J Mengaud

E-Cadherin Is the Receptor for Internalin, a Surface Protein Required for Entry of L. monocytogenes into Epithelial Cells

We report the first identification of a cellular receptor mediating entry of a gram-positive bacterium into nonphagocytotic cells. By an affinity chromatography approach, we identified E-cadherin as the ligand for internalin, an L. monocytogenes protein essential for entry into epithelial cells. Expression of the chicken homolog of E-cadherin (L-CAM) in transfected fibroblasts dramatically increases entry of L. monocytogenes and promotes that of a recombinant L. innocua strain expressing internalin but does not promote entry of the wild-type noninvasive L. innocua or that of an internalin-deficient mutant of L. monocytogenes. Furthermore, L-CAM-specific antibodies block internalin-mediated entry. In contrast to Salmonella, Listeria enters cells by a mechanism of induced phagocytosis occurring without membrane ruffling. This work reveals a novel type of heterophilic interactions for E-cadherin.

Pleiotropic control of Listeria monocytogenes virulence factors by a gene that is autoregulated

Evidence for ptelotropic activation of virulence genes in Listeria monocytogenes is presented. A complementation study of a spontaneous prfA‐deletion mutant and analysis of cassette and transposon insertion mutants showed that the gene prfA activates the transcription of four independent genes which code for a phosphatidyl‐inositol‐specific phospholipase C (gene plcA), listeriolysin O (gene hlyA), a metallo‐protease (gene prtA) and a lecithinase (gene prtC). Transcription of prfA is not constitutive. During the growth phase, two peaks of prfA transcript accumulation were observed: the first was during exponential growth, and the second was at the beginning of the stationary phase. In addition, two prf4‐specific transcripts of 2.2 kb and 1 kb are detected. Early in exponential growth, prfA is co‐transcribed with plcA which lies upstream prfA, giving rise to the 2.2 kb plcA‐prfA transcript. In late‐exponential growth and at the beginning of the stationary phase, prfA transcripts of 1 kb are predominantly detected. Our results demonstrate that since prfA controls plcA transcription, it also regulates its own synthesis.

Molecular and Genetic Determinants of the Listeria monocytogenes Infectious Process

Listeria monocytogenes was first characterized in 1926 following an outbreak of listeriosis in laboratory animals (MURRAY et al. 1926). However, it was not until the 1980s that an unambiguous link was established between the human disease and the consumption of Listeria-contaminated foodstuffs (SCHLECH et al. 1983). Immunosuppressed individuals, pregnant women, foetuses and neonates are most susceptible to Listeria infection. Human listeriosis is characterized by a high mortality rate, with clinical features including meningitis or meningo-encephalitis, septicemia, abortion, and perinatal infections (GRAY and KILLINGER 1966). If diagnosed early, listeriosis can be successfully treated by the administration of high doses of antibiotics, most frequently ampicillin or penicillin, either alone or in combination with aminoglycosides.

Identification of phosphatidylinositol-specific phospholipase C activity in Listeria monocytogenes: a novel type of virulence factor?

A phospholipase C which cleaves phosphatidylinositol and glycosylphosphatidylinositol (GPI) anchors was identified in Listeria monocytogenes. This 36kDa protein is encoded by the gene plcA, and is homologous to the Bacillus cereus, Bacillus thuringiensis and eukaryotic phosphatidylinositol‐specific phospholipases C (PI‐PLC). Expression of the plcA gene in Escherichia coli correlates with the appearance of PI‐PLC activity in the cells. In Listeria monocytogenes, the activity is secreted to the culture medium. PI‐PLC activity was only found in the two pathogenic species of the genus Listeria, namely L. monocytogenes and L. ivanovii. PI‐PLC activity was lost and virulence decreased when the plcA gene was disrupted in the chromosome. This suggests that the PI‐PLC of L. monocytogenes might be involved in virulence.

Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells

Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram‐positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C‐terminal end. By immunofluorescence and immunogold labelling, we show that in wild‐type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C‐terminal region of internalin is necessary for cell‐surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a‘domain‐swapping’strategy ‐ replacement of the cell wall anchor of InIA by the membrane anchor of ActA ‐ we show that the reduced ability to adhere and enter cells of strains expressing InIA‐ActA correlates with a lower amount of surface‐exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.

Use of specific oligonucleotides for direct enumeration of Listeria monocytogenes in food samples by colony hybridization and rapid detection by PCR

Two 18-mer oligonucleotides derived from the sequence of hly, the gene coding for listeriolysin O, were shown to be specific for Listeria monocytogenes in the genus Listeria in colony hybridization tests. The oligonucleotides did not hybridize with any of the bacterial species found in food and co-isolated with Listeria on selective media. They were used in colony hybridization tests for enumeration of L. monocytogenes present in food samples after direct plating on selective media plates. In addition, two 24-mer oligonucleotides, each including the sequence of one of the 18-mers, were successfully used for the PCR-based detection of L. monocytogenes bacilli present in food samples after 48-h enrichment period. Using this technique, as little as 10(2) bacteria per ml of enrichment broth can be detected.