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PubMed1999

Influence of temperature and growth phase on expression of a 104-kilodalton Listeria adhesion protein in Listeria monocytogenes.

Interaction of Listeria monocytogenes with mammalian intestinal cells is believed to be an important first step in Listeria pathogenesis. Transposon (Tn916) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the Listeria adhesion protein (LAP), was involved in adherence of L. monocytogenes to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117-124, 1999). In this study, expression of LAP in L. monocytogenes at various growth temperatures (25, 37, and 42 degrees C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42 degrees C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25 degrees C. Examination of the adhesion of L. monocytogenes cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37 degrees C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of L. monocytogenes LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells.

Bacterial Adhesion↗

PubMed2005

The increase in mucin exocytosis and the upregulation of MUC genes encoding for membrane-bound mucins induced by the thiol-activated exotoxin listeriolysin O is a host cell defence response that inhibits the cell-entry of Listeria monocytogenes.

In vivo Listeria monocytogenes infection results in the massive release of mucus by goblet cells into the lumen of the intestine. We have previously reported that apical infection by L. monocytogenes is followed by listeriolysin O (LLO)-dependent stimulation of mucus exocytosis, and the upregulation of the MUC genes. Here, we report that L. monocytogenes EGD wild-type bacteria enter cultured human polarized, mucin-secreting, HT29-MTX cells apically by an InlA-dependent mechanism. The LLO-induced increase in mucin secretion together with an increase in transcription of the MCU4 and MUC12 genes encoding for membrane-bound mucins, results in the inhibition of the cell-entry of L. monocytogenes into mucin-secreting, HT29-MTX cells. Moreover, we report that sialic acid residues in mucins are crucial for the inhibition of L. monocytogenes internalization. Based on these findings, we suggest that the LLO-induced mucin exocytosis and upregulation of the MUC genes encoding for membrane-bound mucins constitute a host cell defence response that inhibits the cell-entry of L. monocytogenes.

Bacterial Toxins↗

PubMed2000

Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility.

The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH(2)-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer-binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell-cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.

Actin-Related Protein 2↗

PubMed1995

Sets of EcoRI fragments containing ribosomal RNA sequences are conserved among different strains of Listeria monocytogenes.

To classify Listeria monocytogenes using taxonomic characters derived from the rRNA operons and their flanking sequences, we studied a sample of 1346 strains within the taxon. DNA from each strain was digested with a restriction endonuclease, EcoRI. The fragments were separated by gel electrophoresis, immobilized on a membrane, and hybridized with a labeled rRNA operon from Escherichia coli. The pattern of bands, positions, and intensities of hybridized fragments were electronically captured. Software was used to normalize the band positions relative to standards, scale the signal intensity, and reduce the background so that each strain was reproducibly represented in a data base as a pattern. With these methods, L. monocytogenes was resolved into 50 pattern types differing in the length of at least one polymorphic fragment. Pattern types representing multiple strains were recorded as the mathematical average of the strain patterns. Pattern types were arranged by size polymorphisms of assigned rRNA regions into subsets, which revealed the branching genetic structure of the species. Subtracting the polymorphic variants of a specific assigned region from the pattern types and averaging the types within each subset resulted in reduced sets of conserved fragments that could be used to recognize strains of the species. Pattern types and reduced sets of conserved fragments were conserved among different strains of L. monocytogenes but were not observed in total among strains of other species.

Base Sequence↗

PubMed2004

FbpA, a novel multifunctional Listeria monocytogenes virulence factor.

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.

Adhesins, Bacterial↗

PubMed1990

Penicillin binding proteins in Listeria monocytogenes.

Membranes of Listeria were obtained from protoplasts and treated with 125I-ampicillin as probe, at different concentrations. Eight bands, corresponding to proteins labelled with the probe were detected their molecular weight ranged from 38,000 to 100,000, being the predominant ones at 95,000; 85,000; 60,000; 49,000 and 38,000. The copy number of each PBP was also estimated. By means of competitive experiments the binding pattern of ampicillin, mecillinam, piperacillin, cefalotin, cefaloridine, cefoxitin, cefotaxime, azthreonam and imipenem was studied. The most effective binding was obtained with ampicillin, piperacillin and imipenem. Cefotaxime, and particularly cefoxitin, present an extremely low binding ability. The amount of antibiotic concentration preventing an effective label by the radioactive probe to the detected penicillin binding proteins seems to correlate with the lethal concentration of the different antibiotics on Listeria monocytogenes and explains the natural resistance of this genus to certain beta-lactamic compounds.

Anti-Bacterial Agents↗

PubMed1990

Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization.

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.

Anaerobiosis↗

PubMed2001

Food-borne Listeria monocytogenes risk assessment.

Listeria monocytogenes is ubiquitous in the environment and in food processing plants. Consequently, foods are frequently contaminated. However, the occurrence rate of listeriosis is only about five cases per million people per year. Listeriosis primarily strikes immunocompromised individuals, pregnant women and the elderly with a fatality rate of 20-25%. The FDA is in the process of finishing a risk assessment that is being conducted as an initial step in reviewing its approach to maximizing the public protection from foodborne L. monocytogenes. The risk assessment evaluated the presence and quantitative levels of L. monocytogenes in 21 groups of ready-to-eat foods. The potential growth of L. monocytogenes between retail point-of-sale, where contamination data originated, and consumption was modelled. The frequency and amount of consumption of these foods completed the data for the exposure assessment. For the hazard characterization or dose response part of the risk assessment, data from animal studies, virulence assays and epidemiological investigations were used to estimate the likelihood of illness for different human groups from consuming different numbers of L. monocytogenes. This risk assessment is a virtual review of current scientific knowledge. Quantitative modelling provides greater insight than a qualitative review and also indicates the uncertainty about our knowledge. The risk assessment does not attempt to define an acceptable or tolerable level of L. monocytogenes consumption or propose changes in regulations. These decisions are the responsibility of risk managers who consider additional factors such as food preferences, technical feasibility and societal values when evaluating regulatory policies.

Adult↗

PubMed2004

Classes and functions of Listeria monocytogenes surface proteins.

Listeria monocytogenes is an opportunistic pathogen that causes infections collectively termed listeriosis, which are related to the ingestion of food contaminated with these gram-positive rods. The pathogenicity of L. monocytogenes is determined by the following virulence factors: listeriolysin O, protein ActA, two phospholipases C, internalins (In1A and In1B), protein CwhA and a metalloprotease. The bacterium is a model organism in studies on the pathogenesis of intracellular parasites. It is able to penetrate, multiply and propagate in various types of eukaryotic cells and is also able to overcome the three main barriers encountered in the host: the intestinal barrier, the blood-brain barrier and the placenta. Based on L. monocytogenes genome sequence analysis 133 surface proteins have been identified. In particular, the large number of proteins covalently bound to murein sets L. monocytogenes apart from other gram-positive bacteria. The ability of this pathogen to multiply in various environments as well as the possibility of its interaction with many kinds of eukaryotic cells is, in fact, made possible by the large number of surface proteins.

Animals↗

PubMed2001

Met, the HGF-SF receptor: another receptor for Listeria monocytogenes.

The bacterium Listeria monocytogenes invades a variety of cells in vitro and in vivo. Two proteins are crucial in this process: internalin, which interacts with E-cadherin, and InlB. The first identified ligand for InlB was gC1qR, which has no cytoplasmic domain. The newly discovered InlB receptor, Met, fits with the known InlB-induced signals.

Animals↗

PubMed2001

Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility.

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility.

Actin-Related Protein 2↗

PubMed1996

Recombinant Listeria monocytogenes cancer vaccines.

Listeria monocytogenes has recently been exploited as a vector for targeting antigens to the immune system. In the past year it has been shown to be particularly effective as a tumor antigen vector. Here we set its potency as a cancer vaccine in the context of the type of immunity induced by this facultative intracellular bacterium.

Animals↗

PubMed2004

Use of PFGE to characterize clonal relationships among Belgian clinical isolates of Listeria monocytogenes.

The Belgian Listeria Reference Centre receives between 30 and 50 human clinical strains of Listeria monocytogenes per year. In general, epidemiological data are absent or incomplete, preventing recognition of episodes of listeriosis. However, data on a clonal relationship between strains can indirectly give an idea of the occurrence of episodes. Human isolates of L. monocytogenes from 2001 were serotyped, their arsenic-cadmium resistance profiles were determined, and they were pulsotyped with the application of pulsed-field gel electrophoresis using AscI and ApaI restriction endonucleases. On five occasions, two or more strains presented the same serovar, metal-resistance profile and pulsovar, suggesting a clonal relationship. This is the first report to identify accurately potential listeriosis episodes occurring in Belgium.

Arsenic↗

PubMed2006

Genetic and phenotypic characterization of Listeria monocytogenes lineage III.

Listeria monocytogenes has been previously grouped into three evolutionary groups, termed lineages I, II and III. While lineages I and II are commonly isolated from various sources, lineage III isolates are rare and have several atypical and unique phenotypic characteristics. Relative to their prevalence in other sources, lineage III strains are overrepresented among isolates from food-production animals, and underrepresented among isolates from human clinical cases and foods. This work describes an extensive genotypic and phenotypic characterization of 46 lineage III isolates. Phylogenetic analyses of partial sigB and actA sequences showed that lineage III represents three distinct subgroups, which were termed IIIA, IIIB and IIIC. Each of these lineage III subgroups is characterized by differentiating genotypic and phenotypic characteristics. Unlike typical L. monocytogenes, all subgroup IIIB and IIIC isolates lack the ability to ferment rhamnose. While all IIIC and most IIIB isolates carry the putative virulence gene lmaA, the majority of subgroup IIIA isolates lack this gene. All three lineage III subgroups contain isolates from human clinical cases as well as isolates that are cytopathogenic in a cell culture plaque assay, indicating that lineage III isolates have the potential to cause human disease. The identification of specific genotypic and phenotypic characteristics among the three lineage III subgroups suggests that these subgroups may occupy different ecological niches and, therefore, may be transmitted by different pathways.

Animals↗

PubMed2006

Interference of components of the phosphoenolpyruvate phosphotransferase system with the central virulence gene regulator PrfA of Listeria monocytogenes.

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.

Bacterial Proteins↗

PubMed2002

Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation.

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32 degrees C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.

Bacterial Adhesion↗

PubMed2004

Characterization of the calcium-binding sites of Listeria monocytogenes InlB.

The Listeria monocytogenes protein InlB promotes invasion of mammalian cells through activation of the receptor tyrosine kinase Met. The InlB N-cap, a approximately 40 residue part of the domain that binds Met, was previously observed to bind two calcium ions in a novel and unusually exposed manner. Because subsequent work raised questions about the existence of these calcium-binding sites, we assayed calcium binding in solution to the InlB N-cap. We show that calcium ions are bound with dissociation constants in the low micromolar range at the two identified sites, and that the sites interact with one another. We demonstrate that the calcium ions are not required for structure, and also find that they have no appreciable effect on Met activation or intracellular invasion. Therefore, our results indicate that the sites are fortuitous in InlB, but also suggest that the simple architecture of the sites may be adaptable for protein engineering purposes.

Bacterial Proteins↗

PubMed2000

Osmotic and chill activation of glycine betaine porter II in Listeria monocytogenes membrane vesicles.

Listeria monocytogenes is a foodborne pathogen known for its tolerance to conditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organism's tolerance to both of these stresses. A procedure was developed for the purification of membranes from L. monocytogenes cells in which the putative ATP-driven glycine betaine permease glycine betaine porter II (Gbu) is functional. As is the case for the L. monocytogenes sodium-driven glycine betaine uptake system (glycine betaine porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the gbu gene product had no uptake activity. Transport by this porter did not require sodium ion and could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically activated and cryoactivated. Under conditions of osmotic activation, the system exhibited Arrhenius-type behavior although the uptake rates were profoundly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18 degrees C. In the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4 degrees C. Kinetic analyses of the permease at 30 degrees C revealed K(m) values for glycine betaine of 1.2 and 2.9 microM with V(max) values of 2,200 and 3,700 pmol/min. mg of protein under conditions of optimal osmotic activation as mediated by KCl and sucrose, respectively.

ATP-Binding Cassette Transporters↗