Correlation between the Presence of Virulence-Associated Genes as Determined by PCR and Actual Virulence to Mice in Various Strains of Listeria Spp.

Five chromosomal genes, prfA, plcA, hlyA, mpl and plcB, are implicated in the virulence of Listeria monocytogenes and some of these genes have been used for the identification of bacteria by polymerase chain reaction (PCR). Using 6 strains of L. monocytogenes and 3 L. innocua strains, the relationship was examined between the presence of five virulence-associated genes and actual virulence to mice in terms of 50% lethal dose (LD₅₀), bacterial viability in the organ of infected mice and the intracellular growth in cultured macrophages. None of the five genes could be amplified by PCR in all the L. innocua strains and they were actually avirulent to mice. All L. monocytogenes strains were shown to be virulent and to have intact virulence-associated genes except for the strain ATCC15313. This particular strain was revealed to be avirulent and defective in hlyA and plcA in PCR amplification. It was suggested that PCR detection of genes prfA, mpl, or plcB may not be sufficient to detect virulent strains of L. monocytogenes. It appeared that the ability to produce listeriolysin O (LLO), which is encoded by hlyA, was critical for the expression of virulence regardless of the amount of LLO produced.     

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.     

Differential expression of InlB and ActA in Listeria monocytogenes in selective and nonselective enrichment broths

Aim: To investigate the effect of selective and nonselective media on the expression of ActA and InlB proteins in Listeria monocytogenes. Methods and Results: Polyclonal antibodies to InlB and ActA were used in western blotting to determine the effect of selective (BLEB, UVM, and FB) or nonselective (BHI and LB) enrichment broths or hotdog exudates. Of the 13 L. monocytogenes serotypes tested, 11 and 12 serotypes showed a strong InlB expression in brain heart infusion (BHI) and Luria‐Bertani (LB), respectively, while only seven and one serotypes showed a strong ActA expression in these two respective broths, and others showed a weaker or no expression. On the contrary, in selective broths, expression of InlB was either very weak or undetectable. However, ActA expression was stronger in 12 serotypes when grown in buffered Listeria enrichment broth (BLEB), 11 in University of Vermont medium (UVM), and 10 in Fraser broth (FB). When tested in hotdog exudates, InlB and ActA were detected in serotypes grown at 37°C but not at 4°C. Transmission electron microscopy, enzyme‐linked immunosorbent assay, and mRNA analysis further supported these observations. Conclusion: Overall, selective enrichment broths promote ActA while nonselective broths promote InlB expression. Significance and Impact of the study: As commonly recommended enrichment broths show differential InlB and ActA expression, proper media must be selected to avoid false results during antibody‐based detection of L. monocytogenes.     

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.     

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.     

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.     

Expression of ActA, Ami, InlB, and Listeriolysin O in Listeria monocytogenes of Human and Food Origin

Expression of proteins involved in the adhesion of Listeria monocytogenes to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of L. monocytogenes strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90; P = 1 × 10⁻⁴). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89; P = 1 × 10⁻²) and central nervous system infections were associated with group ActA2 strains (OR = 3.04; P = 1 × 10⁻³) and group ActA3 strains (OR = 3.91; P = 1 × 10⁻³).     

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.     

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.     

Mutations affecting hemolysin production in Listeria monocytogenes located outside the listeriolysin gene

We have investigated the molecular basis of spontaneous mutations leading to non-hemolytic and avirulent variants of the Listeria monocytogenes serotype 1/2a strain NCTC 7973 using Southern hybridization to DNA fragments that harbor the listeriolysin gene (hlyA) and adjacent regions cloned from a L. monocytogenes serotype 1/2a strain. The analysis of such non-hemolytic variants revealed the presence of a deletion of 300 base pairs, located 1.6 kb upstream of an otherwise intact listeriolysin gene. The importance of regions upstream of the hlyA gene in controlling the expression of the listeriolysin gene was further emphasized by the detection of a transposon-derived nonhemolytic mutant in which the transposon had inserted approximately 200 bp upstream of the listeriolysin gene. We conclude that at least two elements, contained within a region encompassing 1.6 kb of sequences upstream of the hlyA gene, may be required for expression of the listeriolysin gene.     

Characteristics of cell-mediated,anti-listerial immunity induced by a naturally avirulent <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> serotype 4a strain HCC23

The characteristics of cell-mediated, anti-listerial immune response initiated by an avirulent Listeria monocytogenes serotype 4a strain HCC23 was assessed. Similar to virulent strain EGD, avirulent strain HCC23 grew readily within macrophage-like J774 cells, but nonhemolytic strain ATCC 15313 did not. Compared with EGD, HCC23 induced a relatively low level of gamma interferon (IFN-γ) in mice, and ATCC 15313 stimulated no detectable IFN-γ. The percentages of gated CD4 T cells from mice immunized with EGD and HCC23 showed a notable drop (to 30%) at 21 days post exposure in comparison with that (about 50%) from ATCC 15313-injected or untreated mice; and the percentage of gated NK cells from EGD-immunized group was markedly higher than those from other treatment groups. Mice immunized with HCC23 and EGD developed an equally strong protective immunity against listeriosis that was effective in both short and long terms, but those injected with ATCC 15313 or saline succumbed to listeriosis within 6 days of challenge.     

The molecular mechanisms of listeriolysin O-induced lipid membrane damage

Listeria monocytogenes is an intracellular food-borne pathogen that causes listeriosis, a severe and potentially life-threatening disease. Listeria uses a number of virulence factors to proliferate and spread to various cells and tissues. In this process, three bacterial virulence factors, the pore-forming protein listeriolysin O and phospholipases PlcA and PlcB, play a crucial role. Listeriolysin O belongs to a family of cholesterol-dependent cytolysins that are mostly expressed by gram-positive bacteria. Its unique structural features in an otherwise conserved three-dimensional fold, such as the acidic triad and proline-glutamate-serine-threonine-like sequence, enable the regulation of its intracellular activity as well as distinct extracellular functions. The stability of listeriolysin O is pH- and temperature-dependent, and this provides another layer of control of its activity in cells. Moreover, many recent studies have demonstrated a unique mechanism of pore formation by listeriolysin O, i.e., the formation of arc-shaped oligomers that can subsequently fuse to form membrane defects of various shapes and sizes. During listerial invasion of host cells, these membrane defects can disrupt phagosome membranes, allowing bacteria to escape into the cytosol and rapidly multiply. The activity of listeriolysin O is profoundly dependent on the amount and accessibility of cholesterol in the lipid membrane, which can be modulated by the phospholipase PlcB. All these prominent features of listeriolysin O play a role during different stages of the L. monocytogenes life cycle by promoting the proliferation of the pathogen while mitigating excessive damage to its replicative niche in the cytosol of the host cell.     

Comparative Proteomic Analysis of Listeria monocytogenes Strains F2365 and EGD

Listeria monocytogenes is a gram-positive, food-borne pathogen that causes disease in both humans and animals. There are three major genetic lineages of L. monocytogenes and 13 serovars. To further our understanding of the differences that exist between different genetic lineages/serovars of L. monocytogenes, we analyzed the global protein expression of the serotype 1/2a strain EGD and the serotype 4b strain F2365 during early-stationary-phase growth at 37°C. Using multidimensional protein identification technology with electrospray ionization tandem mass spectrometry, we identified 1,754 proteins from EGD and 1,427 proteins from F2365, of which 1,077 were common to both. Analysis of proteins that had significantly altered expression between strains revealed potential biological differences between these two L. monocytogenes strains. In particular, the strains differed in expression of proteins involved in cell wall physiology and flagellar biosynthesis, as well as DNA repair proteins and stress response proteins.     

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.