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P Cossart

Publications and source records attributed to P Cossart.

At least 19 recordsLinked to original sources
PubMed1997

ActA is a dimer.

ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells. Its N-terminal domain is sufficient to induce actin tail formation and movement. Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers. By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer. Cross-linking experiments on various L. monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97-126, and previously identified as critical for actin tail formation, is also critical for dimer formation. A model of actin polymerization by L. monocytogenes, involving the ActA dimer, is presented.

Bacterial Proteins↗
PubMed1997

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.

Animals↗
PubMed1997

Identification of two regions in the N-terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes.

The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface. The continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts. Here, by fusing the N-terminus of ActA (residues 1-234) to the omega fragment of beta-galactosidase, we present the first evidence that this domain contains all the necessary elements for actin tail formation. A detailed analysis of ActA variants, in which small fragments of the N-terminal region were deleted, allowed the identification of two critical regions. Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process. The first region (region T, amino acids 117-121) is critical for filament elongation, as shown by the absence of actin tail in a 117-121 deletion mutant or when motility assays are performed in the presence of anti-region T antibodies. The second region (region C, amino acids 21-97), is more specifically involved in maintenance of the continuity of the process, probably by F-actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21-97) leading to 'discontinuous' actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33-74 can interact with F-actin. Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N-terminal domain of ActA.

Actins↗
PubMed1997

InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association.

Listeria monocytogenes is an intracellular bacterial pathogen that expresses several surface proteins critical for the infectious process. Such proteins include InlA (internalin) and InlB, involved in bacterial entry into the host cell, and ActA, required for bacterially induced actin-based motility. Although the molecular mechanisms of attachment of InlA and ActA have been characterized, essentially nothing is known about how InlB is anchored to the bacterial surface. Using a genetic approach, we demonstrate that the last 232 amino acids of InlB are both necessary and sufficient for anchoring this protein to the bacterial surface. An InlB mutant protein deleted for the last 232 amino acids was secreted and not detected at the cell surface. A 'domain-swapping' strategy in which these 232 amino acids were used to replace the normal cell wall-anchoring domain of InlA resulted in a chimeric protein that was anchored to the cell surface and able to confer entry. Interestingly, surface association of InlB also occurred when InlB was added externally to bacteria, suggesting that association may be able to occur after secretion. This association was productive for invasion, as it conferred bacterial entry into host cells. The C-terminal anchoring region in InlB contains 80-amino-acid repeats beginning with the sequence GW that is also present in a newly identified surface-associated bacteriolysin of L. monocytogenes, called Ami. Addition of GW repeats to the C-terminal of InlB improves anchoring of the protein to the cell surface. These and other data suggest that such 'GW' repeats may constitute a novel motif for cell-surface anchoring in Listeria and other Gram-positive bacteria. This motif may have important consequences for the release of surface proteins involved in interactions with eukaryotic cells.

Amino Acid Sequence↗
PubMed1997

Evidence that PrfA, the pleiotropic activator of virulence genes in Listeria monocytogenes, can be present but inactive.

All virulence genes of Listeria monocytogenes identified to date are positively regulated by PrfA, a transcriptional activator belonging to the Crp-Fnr family. Low temperature and cellobiose are two environmental signals known to repress expression of virulence genes in L. monocytogenes. In the present work, we analyzed the effect of temperature and cellobiose on the expression of the PrfA protein. At low temperature, PrfA was undetected, although prfA monocistronic transcripts are present. In contrast, PrfA was fully expressed in the presence of cellobiose. These results strongly suggest that virulence gene activation depends on both the presence of PrfA and additional regulatory pathways that either modify PrfA or act synergistically with PrfA.

Bacterial Proteins↗
PubMed1997

Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD.

Listeria monocytogenes is a bacterial pathogen that is able to invade nonphagocytic cells. Two surface proteins, internalin, the inlA gene product, and InlB, play important roles in the entry into cultured mammalian cells. These proteins also have extensive sequence similarities. Previously, Southern hybridization predicted the existence of an internalin multigene family. Recently, InlC, a secreted protein of 30 kDa homologous to InlA and InlB, was identified. In this work, we identified and characterized four new members of the internalin multigene family, inlC2, inlD, inlE, and inlF which encode proteins of 548, 567, 499, and 821 amino acids respectively. inlC2, inlD, and inlE are contiguous on the chromosome of L. monocytogenes EGD, whereas inlF is located in a different chromosomal region. These four inl gene products display the principal features of internalin, namely, a signal sequence, two regions of repeats (or LRR and B repeats), and a putative cell wall anchor sequence containing the sorting motif LPXTG. The four inl genes were maximally expressed albeit at a low level during early exponential growth in bacterial medium at 37 degrees C. The role of these inl genes in L. monocytogenes invasion was assessed by constructing isogenic chromosomal deletion mutants and testing them for entry into various nonphagocytic cells. Unexpectedly, the inlC2, inlD, inlE, and inlF null mutants were not affected for entry into any of the cell lines tested, raising the possibility that these genes are needed for an aspect of pathogenicity other than invasion. The identity of such an aspect remains to be determined.

Amino Acid Sequence↗
PubMed1996

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.

Androstadienes↗
PubMed1996

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.

Animals↗
PubMed1996

A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence.

PrfA, the regulator of virulence-gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP-FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of PrfA predicted to contact DNA, i.e. Ser-184 and Ser-183. Substitution of Ser-184 by Ala reduced DNA binding and virulence-gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser-183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target-site recognition by PrfA and PrfA-S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence-gene activation, and virulence.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Antibodies to the leucine-rich repeat region of internalin block entry of Listeria monocytogenes into cells expressing E-cadherin.

Internalin, a surface protein essential for entry of Listeria monocytogenes EGD into epithelial cells, was used as an antigen to raise nine monoclonal antibodies. These monoclonal antibodies recognized seven distinct epitopes which were located in three different regions of the protein. Three of them inhibited internalin-mediated entry and recognized the amino-terminal leucine-rich repeat region of the protein, suggesting that this region is essential for entry.

Animals↗
PubMed1996

Listeriolysin O activates mitogen-activated protein kinase in eucaryotic cells.

Infection with Listeria monocytogenes induces the activation of mitogen-activated protein (MAP) kinase in several tissue culture cell lines (P.Tang, I. Rosenshine, and B. B. Finlay, Mol. Biol. Cell 5:455-464, 1994). After various mutants were examined, the bacterial factor responsible for MAP kinase activation was identified as listeriolysin O (LLO). Growth supernatant containing LLO or purified LLO alone can induce MAP kinase tyrosine phosphorylation in HeLa cells. Single-amino-acid mutations in LLO that do not affect its membrane binding capacity but reduce its cytolytic activity also reduced its ability to induce MAP kinase activity in HeLa cells. Streptolysin O, another sulfhydryl-activated hemolysin, and the detergent saponin are also able to activate MAP kinase in target cells. Thus, the increased MAP kinase activity observed in L. monocytogenes-infected cells is most likely a result of the permeabilization of the host cell membrane by LLO and may not be linked with invasion.

Bacterial Toxins↗
PubMed1995

Actin-based motility of vaccinia virus.

The role of the cytoskeleton during viral infection is poorly understood. Here we show, using a combination of mutant and drug studies, that the intracellular enveloped form of vaccinia virus is capable of inducing the formation of actin tails that are strikingly similar to those seen in Listeria, Shigella and Rickettsia infections. Analysis using video microscopy reveals that single viral particles are propelled in vivo on the tip of actin tails, at a speed of 2.8 mumol min-1. On contact with the cell surface, virus particles extend outwards on actin projections at a similar rate, to contact and infect neighboring cells. Given the similarities between the motility of vaccinia virus and bacterial pathogens, we suggest that intracellular pathogens have developed a common mechanism to exploit the actin cytoskeleton as a means to facilitate their direct spread between cells.

Actins↗
PubMed1995

Targeting of Listeria monocytogenes ActA protein to the plasma membrane as a tool to dissect both actin-based cell morphogenesis and ActA function.

Actin assembly on the surface of Listeria monocytogenes in the cytoplasm of infected cells provides a model to study actin-based motility and changes in cell shape. We have shown previously that the ActA protein, exposed on the bacterial surface, is required for polarized nucleation of actin filaments. To investigate whether plasma membrane-associated ActA can control the organization of microfilaments and cell shape, variants of ActA, in which the bacterial membrane signal had been replaced by a plasma membrane anchor sequence, were produced in mammalian cells. While both cytoplasmic and membrane-bound forms of ActA increased the F-actin content, only membrane-associated ActA caused the formation of plasma membrane extensions. This finding suggests that ActA acts as an actin filament nucleator and shows that permanent association with the inner face of the plasma membrane is required for changes in cell shape. Based on the observation that the amino-terminal segment of ActA and the remaining portion which includes the proline-rich repeats cause distinct phenotypic modifications in transfected cells, we propose a model in which two functional domains of ActA cooperate in the nucleation and dynamic turnover of actin filaments. The present approach is a new model system to dissect the mechanism of action of ActA and to further investigate interactions of the plasma membrane and the actin cytoskeleton during dynamic changes of cell shape.

Actins↗