulates ABA response in Arabidopsis by affecting ABA response of some ABA signaling component genes.

scholarly journals A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents

2021 ◽  
Vol 9 (6) ◽  
pp. 1323
Author(s):  
Etai Boichis ◽  
Nadejda Sigal ◽  
Ilya Borovok ◽  
Anat A. Herskovits
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Protein Pair ◽  
Growth Conditions ◽  
Wall Structure ◽  
Antibiotic Resistant ◽  
Extracellular Milieu ◽  
Virion Release ◽  
Genes Encoding

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.

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