Transport and Catabolism of Pentitols by Listeria monocytogenes

Transposon insertion into Listeria monocytogenes lmo2665, which encodes an EIIC of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), was found to prevent <smlcap>D</smlcap>-arabitol utilization. We confirm this result with a deletion mutant and show that Lmo2665 is also required for <smlcap>D</smlcap>-xylitol utilization. We therefore called this protein EIICAxl. Both pentitols are probably catabolized via the pentose phosphate pathway (PPP) because lmo2665 belongs to an operon, which encodes the three PTSAxl components, two sugar-P dehydrogenases, and most PPP enzymes. The two dehydrogenases oxidize the pentitol-phosphates produced during PTS-catalyzed transport to the PPP intermediate xylulose-5-P. L. monocytogenes contains another PTS, which exhibits significant sequence identity to PTSAxl. Its genes are also part of an operon encoding PPP enzymes. Deletion of the EIIC-encoding gene (lmo0508) affected neither <smlcap>D</smlcap>-arabitol nor <smlcap>D</smlcap>-xylitol utilization, although <smlcap>D</smlcap>-arabitol induces the expression of this operon. Both operons are controlled by MtlR/LicR-type transcription activators (Lmo2668 and Lmo0501, respectively). Phosphorylation of Lmo0501 by the soluble PTSAxl components probably explains why <smlcap>D</smlcap>-arabitol also induces the second pentitol operon. Listerial virulence genes are submitted to strong repression by PTS sugars, such as glucose. However, <smlcap>D</smlcap>-arabitol inhibited virulence gene expression only at high concentrations, probably owing to its less efficient utilization compared to glucose.

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The bvr Locus of Listeria monocytogenes Mediates Virulence Gene Repression by β-Glucosides

Journal of Bacteriology ◽

10.1128/jb.181.16.5024-5032.1999 ◽

1999 ◽

Vol 181 (16) ◽

pp. 5024-5032 ◽

Cited By ~ 77

Author(s):

Klaus Brehm ◽

María-Teresa Ripio ◽

Jürgen Kreft ◽

José-Antonio Vázquez-Boland

Keyword(s):

Listeria Monocytogenes ◽

Virulence Genes ◽

Natural Habitat ◽

Sensory System ◽

Virulence Gene ◽

Gene Repression ◽

Signal Molecules ◽

Wild Type ◽

Phosphotransferase System ◽

Virulence Gene Expression

ABSTRACT The β-glucoside cellobiose has been reported to specifically repress the PrfA-dependent virulence genes hly andplcA in Listeria monocytogenes NCTC 7973. This led to the hypothesis that β-glucosides, sugars of plant origin, may act as signal molecules, preventing the expression of virulence genes if L. monocytogenes is living in its natural habitat (soil). In three other laboratory strains (EGD, L028, and 10403S), however, the effect of cellobiose was not unique, and all fermentable carbohydrates repressed hly. This suggested that the downregulation of virulence genes by β-glucosides is not a specific phenomenon but, rather, an aspect of a global regulatory mechanism of catabolite repression (CR). We assessed the effect of carbohydrates on virulence gene expression in a panel of wild-type isolates of L. monocytogenes by using the PrfA-dependent phospholipase C geneplcB as a reporter. Utilization of any fermentable sugar caused plcB repression in wild-type L. monocytogenes. However, an EGD variant was identified in which, as in NCTC 7973, plcB was only repressed by β-glucosides. Thus, the regulation of L. monocytogenes virulence genes by sugars appears to be mediated by two separate mechanisms, one presumably involving a CR pathway and another specifically responding to β-glucosides. We have identified in L. monocytogenes a 4-kb operon, bvrABC, encoding an antiterminator of the BglG family (bvrA), a β-glucoside-specific enzyme II permease component of the phosphoenolpyruvate-sugar phosphotransferase system (bvrB), and a putative ADP-ribosylglycohydrolase (bvrC). Low-stringency Southern blots showed that this locus is absent from other Listeria spp. Transcription ofbvrB was induced by cellobiose and salicin but not by arbutin. Disruption of the bvr operon by replacing part ofbvrAB with an interposon abolished the repression by cellobiose and salicin but not that by arbutin. Our data indicate that the bvr locus encodes a β-glucoside-specific sensor that mediates virulence gene repression upon detection of cellobiose and salicin. Bvr is the first sensory system found in L. monocytogenes that is involved in environmental regulation of virulence genes.

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Studies of the Listeria monocytogenes Cellobiose Transport Components and Their Impact on Virulence Gene Repression

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000500090 ◽

2019 ◽

Vol 29 (1-6) ◽

pp. 10-26 ◽

Cited By ~ 2

Author(s):

Thanh Nguyen Cao ◽

Philippe Joyet ◽

Francine Moussan Désirée Aké ◽

Eliane Milohanic ◽

Josef Deutscher

Keyword(s):

Listeria Monocytogenes ◽

Double Mutant ◽

Virulence Gene ◽

Gene Repression ◽

Phosphotransferase System ◽

Bacteria Transport ◽

Virulence Gene Expression ◽

Transcription Regulator ◽

Related Phenotype

Background: Many bacteria transport cellobiose via a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). In Listeria monocytogenes, two pairs of soluble PTS components (EIIACel1/EIIBCel1 and EIIACel2/EIIBCel2) and the permease EIICCel1 were suggested to contribute to cellobiose uptake. Interestingly, utilization of several carbohydrates, including cellobiose, strongly represses virulence gene expression by inhibiting PrfA, the virulence gene activator. Results: The LevR-like transcription regulator CelR activates expression of the cellobiose-induced PTS operons celB1-celC1-celA1, celB2-celA2, and the EIIC-encoding monocistronic celC2. Phosphorylation by P∼His-HPr at His550 activates CelR, whereas phosphorylation by P∼EIIBCel1 or P∼EIIBCel2 at His823 inhibits it. Replacement of His823 with Ala or deletion of both celA or celB genes caused constitutive CelR regulon expression. Mutants lacking EIICCel1, CelR or both EIIACel exhibitedslow cellobiose consumption. Deletion of celC1 or celR prevented virulence gene repression by the disaccharide, but not by glucose and fructose. Surprisingly, deletion of both celA genes caused virulence gene repression even during growth on non-repressing carbohydrates. No cellobiose-related phenotype was found for the celC2 mutant. Conclusion: The two EIIA/BCel pairs are similarly efficient as phosphoryl donors in EIICCel1-catalyzed cellobiose transport and CelR regulation. The permanent virulence gene repression in the celA double mutant further supports a role of PTSCel components in PrfA regulation.

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Influence of Sublethal Concentrations of Common Disinfectants on Expression of Virulence Genes in Listeria monocytogenes

Applied and Environmental Microbiology ◽

10.1128/aem.00925-09 ◽

2009 ◽

Vol 76 (1) ◽

pp. 303-309 ◽

Cited By ~ 37

Author(s):

Vicky G. Kastbjerg ◽

Marianne Halberg Larsen ◽

Lone Gram ◽

Hanne Ingmer

Keyword(s):

Gene Expression ◽

Listeria Monocytogenes ◽

Northern Blot ◽

Food Industry ◽

Virulence Genes ◽

Virulence Gene ◽

Human Pathogen ◽

Active Components ◽

Virulence Gene Expression ◽

Sublethal Concentrations

ABSTRACT Listeria monocytogenes is a food-borne human pathogen that causes listeriosis, a relatively rare infection with a high fatality rate. The regulation of virulence gene expression is influenced by several environmental factors, and the aim of the present study was to determine how disinfectants used routinely in the food industry affect the expression of different virulence genes in L. monocytogenes when added at sublethal concentrations. An agar-based assay was developed to screen the effect of disinfectants on virulence gene promoter expression and was validated at the transcriptional level by Northern blot analysis. Eleven disinfectants representing four different groups of active components were evaluated in this study. Disinfectants with the same active ingredients had a similar effect on gene expression. Peroxy and chlorine compounds reduced the expression of the virulence genes, and quaternary ammonium compounds (QAC) induced the expression of the virulence genes. In general, a disinfectant had similar effects on the expression of all four virulence genes examined. Northern blot analyses confirmed the downregulation of prfA and inlA expression by Incimaxx DES (a peroxy compound) and their upregulation by Triquart Super (a QAC) in L. monocytogenes EGD. Hence, sublethal concentrations of disinfectants routinely used in the food industry affect virulence gene expression in the human pathogen L. monocytogenes, and the effect depends on the active components of the disinfectant. From a practical perspective, the study underlines that disinfectants should be used at the lethal concentrations recommended by the manufacturers. Further studies are needed to elucidate whether the changes in virulence gene expression induced by the disinfectants have impact on virulence or other biological properties, such as antibiotic resistance.

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The Posttranslocation Chaperone PrsA2 Contributes to Multiple Facets of Listeria monocytogenes Pathogenesis

Infection and Immunity ◽

10.1128/iai.00280-09 ◽

2009 ◽

Vol 77 (7) ◽

pp. 2612-2623 ◽

Cited By ~ 45

Author(s):

Francis Alonzo ◽

Gary C. Port ◽

Min Cao ◽

Nancy E. Freitag

Keyword(s):

Listeria Monocytogenes ◽

Gene Product ◽

Bacterial Pathogen ◽

Virulence Gene ◽

Host Cells ◽

Listeriolysin O ◽

Virulence Gene Expression ◽

High Concentrations ◽

Host Infection ◽

Cell Spread

ABSTRACT Listeria monocytogenes is an intracellular bacterial pathogen whose virulence depends on the regulated expression of numerous secreted bacterial factors. As for other gram-positive bacteria, many proteins secreted by L. monocytogenes are translocated across the bacterial membrane in an unfolded state to the compartment existing between the membrane and the cell wall. This compartment presents a challenging environment for protein folding due to its high density of negative charge, high concentrations of cations, and low pH. We recently identified PrsA2 as a gene product required for L. monocytogenes virulence. PrsA2 was identified based on its increased secretion by strains containing a mutationally activated form of prfA, the key regulator of L. monocytogenes virulence gene expression. The prsA2 gene product is one of at least two predicted peptidyl-prolyl cis/trans-isomerases encoded by L. monocytogenes; these proteins function as posttranslocation protein chaperones and/or foldases. In this study, we demonstrate that PrsA2 plays a unique and important role in L. monocytogenes pathogenesis by promoting the activity and stability of at least two critical secreted virulence factors: listeriolysin O (LLO) and a broad-specificity phospholipase. Loss of PrsA2 activity severely attenuated virulence in mice and impaired bacterial cell-to-cell spread in host cells. In contrast, mutants lacking prsA1 resembled wild-type bacteria with respect to intracellular growth and cell-to-cell spread as well as virulence in mice. PrsA2 is thus distinct from PrsA1 in its unique requirement for the stability and full activity of L. monocytogenes-secreted factors that contribute to host infection.

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Interference of Components of the Phosphoenolpyruvate Phosphotransferase System with the Central Virulence Gene Regulator PrfA of Listeria monocytogenes

Journal of Bacteriology ◽

10.1128/jb.00972-06 ◽

2006 ◽

Vol 189 (2) ◽

pp. 473-490 ◽

Cited By ~ 75

Author(s):

Sonja Mertins ◽

Biju Joseph ◽

Monika Goetz ◽

Regina Ecke ◽

Gerald Seidel ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Type Strain ◽

Virulence Genes ◽

Wild Type Strain ◽

Virulence Gene ◽

Wild Type ◽

Phosphotransferase System ◽

In The Wild ◽

The Common ◽

Transcript Profiles

ABSTRACT Analysis of Listeria monocytogenes ptsH, hprK, and ccpA mutants defective in carbon catabolite repression (CCR) control revealed significant alterations in the expression of PrfA-dependent genes. The hprK mutant showed high up-regulation of PrfA-dependent virulence genes upon growth in glucose-containing medium whereas expression of these genes was even slightly down-regulated in the ccpA mutant compared to the wild-type strain. The ptsH mutant could only grow in a rich culture medium, and here the PrfA-dependent genes were up-regulated as in the hprK mutant. As expected, HPr-Ser-P was not produced in the hprK and ptsH mutants and synthesized at a similar level in the ccpA mutant as in the wild-type strain. However, no direct correlation was found between the level of HPr-Ser-P or HPr-His-P and PrfA activity when L. monocytogenes was grown in minimal medium with different phosphotransferase system (PTS) carbohydrates. Comparison of the transcript profiles of the hprK and ccpA mutants with that of the wild-type strain indicates that the up-regulation of the PrfA-dependent virulence genes in the hprK mutant correlates with the down-regulation of genes known to be controlled by the efficiency of PTS-mediated glucose transport. Furthermore, growth in the presence of the non-PTS substrate glycerol results in high PrfA activity. These data suggest that it is not the component(s) of the CCR or the common PTS pathway but, rather, the component(s) of subsequent steps that seem to be involved in the modulation of PrfA activity.

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Tannin-Rich Pomegranate Rind Extracts Reduce Adhesion to and Invasion of Caco-2 Cells by Listeria monocytogenes and Decrease Its Expression of Virulence Genes

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-174 ◽

2015 ◽

Vol 78 (1) ◽

pp. 128-133 ◽

Cited By ~ 9

Author(s):

YUNFENG XU ◽

GUANGHUI LI ◽

BAIGANG ZHANG ◽

QIAN WU ◽

XIN WANG ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Epithelial Cells ◽

Virulence Genes ◽

Growth Curves ◽

Virulence Gene ◽

Antimicrobial Activities ◽

Pcr Analysis ◽

Mrna Levels ◽

Dependent Manner ◽

Virulence Gene Expression

Pomegranate rind is rich in tannins that have remarkable antimicrobial activities. This study aimed to evaluate the effects of a tannin-rich fraction from pomegranate rind (TFPR) on Listeria monocytogenes virulence gene expression and on the pathogen's interaction with human epithelial cells. Growth curves were monitored to determine the effect of TFPR on L. monocytogenes growth. The 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide and fluorescence staining assays were used to examine the cytotoxicity of TFPR. The effects of TFPR on L. monocytogenes adhesion to and invasion of epithelial cells were investigated using Caco-2 cells. Real-time quantitative PCR analysis was conducted to quantify mRNA levels of three virulence genes in L. monocytogenes. Results showed that a MIC of TFPR against L. monocytogenes was 5 mg/ml in this study. TFPR exhibited cytotoxicity against Caco-2 cells when the concentration was 2.5 mg/ml. Subinhibitory concentrations of TFPR significantly reduced, in a dose-dependent manner, adhesion to and invasion of Caco-2 cells by L. monocytogenes. When L. monocytogenes was grown in the presence of 2.5 mg/ml TFPR, the transcriptional levels of prfA, inlA, and hly decreased by 17-, 34-, and 28-fold, respectively.

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The Alternative Sigma Factor σB and the Virulence Gene Regulator PrfA Both Regulate Transcription of Listeria monocytogenes Internalins

Applied and Environmental Microbiology ◽

10.1128/aem.02664-06 ◽

2007 ◽

Vol 73 (9) ◽

pp. 2919-2930 ◽

Cited By ~ 47

Author(s):

Patrick McGann ◽

Martin Wiedmann ◽

Kathryn J. Boor

Keyword(s):

Listeria Monocytogenes ◽

Sigma Factor ◽

Virulence Genes ◽

Virulence Gene ◽

Alternative Sigma Factor ◽

Transcript Levels ◽

Virulence Gene Expression ◽

Reverse Transcription Pcr ◽

Culture Cells ◽

Tissue Culture Cells

ABSTRACT Some Listeria monocytogenes internalins are recognized as contributing to invasion of mammalian tissue culture cells. While PrfA is well established as a positive regulator of L. monocytogenes virulence gene expression, the stress-responsive σB has been recognized only recently as contributing to expression of virulence genes, including some that encode internalins. To measure the relative contributions of PrfA and σB to internalin gene transcription, we used reverse transcription-PCR to quantify transcript levels for eight internalin genes (inlA, inlB, inlC, inlC2, inlD, inlE, inlF, and inlG) in L. monocytogenes 10403S and in isogenic ΔprfA, ΔsigB, and ΔsigB ΔprfA strains. Strains were grown under defined conditions to produce (i) active PrfA, (ii) active σB and active PrfA, (iii) inactive PrfA, and (iv) active σB and inactive PrfA. Under the conditions tested, σB and PrfA contributed differentially to the expression of the various internalins such that (i) both σB and PrfA contributed to inlA and inlB transcription, (ii) only PrfA contributed to inlC transcription, (iii) only σB contributed to inlC2 and inlD transcription, and (iv) neither σB nor PrfA contributed to inlF and inlG transcription. inlE transcript levels were undetectable. The important role for σB in regulating expression of L. monocytogenes internalins suggests that exposure of this organism to environmental stress conditions, such as those encountered in the gastrointestinal tract, may activate internalin transcription. Interplay between σB and PrfA also appears to be critical for regulating transcription of some virulence genes, including inlA, inlB, and prfA.

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Sequence Variations within PrfA DNA Binding Sites and Effects on Listeria monocytogenes Virulence Gene Expression

Journal of Bacteriology ◽

10.1128/jb.182.3.837-841.2000 ◽

2000 ◽

Vol 182 (3) ◽

pp. 837-841 ◽

Cited By ~ 29

Author(s):

James R. Williams ◽

Chetna Thayyullathil ◽

Nancy E. Freitag

Keyword(s):

Gene Expression ◽

Listeria Monocytogenes ◽

Dna Binding ◽

Binding Sites ◽

Virulence Genes ◽

Regulation Of Gene Expression ◽

Virulence Gene ◽

Virulence Gene Expression ◽

Dna Binding Sites ◽

Sequence Variations

ABSTRACT Reporter gene fusions were used to investigate the contributions of PrfA DNA binding sites to Listeria monocytogenes virulence gene expression. Our results suggest that the DNA sequence of PrfA binding sites determines the levels of expression of certain virulence genes, such as hly and mpl. Other virulence genes, such as actA and plcB, may depend upon additional factors for full regulation of gene expression.

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Regulation of Mannose Phosphotransferase System Permease and Virulence Gene Expression in Listeria monocytogenes by the EIItMan Transporter

Applied and Environmental Microbiology ◽

10.1128/aem.01104-09 ◽

2009 ◽

Vol 75 (21) ◽

pp. 6671-6678 ◽

Cited By ~ 22

Author(s):

Hung Vu-Khac ◽

Kurt W. Miller

Keyword(s):

Gene Expression ◽

Listeria Monocytogenes ◽

Deletion Mutant ◽

Glucose Transporter ◽

Mrna Level ◽

Virulence Gene ◽

Pcr Analysis ◽

Mrna Levels ◽

Phosphotransferase System ◽

Virulence Gene Expression

ABSTRACT The EIIt Man phosphotransferase system (PTS) permease encoded by the mpt operon is the principal glucose transporter in Listeria monocytogenes. EIIt Man participates in glucose-mediated carbon catabolite repression (CCR) and downregulation of virulence gene expression, and it is the receptor for class IIa bacteriocins. The regulation of this important protein and its roles in gene control were examined using derivatives of strain EGD-e in which the mpt operon or its regulatory genes, manR and lmo0095, were deleted. Real-time reverse transcription-PCR analysis showed that the mpt mRNA level was 10- and 100-fold lower in the lmo0095 and manR deletion strains, respectively. The manR mRNA level was higher in the mpt deletion mutant in medium lacking glucose, possibly due to disruption of a regulatory process that normally downregulates manR transcription in the absence of this sugar. Analysis of the mpt deletion mutant also showed that EIIt Man participates to various degrees in glucose-mediated CCR of PTS operons. CCR of the lmo0027 gene, which encodes a β-glucoside PTS transporter, required expression of EIIt Man. In contrast, genes in two mannose PTS operons (lmo0024, lmo1997, and lmo2002) were repressed by glucose even when EIIt Man was not synthesized. A third mannose PTS operon, mpo, was not regulated by glucose or by the level of EIIt Man. Finally, the mRNA levels for five genes in the prfA virulence gene cluster were two- to fourfold higher in the mpt deletion mutant. The results show that EIIt Man participates to various extents in glucose-mediated CCR of PTS operons and makes a small, albeit significant, contribution to downregulation of virulence gene transcription by glucose in strain EGD-e.

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A Homolog of CcpA Mediates Catabolite Control in Listeria monocytogenes but Not Carbon Source Regulation of Virulence Genes

Journal of Bacteriology ◽

10.1128/jb.180.23.6316-6324.1998 ◽

1998 ◽

Vol 180 (23) ◽

pp. 6316-6324 ◽

Cited By ~ 51

Author(s):

Jaideep Behari ◽

Philip Youngman

Keyword(s):

Listeria Monocytogenes ◽

Carbon Source ◽

Virulence Genes ◽

Virulence Gene ◽

Metabolic Enzyme ◽

Virulence Gene Expression ◽

Gram Positive Bacteria ◽

Glucosidase Activity ◽

Acid Protein ◽

Catabolite Regulation

ABSTRACT Readily utilizable sugars down-regulate virulence gene expression in Listeria monocytogenes, which has led to the proposal that this regulation may be an aspect of global catabolite regulation (CR). We recently demonstrated that the metabolic enzyme α-glucosidase is under CR in L. monocytogenes. Here, we report the cloning and characterization from L. monocytogenes of an apparent ortholog of ccpA, which encodes an important mediator of CR in several low-G+C-content gram-positive bacteria. L. monocytogenes ccpA(ccpA Lm) is predicted to encode a 335-amino-acid protein with nearly 65% identity to the gene product ofBacillus subtilis ccpA (ccpA Bs). Southern blot analysis with a probe derived fromccpA Lm revealed a single strongly hybridizing band and also a second band of much lower intensity, suggesting that there may be other closely related sequences in the L. monocytogenes chromosome, as is the case in B. subtilis. Disruption of ccpA Lm resulted in the inability of the mutant to grow on glucose-containing minimal medium or increase its growth rate in the presence of preferred sugars, and it completely eliminated CR of α-glucosidase activity in liquid medium. However, α-glucosidase activity was only partially relieved from CR on solid medium. These results suggest that ccpA is an important element of carbon source regulation in L. monocytogenes. Nevertheless, utilizable sugars still down-regulate the expression of hly, which encodes the virulence factor hemolysin, in a ccpA Lm mutant, indicating that CcpA is not involved in carbon source regulation of virulence genes.

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