A mutant of Listeria monocytogenes EGD was constructed that carries an extended deletion removing the entire PrfA-regulated gene cluster from plcA to plcB and a second deletion inactivating the inlA gene. Upon supplementation of this mutant with multiple gene copies of prfA, a protein of 30 kDa was detected in the supernatant of the mutant strain. The gene encoding this protein was obtained by direct and inverse polymerase chain reaction using oligonucleotide primers that were deduced from partial amino acid sequences of the purified 30 kDa protein. The amino acid sequence of the gene product revealed a protein of 297 amino acids that carried eight repeat units with high homology to those of the two known internalin proteins A and B. This secretory protein, termed internalin C, is much smaller than InlA or InlB and its complete sequence is related to the two known internalins. The gene InlC is transcribed into a monocistronic mRNA from a single promoter which shows a typical consensus sequence for PrfA-binding at the position -40. In contrast to the transcription of the InlAB operon, which is downregulated after shift of an L. monocytogenes EGD culture from brain-heart infusion into minimum essential medium (MEM), transcription of inlC is induced in MEM like most of the other known PrfA-regulated virulence genes. In addition, InlC is strongly transcribed in the cytoplasm of phagocytic J774 cells whereas inlA is poorly transcribed under these conditions, suggesting that internalin C may play a role in a late stage of L. monocytogenes infection rather than in the uptake of L. monocytogenes by non-professional phagocytic cells. An InlC deletion mutant shows reduced virulence when tested in an intravenous mouse model, but intracellular replication of the mutant in Caco-2 and J774 cells appears to be comparable with that of the wild-type strain.

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-beta-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615-1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and the 5'-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA-independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain.

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show thatL. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressingL. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growingL. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D1 macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.

SummarySeveral large, cell wall-associated internalins and one small, secreted internalin (InlC) have been described previously in Listeria monocytogenes. Using degenerate primers derived from sequenced peptides of an L. ivanovii major secreted protein, we identified a new 4.25 kb internalin locus of L. ivanovii, termed i-inl FE. The two proteins encoded by this locus, i-InlE and i-InlF, belong to the group of small, secreted internalins. Southern blot analyses show that the i-inl FE locus does not occur in L. monocytogenes. These data also indicate that six genes encoding small, secreted internalins are present in L. ivanovii, in contrast to L. monocytogenes, in which inl C encodes the only small internalin. The mature i-InlE protein (198 amino acids) is secreted in large amounts into the brain-heart infusion (BHI) culture medium in the stationary growth phase. In minimum essential medium (MEM), which has been used previously to induce PrfA-dependent gene transcription, i-inl E mRNA and i-InlE protein are expressed at high levels. As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i-inl F and i-inl E genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences ('PrfAboxes') were identified in the promoter regions of i-inl F and i-inl E. Non-polar i-inl E and i-inl F deletion mutants and an i-inl FE double deletion mutant were constructed and tested in the mouse infection model. After intravenous infection, all three mutants entirely failed to kill C57BL /6 mice even at high infectious doses of 10 9 bacteria per mouse, whereas the LD 50 for the parental strain was determined as 4 × 10 7 bacteria per mouse. These data suggest an important role for i-InlE and i-InlF in L. ivanovii virulence.