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Toward a Complete Understanding of Human Listeriosis
Summary: Pascale Cossart is investigating infection by the bacterium Listeria monocytogenes, one of the best models for studying intracellular parasitism, host tissue tropism, and crossing of host barriers. She is characterizing the molecular strategies Listeria uses to infect cells, disseminate into tissues, and breach the host's intestinal, placental, and blood-brain barriers.
Over the past several years, we have been analyzing the molecular pathogenesis of infection by the bacterium Listeria monocytogenes, a paradigm for the study of intracellular parasitism and the crossing of host barriers. By combining an increasingly wide range of techniques and approaches, including in vivo imaging techniques, our goal is to provide a spatiotemporal identification and characterization of the cells that are infected, the bacterial factors that are used, the bacterial ligands with which the factors interact, and the signaling pathways that are subverted at each step during the infectious process.
L. monocytogenes is responsible for severe food-borne infections. It is characterized by its ability, during disease, to cross three host barriers: intestinal, blood-brain, and fetal-placental. It also has the ability to survive in macrophages. Moreover, it invades a variety of nonphagocytic cells, where it multiplies. L. monocytogenes moves in the cytosol of infected cells and propels itself from cell to cell by using cell-actin polymerization at one pole of the bacteria.
Our present activity focuses on (1) investigation and characterization, using postgenomic approaches, of the complete repertoire of genes involved in virulence and regulation of virulence; (2) the molecular cross-talk between Listeria and host cells during entry and intracellular movements; (3) cellular responses to infection in vitro and in vivo; and (4) the crossing of host barriers. In parallel, we analyze the infection by another cytosolic bacterium, Rickettsia conorii. When possible, we generalize newly discovered concepts to other bacteria or to other pathogens.
During our initial genetic studies, we identified several key genes that are important for various steps of the infectious process, in particular the invasion genes inlA and inlB, which encode internalin and InlB, and a series of virulence genes that are clustered on the chromosome. This virulence gene cluster contains actA, which encodes the protein responsible for actin polymerization, and PrfA, a transcriptional regulator. To invade cells, internalin uses as receptor the cell adhesion molecule E-cadherin, whereas InlB uses three receptors: the tyrosine kinase receptor Met, gC1qR (the receptor for the first component of the complement gC1q), and glycosaminoglycans.
The stringent species specificity of internalin for human E-cadherin led us to generate the first transgenic model used in the study of a bacterial disease—a mouse that expresses human E-cadherin—and to demonstrate how Listeria crosses the intestinal barrier at the level of enterocytes. This is the most relevant animal model to investigate orally acquired listeriosis.
A combination of epidemiologic, in vitro, and ex vivo studies allowed us to demonstrate that Listeria also uses internalin to cross the human maternofetal barrier. During entry via E-cadherin, Listeria exploits the whole junctional complex. One pending question is how the actin cytoskeleton is activated. Our recent results show that alpha-catenin binds to a newly discovered ligand called ARHGAP10 and that Src, cortactin, and Arp2/3 are required for entry. How these molecules are orchestrated to trigger entry or to allow junction formation is under investigation.
Binding of InlB to Met, its major receptor, activates, via Rac and Cdc42, the entire actin cytoskeleton machinery, which is required for efficient bacterial entry. InlB also activates the phosphatidylinositol 3-kinase pathway, although the precise role of phosphatidylinositol 3,4,5-trisphosphate (PIP3) has long remained elusive. Our recent results using fluorescence resonance energy transfer showed that PIP3 production in lipid rafts leads to Rac activation and actin rearrangements. An unexpected recent discovery was that the endocytosis protein clathrin participates in and is absolutely critical to the entry process—until recently, clathrin was considered to be involved only in internalization of molecules or small objects. Thus, this is a shift in paradigm, and we hope to generalize our findings to other bacteria and investigate the real role of clathrin in this new type of clathrin-mediated endocytosis.
After our discovery of ActA and that of other investigators who showed that ActA activates the Arp2/3 complex, we analyzed the actin-based motility of Rickettsia conorii and discovered the gene rickA, which encodes a protein that also activates Arp2/3. We are trying to determine how Rickettsia generates actin tails, where actin filaments are long and bundled and not branched as in Listeria tails. ActA-mediated actin-based motility is one of the best examples of how the study of a bacterial factor can lead to key discoveries in cell biology.
A feature of bacterial pathogens is that they often express their virulence factors in a coordinated manner. In Listeria, this is achieved through activation by PrfA. The prfA gene is itself thermoregulated via an RNA thermosensor, a structure of its untranslated region that forms at low temperature and sequesters the ribosome binding site, thus preventing translation. At higher temperatures, the structure melts and allows translation and activation of the PrfA regulon. The prfA RNA thermosensor can be considered as a special form of riboswitch for which the effector is temperature. Other riboswitches are predicted in other bacteria.
We achieved an important milestone when, in the framework of a European consortium, we determined the complete genome sequence of L. monocytogenes and that of Listeria innocua, a related nonpathogenic species. This paved the way for a series of postgenomic studies, including transcriptomic studies, which helped us find other virulence genes regulated by PrfA, such as a bile salt hydrolase that helps the bacterium survive the hostile bile salts secreted in the intestinal tract during digestion. One of the main results of the sequencing project was the discovery that Listeria encodes a remarkable number of surface proteins, including the internalin family, which has 25 members. One of them, InlJ, is a newly discovered virulence factor that is unique to L. monocytogenes and whose role is unknown. We have embarked on a systematic but targeted study of surface and secreted proteins unique to the pathogenic species but absent from L. innocua. We use two-hybrid screens to find their ligands. We inactivate the corresponding genes and analyze, in vivo and in relevant animal models, the functions of these proteins.
Other important data provided by complete genomes are the sequences of intergenic regions. They often encode small noncoding RNAs. We found nine novel noncoding RNAs, whose role in virulence is under investigation. Another striking discovery was a gene encoding a deacetylase that deacetylates the N-acetylglucosamine residues of the peptidoglycan and, during inflammation, allows Listeria to escape degradation by lysozymes in the intestine and inside macrophages. This was the first demonstration that a peptidoglycan modification can help bacteria escape host innate immune defenses.
We have embarked on several studies aimed at analyzing the host response to Listeria. One of them, carried out in collaboration with Jeff Gordon (Washington University, St. Louis), was to analyze the host response to Listeria in germ-free transgenic mice bearing human E-cadherin. This study led to the unexpected result that invasion of the enterocytes was not critical for the early response of the host, whereas expression of listeriolysin, the protein that was first described as the key element for escape from the internalization vacuole, was the most potent signaling molecule.
Last updated April 2007
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