Fascin-mediated propulsion of Listeria monocytogenes independent of frequent nucleation by the Arp2/3 complex

Actin-dependent propulsion of Listeria monocytogenes is thought to require frequent nucleation of actin polymerization by the Arp2/3 complex. We demonstrate that L. monocytogenes motility can be separated into an Arp2/3-dependent nucleation phase and an Arp2/3-independent elongation phase. Elongation-based propulsion requires a unique set of biochemical factors in addition to those required for Arp2/3-dependent motility. We isolated fascin from brain extracts as the only soluble factor required in addition to actin during the elongation phase for this type of movement. The nucleation reaction assembles a comet tail of branched actin filaments directly behind the bacterium. The elongation-based reaction generates a hollow cylinder of parallel bundles that attach along the sides of the bacterium. Bacteria move faster in the elongation reaction than in the presence of Arp2/3, and the rate is limited by the concentration of G-actin. The biochemical and structural differences between the two motility reactions imply that each operates through distinct biochemical and biophysical mechanisms.     

An iron-dependent mutant of Listeria monocytogenes of attenuated virulence

Abstract A bank of Tn 917 -insertional mutants from the facultative intracellular pathogen Listeria monocytogenes was screened by an original method based on bacterial growth on synthetic medium under iron-limiting conditions. One mutant, whose in vitro growth in synthetic medium was specifically dependent upon the availability of iron in its environment, was isolated and characterized. The insertional event occurred in a non-coding region, upstream of a rrn operon and located within a 1100-kb Not I fragment of the physical map, where the virulence genes already identified in L. monocytogenes were also present. Protein analysis by SDS-PAGE revealed a pleiotropic effect of the insertional event on cell-associated proteins, suggesting a polar effect of the transposon on adjacent unknown gene(s). The virulence in the mouse of this mutant was strongly impaired, although it was capable in vitro of growing intracellularly and of spreading from cell to cell, as shown by the production of lytic plaques on cell culture.     

CD8 T cell immunome analysis of Listeria monocytogenes

The identification of T cell epitopes is crucial for the understanding of the host response during infections with pathogenic microorganisms. Generally, the identification of relevant T cell responses is based on the analysis of T cell lines propagated in vitro. We used an ex vivo approach for the analysis of the CD8 T cell response against Listeria monocytogenes that is based upon the fractionation of naturally processed antigenic peptides and subsequent analysis with T cells in an enzyme-linked immunospot (ELISPOT) assay. Our data indicate that the direct ex vivo ELISPOT analysis of peptides extracted from infected tissues represents a versatile and potent test system for the analysis of the CD8 T cell immunome of microorganisms that furthermore requires neither the knowledge of the microbial genome nor of the specificity of responding T cells.     

Autolysis of Listeria monocytogenes

Autolytic curves of five representative strains of Listeria monocytogenes are described. Of 24 strains so far examined, the majority are unstable in vitro.     

Autolysis of Listeria monocytogenes

Physiological conditions that could provide maximal rates of autolysis of Listeria monocytogenes were examined. L. monocytogenes was found to be refractory to most treatments that promote rapid autolysis in other bacteria. Best rates of autolysis were obtained after resuspending the cells in Tris-hydrochloride buffer at 37 degrees C with the pH optimum at 8.0. Autolysis was also efficiently promoted by the surfactant Triton X-100. Antibiotics that interfere with the biosynthesis of the cell wall murein (peptidoglycan) caused death of the cells without autolysis after prolonged incubation in the presence of the drug. Only nisin, which has been shown to bind in vitro to the murein precursors lipid I and lipid II brings about autolysis of L. monocytogenes cells, although with slower kinetics than in the case of Tris-HCl and Triton.     

Mutational analysis of the role of HPr in Listeria monocytogenes

The regulatory role of HPr, a protein of the phosphotransferase system (PTS), was investigated in Listeria monocytogenes. By constructing mutations in the conserved histidine 15 and serine 46 residues of HPr, we were able to examine how HPr regulates PTS activity. The results indicated that histidine 15 was phosphorylated in a phosphoenolpyruvate (PEP)-dependent manner and was essential for PTS activity. Serine 46 was phosphorylated in an ATP-dependent manner by a membrane-associated kinase. ATP-dependent phosphorylation of serine 46 was significantly enhanced in the presence of fructose 1,6-diphosphate and resulted in a reduction of PTS activity. The presence of a charge at position 15 did not inhibit ATP-dependent phosphorylation of serine 46, a finding unique to gram-positive PEP-dependent PTSs studied to this point. Finally, HPr phosphorylated at serine 46 does not appear to possess self-phosphatase activity, suggesting a specific phosphatase protein may be essential for the recycling of HPr to its active form.     

Antimicrobial resistance of Listeria monocytogenes

Listeria monocytogenes is an opportunistic pathogen that causes rare but frequently fatal infections, termed listerioses. In general, strains of L. monocytogenes are susceptible to a wide range of antibiotics, except for the cephalosporins, fluorochinolones and fosfomycin (Hof, 1991). The current therapy of choice is a combination of ampicillin and aminoglycoside, usually gentamicin (Lorber, 1997). In cases when it is not possible to use a beta-lactam antibiotic, second-choice therapy involves the use of an association of trimethoprim with a sulfonamide, such as in co-trimoxazole, in which the more active in the combination seems trimethoprim, synergized by the sulfa compound. Other second line agents for listeriosis include erythromycin and vancomycin (Temple and Nahata, 2000). The first strains of L. monocytogenes resistant to antibiotics were reported in 1988 (Poyart-Salmeron et al. 1990) The present paper reviews the current state of affairs with regard to the resistance of L. monocytogenes isolated from food products and clinical material to different antibiotics, with particular emphasis on those used in the therapy of listeriosis.     

Comparative proteomic analysis of Listeria monocytogenes tolerance to bile stress

The ability of the food-borne pathogen Listeria monocytogenes to tolerate bile is critical to its successful infection and colonization in the human gastrointestinal tract. Using comparative proteomics, a total of 48 proteins were identified in this study in the presence of moderate (0.3 %) or high (3 %) level of bile salts in the wild-type strain EGD. Identified proteins fell into 14 functional categories covering most of the biochemical functions of bacterial cells, indicating that there were complex physiological mechanisms involved in L. monocytogenes tolerance of bile stress. Among them, 16, 14, and 18 proteins were expressed differently in the isogenic deletion mutants of L. monocytogenes EGDΔsigB, EGDΔprfA, and EGDΔprfAΔsigB, respectively, compared with their parent strain EGD at corresponding concentrations of bile salts. All proteins identified in EGDΔsigB and EGDΔprfAΔsigB were all down-expressed in the presence of bile salts, whereas several proteins were up-expressed in EGDΔprfA, in particular at the high level of bile (3 %), indicating that SigB plays an essential positive role in L. monocytogenes tolerance of bile stress and that the negative effect of PrfA may facilitate its survival in bile in the gastrointestinal tract before its successful colonization and invasion.     

16S rRNA analysis of Listeria monocytogenes and Brochothrix thermosphacta

Abstract Listeria monocytogenes and Brochothrix thermosphacta were investigated by the 16S rRNA cataloguing approach in order to determine their phylogenetic relationship. Both species are specifically, although moderately related, forming one of several sublines within the Bacillus-Lactobacillus-Streptococcus cluster of the ' Clostridium ' subbranch of Gram-positive eubacteria.     

An investigation of the antibacterial effect of carrot on Listeria monocytogenes

Carrot slices immersed in a potassium phosphate buffer (0.1 mol/l, pH 7.0) or carrot tissue macerated in the buffer had a lethal effect on Listeria monocytogenes. This antilisterial activity was suppressed by anaerobiosis, thiol compounds (1 mmol/l) and bovine serum albumin (0.05%) but was not affected by sodium ascorbate (200 mmol/l), propyl gallate (25 mmol/l), catalase (1100 U/ml), superoxide dismutase (357 U/ml), or chelating agents (10 mmol/l). Free-radical scavengers had no effect at 10 mmol or 50 mmol/l but histidine and diazabenzocyclooctane at 100 mmol/l reduced the antilisterial activity. The addition of Tween 20, 0.05% (v/v), to carrot macerates improved the recovery of the activity in the supernatant liquid after centrifugation at 10 000 g for 2 min. The addition of higher concentrations of the detergent to the macerate reduced the antilisterial activity.     

Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria

Minimum growth temperatures and those of decreased growth were determined for 100 strains of listerias. The ability of 78 strains of Listeria monocytogenes isolated from animals and 22 non-haemolytic strains to grow at low temperatures was studied, using a flooding technique, in a plate-type continuous temperature gradient incubator at temperatures between -1.6 and 14.5 degrees C. The mean minimum temperature for L. monocytogenes was +1.7 +/- 0.5 degrees C. The growth of non-haemolytic listerias was unobservable at +1.7 +/- 0.5 degrees C. The L. monocytogenes strains grew at about 0.6 degrees C lower than the non-pathogenic strains. No differences in growth temperatures were observed among L. monocytogenes strains isolated from different sources. The serovars with the OI antigen grew at lower temperatures (+1.0 +/- 0.3 degrees C) than the other common serovar 4b (+1.3 +/- 0.4 degrees C). The results indicate that L. monocytogenes grows better than non-haemolytic strains under cold conditions. The possible role of haemolysins as growth factors is also discussed.     

Neuroinfections due to Listeria monocytogenes

Listeria monocytogenes is not a rare pathogen causing meningitis, mainly in small children and in close contacts to livestock. The pathogen is naturally resistant to cephalosporins and some glycopeptides as well, therefore despite of syndromologic diagnosis of meningitis and initial therapy with 3rd generation cephalosporins according to the guidelines therapeutic failures with clinical consequences may occur.     

Chemiluminescence by Listeria monocytogenes.

Listeria monocytogenes cells suspended in brain heart infusion broth or in carbonated saline solution emitted light (chemiluminescence) that could be detected by a liquid scintillation spectrometer. This chemiluminescence was inhibited by superoxide dismutase and catalase but not by the hydroxyl radical scavengers mannitol and benzoate; it was also dependent upon and proportional to the carbonate ion concentration in the medium. Organisms suspended in carbonated saline solution which had ceased to chemiluminesce immediately began to chemiluminesce again when acetaldehyde was added but not when glucose, sucrose, or xanthine was added. Acetaldehyde-induced chemiluminescence was inhibited by suproxide dismutase and catalase but not by allopurinol. Our data indicate that the superoxide anion, hydrogen peroxide, and the carbonate ion are involved in chemiluminescence by L. monocytogenes. Chemiluminescence is apparently initiated by the extracellular generation of superoxide anon by this organism. The mechanism for the production of the superoxide anion is not known, but xanthine oxidase does not appear to be involved.     

Listeria monocytogenes in pasteurized milk

An haemolytic Listeria monocytogenes strain pathogenic to mice was isolated from 6 out of 28 (21.4%) pasteurized milk samples (3.2% fat milk treated at 78 degrees C for 15 s) marketed by a Madrid processing plant. Listeria grayi was recovered from 25 of the samples (89.2%) and L. innocua from 3 samples (10.7%). One milk sample was contaminated with L. welshimeri. No strains of L. ivanovii, L. seeligeri, L. murrayi, or L. denitrificans were isolated. These results show that pathogenic Listeria strains can be isolated from pasteurized milk and reinforce the hypothesis that this food product may be the source of numerous human listeriosis.