Surface-associated, PrfA-regulated proteins of Listeria monocytogenes synthesized under stress conditions

The expression of the five clustered genes of Listeria monocytogenes: plcA, hly, mpl, actA and plcB is under the control of the positive regulation factor PrfA. Listeriolysin, encoded by the hly gene, is the only prominent PrfA-controlled gene product observed when L. monocytogenes strain NCTC 7973 is cultured in a rich medium at 37°C to the logarithmic growth phase. Stress conditions such as heat-shock or stationary culture conditions lead to the induction of additional PrfA-dependent proteins (PdPs): ActA (92 kDa), a 38kDa protein of unknown function and a 34kDa protein which probably represents PlcA. Under nutrient-stress conditions PdPs are preferentially synthesized and in addition to the already known PdPs at least five new, not yet functionally identified PdPs are detected. All PdPs are either secreted or are localized at the cell surface. Differences in the amount as well as the sizes of the PdPs are observed in different L. monocytogenes strains.     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow＇s Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow＇s milk, as screened in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Corresponding virulence genes （iap, prfA, picA, hly, mpl, actA, plcB, InlA and lnlB） were compared with L. monocytogenes reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S in mouse and chicken embryo models （50% lethal dose： 10^8.14 VS. 10^5.49 and 10^6.73 VS. 10^1.9, respectively）. The genes prfA, picA and mpl were homologous among L. monocytogenes strains H4, 10403S and EGD （〉98%）. Genes iap, hly, plcB, lnlA and lnlB of L. monocytogenes 10403S had higher homology to those of strain EGD （〉98%） than isolate H4. The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level, but 98.7% at the amino acid level. The actA gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acid deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow's Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow's milk,as screened in mouseand chicken embryonated egg models,was examined for virulence-related phenotypic traits.Correspondingvirulence genes (iap,prfA,plcA,hly,mpl,actA,plcB,InlA and InlB) were compared with L.monocytogenesreference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence.Al-though L.monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity,invitro growth and invasiveness and even had higher adhesiveness,faster intracellular growth and higherphospholipase activity in vitro,it was substantially less virulent than the strain 10403S in mouse and chickenembryo models (50% lethal dose:10~(8.14) vs.10~(5.49) and 10~(6.73) vs.10~(1.9),respectively).The genes prfA,plcA andmpl were homologous among L.monocytogenes strains H4,10403S and EGD (>98%).Genes iap,hly,plcB,InlA and InIB of L.monocytogenes 10403S had higher homology to those of strain EGD (>98%) than isolateH4.The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level,but 98.7% at the amino acid level.The actA gene of isolate H4 had deletions of 105 nucleotides correspondingto 35 amino acid deletions falling Within the proline-rich region.Taken together,this study presents someclues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletionmutations of actA.     

Type I IFN are host modulators of strain-specific Listeria monocytogenes virulence

Type I IFN (IFN-I) increase the sensitivity of cells and mice to lethal infection with Listeria monocytogenes . Therefore the amount of IFN-I produced during infection might be an important factor determining Listeria virulence. Two commonly used strains of L. monocytogenes , EGD and LO28, were identified as, respectively, low and high inducers of IFN-I synthesis in infected macrophages. Increased IFN-I production resulted from the stronger ability of the LO28 strain to trigger the IRF3 signalling pathway and correlated with an increased sensitization of macrophages to lethal infection. In contrast, stimulation of NFκB, MAPK, or inflammasome signalling by the LO28 and EGD strains did not differ significantly. The LO28 strain was more virulent in wild-type (wt) C57/BL6 mice than the EGD strain whereas both strains were similarly virulent in IFN-I receptor-deficient C57/BL6 mice. Together our data suggest that isolates of wt L. monocytogenes differ in their ability to trigger the IRF3 signalling pathway and IFN-I production, and that the amount of IFN-I produced during infection is an important determinant of Listeria virulence.     

Protective immunity and granulomatous inflammation is mediated in vivo by T cells reactive to epitopes common to avirulent and listeriolysin-negative mutants of Listeria monocytogenes.

The ability of several listeriolysin O-negative mutants of the EGD and NCTC 7973 strains of Listeria monocytogenes to activate specific T cell responses in vitro and in vivo was determined. T cell lines from different inbred mouse strains and derived T cell clones elicited by L. monocytogenes, strain EGD, which are able to adoptively transfer protection and granuloma formation were examined. Specificity testing revealed no differences between listeriolysin-positive and -negative strains to induce proliferation of the T cell lines and clones. Similar results were obtained when we examined CD4+ T cell-mediated granuloma formation in the livers of mice previously immunized with viable bacteria of the virulent strain. Granulomatous inflammation could be elicited by iv application of heat-killed bacteria of listeriolysin-positive and of -negative bacteria. Protective immunity to listerial infections and granulomatous inflammation therefore appears to be mediated by T cells recognizing epitopes on listerial antigens that are shared by both pathogenic and nonpathogenic Listeria strains.     

Isolation of Listeria monocytogenes mutants with high-level in vitro expression of host cytosol-induced gene products

The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol. actA, the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L. monocytogenes escape from the phagosome. To identify bacterial factors that participate in the intracellular induction of actA expression, L. monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis. The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression. Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression. Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L. monocytogenes virulence gene expression. PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfA-dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice. Both prfA mutant strains were significantly less motile than wild-type L. monocytogenes. These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L. monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells.