The Host Response to Listeria monocytogenes Mutants Defective in Genes Encoding Phospholipases C (plcA,plcB) and Actin Assembly (actA)

Several genes involved in the determination of Listeria monocytogenes pathogenesis have been identified. Among them, plcA gene encodes phosphatidylinositol-specific phospholipase C (PI-PLC), plcB gene encodes a broad-range phospholipase C (PC-PLC), and actA encodes a protein contributing to actin assembly in infected cells. The interaction of L. monocytogenes wild type (LO 28) strain and two derivative mutants, plcA^? (BUG 206) and actA^?/plcB^? (LUT 12), with macrophages and T lymphocytes was investigated in a mouse model of listeriosis. Both mutants showed evidence of attenuation. The plcA^? mutant, but not the plcB^? mutant, expressed an increase in susceptibility to the anti-listerial activity of macrophages. Both mutants showed a decreased ability to induce IL-12 production by bone marrow macrophages when co-stimulated with E. coli LPS or IFN-γ. In vivo, L. monocytogenes plcA^? mutant was found to be a more effective stimulator of T cells than the wild LO 28 strain.     

A contingency locus in prfA in a Listeria monocytogenes subgroup allows reactivation of the PrfA virulence regulator during infection in mice

A nonhemolytic Listeria monocytogenes strain isolated from a fish processing plant was avirulent in a plaque-forming assay and in a subcutaneous mouse virulence assay. However, it showed 60% lethality (9/15 mice) when 10⁹ CFU were intraperitoneally injected into mice. Hemolytic L. monocytogenes bacteria were recovered from liver and spleen of the deceased mice, and the pulsed-field gel electrophoresis patterns were indistinguishable for the nonhemolytic and the hemolytic isolates. Sequencing of prfA from the nonhemolytic strain revealed a duplication of 7 bp in the helix-turn-helix region, resulting in a truncated PrfA protein. We propose that the direct repeat of 7 bp causes a reversible inactivation of prfA and that slipped-strand mispairing regulates the phase variation in hemolytic activity and virulence. Nonhemolytic L. monocytogenes strains with identical duplications in prfA were isolated from several sources in France, as well as in Norway, suggesting that the reversible inactivation described in this study is not an isolated event.     

Isolation and characterization of Listeria monocytogenes from tropical seafood of Kerala, India

Listeria monocytogenes, which is an intracellular pathogen, causes various illnesses in human as well as in animals. The pathogenicity of this organism depends upon the presence of different virulence genes. A total of 324 tropical seafood and fishery environmental samples were screened for L. monocytogenes. The incidence of the human pathogenic species L. monocytogenes was 1.2 % of the samples. Listeria spp. was detected in 32.3, 27.1, and 5 % of fresh, frozen, and dry fish samples, respectively. Listeria innocua was found to be the most prevalent species of Listeria in the tropical seafood and environmental samples of Kerala. Listeria monocytogenes and L. innocua isolates were confirmed by multiplex PCR. L. monocytogenes isolates from the four positive samples showed phosphatidylinositol-specific phospholipase C reaction on Chromocult® Listeria selective agar. Molecular characterization of L. monocytogenes isolates for virulence genes revealed the presence of β-hemolysin (hly), plcA, actA, metalloprotease (mpl), iap and prfA genes in all the isolates recovered from the positive samples.     

Characterization of the prfA Virulence Gene Cluster Insertion Site in Non-Hemolytic Listeria spp.: Probing the Evolution of the Listeria Virulence Gene Island

The prfA virulence gene cluster is present between prs and ldh in the pathogenic L. monocytogenes and L. ivanovii, but absent from the non-pathogenic L. innocua and L. welshimeri. To probe the evolution of this virulence gene cluster, we sequenced the prs-ldh intergenic region in L. welshimeri and L. innocua. Two ORFs (ORFA and ORFB) were found in both species as well as in L. monocytogenes. Another ORF of unknown function (ORFZ) was found in L. monocytogenes and L. innocua, while two unique ORFs were present in L. welshimeri. ORFA and ORFB showed significant functional constraint, suggesting that further investigations in the functions of these genes, including possible roles in horizontal gene transfer or sequence deletion, are warranted. DNA sequences homologous to Tn1545 integration consensus sequences were found downstream of prs and ORFB, thus defining the likely junctions of the virulence gene island and indicating that the prs-ldh intergenic region may represent a Tn insertion hot spot. Our results are consistent with the hypothesis that a combination of horizontal gene transfer and deletion events may have been involved in the evolution of the prfA virulence gene cluster in Listeria. Received: 27 November 2000 / Accepted: 20 February 2001     

A study on the effects of some laboratory-derived genetic mutations on biofilm formation by Listeria monocytogenes

Biofilms formed by the human pathogen Listeria monocytogenes in food-processing environments can be a potential source of contamination. In this study, we investigated the ability of L. monocytogenes wild type and its laboratory-derived isogenic mutants in cwhA, prfA, agrA, flaA, degU, ami and sigB to adhere to and form biofilms on abiotic surfaces. The results suggest that inactivation of the two component regulatory system degU completely abolished biofilm formation, while inactivation of the flagellar gene flaA, two component response regulator agrA and the autolysin-adhesin gene ami lead to severe impairment of initial attachment and the subsequent development of a mature biofilm by L. monocytogenes. Mutants in the global regulator of virulence prfA and the alternative sigma factor sigB were unaffected and formed biofilms similar to wild type L. monocytogenes.     

Vacuolar H+-ATPase and weak base action in Dictyostelium

Infection of a murine-spleen dendritic cell line by Listeria monocytogenes was found to induce cell death through apoptosis. To characterize the bacterial product(s) involved in induction of apoptosis, dendritic cells were infected with the L. monocytogenes EGD strain and several isogenic mutants deficient in the production of individual listerial virulence factors. The ability to induce cellular apoptosis was retained by all mutants tested, except the prfA and Δhly mutants, both of which are unable to produce listeriolysin. Apoptosis was also induced by purified listeriolysin suggesting that this protein directly induces apoptosis. Purified recombinant listeriolysins rendered either weakly haemolytic by a C-484 to S mutation, or non-haemolytic by a W-491 to A mutation exhibited little or no capacity to induce apoptosis, indicating that both activities are associated within the same protein region. Treatment with purified listeriolysin or L. monocytogenes infection also triggers apoptosis in explanted bone-marrow dendritic cells. Thus invasion of dendritic cells by L. monocytogenes, which results in cell death, may play an important role in the pathogenesis of listerial infections by impairing immune responses, hindering bacterial clearance and promoting spread of the infection.     

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.