Complete genome sequence of the animal pathogen Listeria ivanovii, which provides insights into host specificities and evolution of the genus Listeria.

We report the complete and annotated genome sequence of the animal pathogen Listeria ivanovii subsp. ivanovii strain PAM 55 (serotype 5), isolated in 1997 in Spain from an outbreak of abortion in sheep. The sequence and its analysis are available at an interactive genome browser at the Institut Pasteur (http://genolist.pasteur.fr/LivaList/).

Actin-based bacterial motility.

Listeria monocytogenes and other bacterial pathogens move in the host cell cytoplasm, propelled by continuous actin assembly at one pole of the bacterium. This actin-based motility requires the presence of the bacterial proteins ActA on L. monocytogenes and IcsA on Shigella flexneri. There have been several major discoveries in the past year: the discovery of the polar distribution of ActA and IcsA on the bacterial surface; the demonstration that bacterial ActA is phosphorylated in infected host cells; the involvement of some host cell proteins, particularly profilin; and the dramatic effect of ActA expression in transfected eukaryotic cells. A cell-free system that reconstitutes faithfully the actin-based motility of L. monocytogenes promises to be instrumental in the further dissection of this fascinating phenomenon.

Interaction of invasive bacteria with host signaling pathways.

Many bacterial pathogens exploit mammalian cell functions in order to promote their adherence to or uptake by host cells. Recent work has led to the identification of some of the bacterial and mammalian proteins involved in these processes. Although specific mechanisms differ among pathogens, a common aspect appears to be regulation of signaling pathways that control the actin cytoskeleton.

Actin-based bacterial motility: towards a definition of the minimal requirements.

At the border line between microbiology and cell biology, the spectacular capacity o f some intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and several Rickettsias, to use actin polymerization as a driving force for intracellular movement, cell-to-cell spreading and dissemination within the infected tissue is being increasingly studied. Now that it is possible to manipulate the bacterial surface proteins involved in this process - ActA o f L. monocytogenes and IcsA of S. flexneri - these bacterial systems are providing experimental models in which to investigate the role o f actin filament dynamics in cell motility.

Actin-based movement of Listeria monocytogenes: actin assembly results from the local maintenance of uncapped filament barbed ends at the bacterium surface.

The thermodynamic basis for actin-based motility of Listeria monocytogenes has been investigated using cytoplasmic extracts of Xenopus eggs, initially developed by Theriot et al. (Theriot, J. A., J. Rosenblatt, D. A. Portnoy, P. J. Goldschmidt-Clermont, and T. J. Mitchison. 1994. Cell. 76:505-517) as an in vitro cell-free system. A large proportion (75%) of actin was found unpolymerized in the extracts. The amount of unassembled actin (12 microM) is accounted for by the sequestering functions of T beta 4Xen (20 microM) and profilin (5 microM), the barbed ends being capped. Movement of Listeria was not abolished by depletion of over 99% of the endogenous profilin. The proline-rich sequences of ActA are unlikely to be the target of profilin. All data support the view that actin assembly at the rear of Listeria results from a local shift in steady state due to a factor, keeping filaments uncapped, bound to the surface of the bacterium, while barbed ends are capped in the bulk cytoplasm. Movement is controlled by the energetic difference (i.e., the difference in critical concentration) between the two ends of the filaments, hence a constant ATP supply and the presence of barbed end capped F-actin in the medium are required to buffer free G-actin at a high concentration. The role of membrane components is demonstrated by the facts that: (a) Listeria movement can be reconstituted in the resuspended pellets of high speed-centrifuged extracts that are enriched in membranes; (b) Actin-based motility of endogenous vesicles, exhibiting the same rocketing movement as Listeria, can be observed in the extracts.

A role for cofilin and LIM kinase in Listeria-induced phagocytosis.

The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity. This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization. Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization. By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis. Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization. Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1(-), or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis. Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations. Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin. We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment.

Exploitation of mammalian host cell functions by bacterial pathogens.

Interest in bacterial pathogenesis has recently increased because of antibiotic resistance, the emergence of new pathogens and the resurgence of old ones, and the lack of effective therapeutics. The molecular and cellular mechanisms of microbial pathogenesis are currently being defined, with precise knowledge of both the common strategies used by multiple pathogenic bacteria and the unique tactics evolved by individual species to help establish infection. What is emerging is a new appreciation of how bacterial pathogens interact with host cells. Many host cell functions, including signal transduction pathways, cytoskeletal rearrangements, and vacuolar trafficking, are exploited, and these are the focus of this review. A bonus of this work is that bacterial virulence factors are providing new tools to study various aspects of mammalian cell functions, in addition to mechanisms of bacterial disease. Together these developments may lead to new therapeutic strategies.

A role for phosphoinositide 3-kinase in bacterial invasion.

Listeria monocytogenes is a bacterial pathogen that invades cultured nonphagocytic cells. Inhibitors and a dominant negative mutation were used to demonstrate that efficient entry requires the phosphoinositide (PI) 3-kinase p85alpha-p110. Infection with L. monocytogenes caused rapid increases in cellular amounts of PI(3, 4)P2 and PI(3,4,5)P3, indicating that invading bacteria stimulated PI 3-kinase activity. This stimulation required the bacterial protein InlB, host cell tyrosine phosphorylation, and association of p85alpha with one or more tyrosine-phosphorylated proteins. This role for PI 3-kinase in bacterial entry may have parallels in some endocytic events.

A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.

Listeria monocytogenes is responsible for severe food-borne infections, but the mechanisms by which bacteria cross the intestinal barrier are unknown. Listeria monocytogenes expresses a surface protein, internalin, that interacts with a host receptor, E-cadherin, to promote entry into human epithelial cells. Murine E-cadherin, in contrast to guinea pig E-cadherin, does not interact with internalin, excluding the mouse as a model for addressing internalin function in vivo. In guinea pigs and transgenic mice expressing human E-cadherin, internalin was found to mediate invasion of enterocytes and crossing of the intestinal barrier. These results illustrate how relevant animal models for human infections can be generated.

Mechanisms of evolution in Rickettsia conorii and R. prowazekii.

Rickettsia conorii is an obligate intracellular bacterium that causes Mediterranean spotted fever in humans. We determined the 1,268,755-nucleotide complete genome sequence of R. conorii, containing 1374 open reading frames. This genome exhibits 804 of the 834 genes of the previously determined R. prowazekii genome plus 552 supplementary open reading frames and a 10-fold increase in the number of repetitive elements. Despite these differences, the two genomes exhibit a nearly perfect colinearity that allowed the clear identification of different stages of gene alterations with gene remnants and 37 genes split in 105 fragments, of which 59 are transcribed. A 38-kilobase sequence inversion was dated shortly after the divergence of the genus.

Reassessing the role of internalin B in Listeria monocytogenes virulence using the epidemic strain F2365.

OBJECTIVES: To investigate the contribution to virulence of the surface protein internalin B (InlB) in the Listeria monocytogenes lineage I strain F2365, which caused a deadly listeriosis outbreak in California in 1985. METHODS: The F2365 strain displays a point mutation that hampers expression of InlB. We rescued the expression of InlB in the L. monocytogenes lineage I strain F2365 by introducing a point mutation in the codon 34 (TAA to CAA). We investigated its importance for bacterial virulence using in vitro cell infection systems and a murine intravenous infection model. RESULTS: In HeLa and JEG-3 cells, the F2365 InlB+ strain expressing InlB was ≈9-fold and ≈1.5-fold more invasive than F2365, respectively. In livers and spleens of infected mice at 72 hours after infection, bacterial counts for F2365 InlB+ were significantly higher compared to the F2365 strain (≈1 log more), and histopathologic assessment showed that the F2365 strain displayed a reduced number of necrotic foci compared to the F2365 InlB+ strain (Mann-Whitney test). CONCLUSIONS: InlB plays a critical role during infection of nonpregnant animals by a L. monocytogenes strain from lineage I. A spontaneous mutation in InlB could have prevented more severe human morbidity and mortality during the 1985 California listeriosis outbreak.

Non-classical use of clathrin during bacterial infections.

How invasive bacteria exploit mammalian host cell components to induce their entry into cells has received a lot of attention in the last two decades. Model organisms have emerged and helped understanding the various mechanisms that are used. Among those, Listeria monocytogenes is one of the most documented organisms. It enters into cells via two bacterial proteins, internalin (also called InlA) and InlB, which interact with cell surface receptors, E-cadherin and the hepatocyte growth factor receptor, Met, respectively. These interactions initiate a series of events that leads to actin polymerization, membrane invagination and bacterial internalization. Investigations on internalin- and InlB-mediated entries have repeatedly shown that Listeria fully usurps the host cell machinery. Moreover, they have also shown that previously unknown components discovered during the study of Listeria invasion play a role either in E-cadherin-mediated cell-cell adhesion or Met signalling. Unexpectedly, recent studies have highlighted a role for clathrin in Listeria InlB-mediated actin polymerization and entry, revealing a new role for this endocytic protein, i.e. in bacterial-induced internalization. Furthermore, comparative studies have demonstrated that the clathrin-mediated endocytosis machinery is also used in the internalin-E-cadherin pathway, and for the entry of other bacteria that enter by a 'zipper' mechanism. By contrast, the clathrin-mediated endocytic machinery is not used by bacteria that inject effectors into mammalian cells via the type III secretion system and enter by the so-called trigger mechanism, characterized by enormous membrane ruflles that result in the macropinocytosis of the corresponding bacteria. Finally, adherent bacteria, for example enteropathogenic Escherichia coli (EPEC), also co-opt clathrin to induce the formation of actin-rich pedestals. Together, these new data illuminate our view on how actin rearrangements may be coupled to clathrin recruitment during bacterial infection. They also shed light on a new function for clathrin in mammalian cells, i.e. internalization of objects much larger than previously accepted.

Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation.

BACKGROUND: Cdc42, a GTP-binding protein of the Rho family, controls actin cytoskeletal organization and helps to generate actin-based protruding structures, such as filopodia. In vitro, Cdc42 regulates actin polymerization by facilitating the creation of free barbed ends - the more rapidly growing ends of actin filaments - and subsequent elongation at these ends. The Wiskott- Aldrich syndrome protein, WASP, which has a pleckstrin-homology domain and a Cdc42/Rac-binding motif, has been implicated in cell signaling and cytoskeleton reorganization. We have investigated the consequences of local recruitment of activated Cdc42 or WASP to the plasma membrane. RESULTS: We used an activated Cdc42 protein that could be recruited to an engineered membrane receptor by adding rapamycin as a bridge, and added antibody-coupled beads to aggregate these receptors. Inducible recruitment of Cdc42 to clusters of receptors stimulated actin polymerization, resulting in the formation of membrane protrusions. Cdc42-induced protrusions were enriched in the vasodilator-stimulated phosphoprotein VASP and the focal-adhesion-associated proteins zyxin and ezrin. The Cdc42 effector WASP could also induce the formation of protrusions, albeit of different morphology. CONCLUSIONS: This is the first demonstration that the local recruitment of activated Cdc42 or its downstream effector, WASP, to a membrane receptor in whole cells is sufficient to trigger actin polymerization that results in the formation of membrane protrusions. Our data suggest that Cdc42-induced actin-based protrusions result from the local and serial recruitment of cytoskeletal proteins including zyxin, VASP, and ezrin.

Internalins: a complex family of leucine-rich repeat-containing proteins in Listeria monocytogenes.

The Listeria monocytogenes genome includes a large family of proteins harbouring leucine-rich repeats known as internalins (Inl). The generation of novel mutants and comparative analysis of Inl variability among Listeria and other bacterial genomes suggest that beyond the extensively-studied invasins, InlA and InlB, additional internalins also play important functions in the infectious process.

The use of host cell machinery in the pathogenesis of Listeria monocytogenes.

The bacterial pathogen, Listeria monocytogenes, exploits the host cell's machinery, enabling the pathogen to enter into cells and spread from cell to cell. Three bacterial surface proteins are crucial for these processes: internalin and InlB, which mediate entry into cells, and ActA, which induces actin polymerisation at one pole of the bacterium and promotes intracellular and intercellular motility. Recent studies have identified several of the cellular factors involved in the entry process and major discoveries have unravelled the mechanisms underlying the actin-based motility. Increasing evidence shows that many cellular genes are up- or down-regulated during infection and probably play a role in the establishment of infection, inflammation and induction of the host immune response.

Met, the HGF-SF receptor: another receptor for Listeria monocytogenes.

The bacterium Listeria monocytogenes invades a variety of cells in vitro and in vivo. Two proteins are crucial in this process: internalin, which interacts with E-cadherin, and InlB. The first identified ligand for InlB was gC1qR, which has no cytoplasmic domain. The newly discovered InlB receptor, Met, fits with the known InlB-induced signals.

EVH1/WH1 domains of VASP and WASP proteins belong to a large family including Ran-binding domains of the RanBP1 family.

The two cytoskeletal proteins VASP and WASP and the protein Homer share a conserved domain, currently designated the WHI domain (WASP homology domain 1) or EVH1 domain (ENA/VASP homology domain 1), which could play an important role in various cellular events such as transport, folding of proteins, and signal transduction. We report here additional occurrences of this domain in Ran-binding proteins of the RanBP1 family and various others proteins, or putative proteins of eukaryotic organisms, suggesting that the EVH1/WH1 domain may be more widely used than originally thought.

Interactions between Listeria monocytogenes and host mammalian cells.

Bacterial pathogens have developed a variety of strategies to induce their own internalization into mammalian cells which are normally nonphagocytic. The Gram-positive bacterium Listeria monocytogenes enters into many cultured cell types using two bacterial surface proteins, InlA (internalin) and InlB. In both cases, entry takes place after engagement of a receptor and induction of a series of signaling events.

Sequences homologous to the listeriolysin O gene region of Listeria monocytogenes are present in virulent and avirulent haemolytic species of the genus Listeria.

Various parts of the hlyA gene region of Listeria monocytogenes which encodes a major virulence factor, listeriolysin O, have been used to detect the presence of homologous sequences in other species of the genus Listeria. Under low-stringency hybridization conditions, sequences homologous to the hlyA gene and its 5' adjacent regions were detected in the haemolytic and pathogenic species L. ivanovii, and in the haemolytic but non-pathogenic species L. seeligeri. In contrast, the region located downstream from hlyA appeared specific to L. monocytogenes. None of the probes spanning the region revealed homologies between L. monocytogenes and the non-pathogenic and non-haemolytic members of the genus, L. innocua, L. murrayi and L. welshimeri. Among various strains of L. monocytogenes tested, the gene hlyA and its 3' adjacent region appeared well-conserved. In contrast, a restriction length polymorphism was detected in the region located upstream from hlyA with no obvious correlation with the haemolytic phenotype or the serovar of the strains tested.

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.