Pascale Cossart

Comparative Genomics of Listeria Species

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

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.

Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci

We report the identification of a previously unknown gene, inlA, which is necessary for the gram-positive intracellular pathogen Listeria monocytogenes to invade cultured epithelial cells. The inlA region was localized by transposon mutagenesis, cloned, and sequenced. inlA was introduced into Listeria innocua and shown to confer on this normally noninvasive species the ability to enter cells. Sequencing of inlA predicts an 80 kd protein, internalin. Two-thirds of internalin is made up of two regions of repeats, region A and region B, and the C-terminus of the molecule is similar to that of surface proteins from gram-positive cocci. Internalin has a high content of threonine and serine residues, and the repeat motif of region A has regularly spaced leucine residues. As evidenced by Southern blot analysis, inlA is part of a gene family. One of them is the gene situated directly downstream of inlA, called inlB, which also encodes a leucine-rich repeat protein.

The Listeria transcriptional landscape from saprophytism to virulence

The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5′ and 3′ untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein

The intracellular pathogenic bacterium L. monocytogenes can spread directly from cell to cell without leaving the cytoplasm. The mechanism of this movement, generated through bacterially induced actin polymerization, is not understood. By analyzing an avirulent Tn917-lac mutant defective for actin polymerization, we have identified a bacterial component involved in this process. The transposon had inserted in actA, the second gene of an operon. Gene disruption of downstream genes and transformation of the mutant strain with actA showed that the actA gene encodes a surface protein necessary for bacterially induced actin assembly. Our results indicate that it is a 610 amino acid protein with an apparent molecular weight of 90 kd.

Bacterial Adhesion and Entry into Host Cells

Successful establishment of infection by bacterial pathogens requires adhesion to host cells, colonization of tissues, and in certain cases, cellular invasion-followed by intracellular multiplication, dissemination to other tissues, or persistence. Bacteria use monomeric adhesins/invasins or highly sophisticated macromolecular machines such as type III secretion systems and retractile type IV pili to establish a complex host/pathogen molecular crosstalk that leads to subversion of cellular functions and establishment of disease.

Listeria monocytogenes : a multifaceted model

The opportunistic intracellular pathogen Listeria monocytogenes has become a paradigm for the study of host–pathogen interactions and bacterial adaptation to mammalian hosts. Analysis of L. monocytogenes infection has provided considerable insight into how bacteria invade cells, move intracellularly, and disseminate in tissues, as well as tools to address fundamental processes in cell biology. Moreover, the vast amount of knowledge that has been gathered through in-depth comparative genomic analyses and in vivo studies makes L. monocytogenes one of the most well-studied bacterial pathogens.

Entry of Listeria monocytogenes into hepatocytes requires expression of InIB, a surface protein of the internalin multigene family

The intracellular bacterium Listeria monocytogenes can invade several types of normally non‐phagocytic cells. Entry into cultured epithelial cells requires the expression of inIA, the first gene of an operon, comprising two genes: inIA, which encodes internalin, an 800‐amino‐acid protein, and inIB, which encodes a 630‐amino‐acid protein. Several genes homologous to inIA are detected in the genome of L. monocytogenes; InIB is one of them. We have assessed the role of inIB In invasiveness of L. monocytogenes by constructing isogenic chromosomal deletion mutants in the inIAB locus. Our findings indicate that: i) inIB is required for entry of L. monocytogenes into hepatocytes, but not into intestinal epithelial cells; ii) inIB encodes a surface protein; iii) internalin plays a role for entry into some hepatocyte cell lines. These results provide the first insight into the cell tropism displayed by L. monocytogenes.

A single amino acid in E‐cadherin responsible for host specificity towards the human pathogen Listeria monocytogenes

Human E‐cadherin promotes entry of the bacterial pathogen Listeria monocytogenes into mammalian cells by interacting with internalin (InlA), a bacterial surface protein. Here we show that mouse E‐cadherin, although very similar to human E‐cadherin (85% identity), is not a receptor for internalin. By a series of domain‐swapping and mutagenesis experiments, we identify Pro16 of E‐cadherin as a residue critical for specificity: a Pro→Glu substitution in human E‐cadherin totally abrogates interaction, whereas a Glu→Pro substitution in mouse E‐cadherin results in a complete gain of function. A correlation between cell permissivity and the nature of residue 16 in E‐cadherins from several species is established. The location of this key specificity residue in a region of E‐cadherin not involved in cell–cell adhesion and the stringency of the interaction demonstrated here have important consequences not only for the understanding of internalin function but also for the choice of the animal model to be used to study human listeriosis: mouse, albeit previously widely used, and rat appear as inappropriate animal models to study all aspects of human listeriosis, as opposed to guinea‐pig, which now stands as a small animal of choice for future in vivo studies.

Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity

Transcriptome-wide studies in eukaryotes have been instrumental in the characterization of fundamental regulatory mechanisms for more than a decade. By contrast, in prokaryotes (bacteria and archaea) whole-transcriptome studies have not been performed until recently owing to the general view that microbial gene structures are simple, as well as technical difficulties in enriching for mRNAs that lack poly(A) tails. Deep RNA sequencing and tiling array studies are now revolutionizing our understanding of the complexity, plasticity and regulation of microbial transcriptomes.