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P Cossart

Publications and source records attributed to P Cossart.

At least 91 records · Page 5Linked to original sources
PubMed1995

Attenuated Listeria monocytogenes as a live vector for induction of CD8+ T cells in vivo: a study with the nucleoprotein of the lymphocytic choriomeningitis virus.

Listeria monocytogenes spends most of its intracellular life cycle in the cytosol of the infected eucaryotic cells. Within this cellular compartment originates the endogenous pathway of antigen processing and presentation. We thus assumed that recombinant L. monocytogenes expressing an heterologous protein, the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV), should be able to induce antigen-specific CD8+ T cells in vivo. The LCMV nucleoprotein gene was inserted in phase with the sequence coding for the putative signal sequence of the hemolysin of L. monocytogenes in order to target its secretion into the cytosol of the infected cell. The ability of this recombinant bacterium to induce LCMV-reactive CD8+ T cells was then monitored in BALB/c mice. The immune status of the immunized BALB/c mice was studied on the seventh day after a single i.v. injection of a sublethal dose of the recombinant bacteria: (i) cytotoxic CD8+ T cells were detected in liver; (ii) using in vitro re-stimulation with PMA and ionomycin, secondary cytotoxic CD8+ T cells were detected in spleen; (iii) an early inflammatory reaction dependent on the presence of CD8+ T cells occurred in the footpad after intraplantar inoculation of live LCMV; and (iv) mice were protected against an otherwise lethal intracerebral LCMV challenge; the protection was accompanied by elimination of the virus. When the immune status of the immunized hosts was monitored for a longer period post-immunization, the balance between immune protection and immunopathology described for the anti-LCMV immune responses was observed; two phases of protection were detected, flanking a transitory phase of exacerbation of the lymphocytic choriomeningitis disease (weeks 2-5).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Modulation of DNA topology by flaR, a new gene from Listeria monocytogenes.

We report the identification of a previously unknown Listeria monocytogenes gene, flaR, which modulates DNA topology. Through the analysis of a Tn917 non-motile mutant, LOSC1, in which production of flagellin was abolished, we have identified a bacterial component involved in gene regulation. The transposon had inserted in flaR, an open reading frame of 531 bp, followed by a second open reading frame of 1252 bp in reverse orientation. On the L. monocytogenes physical map, flaR was located in a different region from that of the flaA gene encoding flagellin. Transcriptional analysis showed that the flaR gene product affects the flaA expression and negatively regulates its own expression. When expressed in Escherichia coli, flaR encodes a protein of 18 kDa (FlaR) whose transcription is osmoregulated. In addition, FlaR also influences the expression of reporter genes containing supercoiling-sensitive promoters such as proU or ompC. The data presented here suggest that FlaR is a histone-like bacterial protein which acts at specific sites to influence DNA topology and, therefore, transcription. flaR is the first gene of this class to be described in Gram-positive pathogenic bacteria.

Amino Acid Sequence↗
PubMed1995

The unrelated surface proteins ActA of Listeria monocytogenes and IcsA of Shigella flexneri are sufficient to confer actin-based motility on Listeria innocua and Escherichia coli respectively.

Listeria monocytogenes and Shigella flexneri are two unrelated facultative intracellular pathogens which spread from cell to cell by using a similar mode of intracellular movement based on continuous actin assembly at one pole of the bacterium. This process requires the asymmetrical expression of the ActA surface protein in L. monocytogenes and the IcsA (VirG) surface protein in S. flexneri. ActA and IcsA share no sequence homology. To assess the role of the two proteins in the generation of actin-based movement, we expressed them in the genetic context of two non-actin polymerizing, non-pathogenic bacterial species, Listeria innocua and Escherichia coli. In the absence of any additional bacterial pathogenicity determinants, both proteins induced actin assembly and propulsion of the bacteria in cytoplasmic extracts from Xenopus eggs, as visualized by the formation of characteristic actin comet tails. E. coli expressing IcsA moved about two times faster than Listeria and displayed longer actin tails. However, actin dynamics (actin filament distribution and filament half-lives) were similar in IcsA- and ActA-induced actin tails suggesting that by using unrelated surface molecules, L. monocytogenes and S. flexneri move intracellularly by interacting with the same host cytoskeleton components or by interfering with the same host cell signal transduction pathway.

Actins↗
PubMed1995

The amino-terminal part of ActA is critical for the actin-based motility of Listeria monocytogenes; the central proline-rich region acts as a stimulator.

The intracellular bacterial pathogen Listeria monocytogenes moves inside the host-cell cytoplasm propelled by continuous actin assembly at one pole of the bacterium. This process requires expression of the bacterial surface protein ActA. Recently, in order to identify the regions of ActA which are required for actin assembly, we and others have expressed different domains of ActA by transfection in eukaryotic cells. As this type of approach cannot address the role of ActA in the actin-driven bacterial propulsion, we have now generated several L. monocytogenes strains expressing different domains of ActA and analysed the ability of the different domains to trigger actin assembly and bacterial movement in both infected cells and cytoplasmic extracts. We show here that the amino-terminal part is critical for F-actin assembly and movement. The internal proline-rich repeats and the carboxy-terminal domains are not essential. However, in vitro motility assays have demonstrated that mutants lacking the proline-rich repeats domain of ActA moved two times slower (6+/-2 micrometers min(-1)) than the wild type (13 +/-3 micrometers min(-1)). In addition, phosphatase treatment of protein extracts of cells infected with the L. monocytogenes strains expressing the ActA variants suggested that phosphorylation may not be essential for ActA activity.

Actins↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

iactA of Listeria ivanovii, although distantly related to Listeria monocytogenes actA, restores actin tail formation in an L. monocytogenes actA mutant.

A gene homologous to the actA gene of Listeria monocytogenes was cloned from Listeria ivanovii (strain CLIP257) by chromosome walking starting from the ilo gene that encodes the pore-forming toxin ivanolysin. The nucleotide sequence revealed that this gene, named iactA, encodes a protein of 1,044 amino acids (IactA) comprising a central region with seven highly conserved tandem proline-rich repeats of 47 amino acids. Although IactA and ActA share an overall similar structure, these two proteins are only distantly related. Like ActA, IactA migrates aberrantly on sodium dodecyl sulfate gels. When expressed in an L. monocytogenes actA deletion mutant strain, iactA restored actin polymerization.

Actins↗
PubMed1995

Differential activation of virulence gene expression by PrfA, the Listeria monocytogenes virulence regulator.

PrfA is a pleiotropic activator of virulence gene expression in the pathogenic bacterium Listeria monocytogenes. Several lines of evidence have suggested that a hierarchy of virulence gene activation by PrfA exists. This hypothesis was investigated by assessing the ability of PrfA to activate the expression of virulence gene fusions to lacZ in Bacillus subtilis. Expression of PrfA in this heterologous host was sufficient for activation of transcription at the hly, plcA, mpl, and actA promoters. Activation was most efficient at the divergently transcribed hly and plcA promoters. The putative PrfA binding site shared by these promoters is perfectly symmetrical and appears to represent the optimum sequence for target gene activation by PrfA. The activation of actA and mpl expression was considerably weaker and occurred more slowly than that observed at the hly and plcA promoters, suggesting that greater quantities of PrfA are required for productive interaction at these promoters. Interestingly, expression of an inlA-lacZ transcriptional fusion was very poorly activated by PrfA in B. subtilis, suggesting that other Listeria factors, in addition to PrfA, are required for PrfA-mediated activation at this promoter. Further support for the involvement of such factors was obtained by constructing and analyzing a prfA deletion mutant of L. monocytogenes. We observed that, in contrast to that of the other genes of the PrfA regulon, expression of inlA is only partially dependent on PrfA.

Animals↗
PubMed1994

The actin-based motility of the facultative intracellular pathogen Listeria monocytogenes.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular parasite that invades and multiplies within diverse eukaryotic cell types. An essential pathogenicity determinant is its ability to move in the host cell cytoplasm and to spread within tissues by directly passing from one cell to another. The propulsive force for intracellular movement is thought to be generated by continuous actin assembly at the rear end of the bacterium. Moving bacteria that reach the plasma membrane induce the formation of long membranous protrusions that are internalized by neighbouring cells, thus mediating the spread of infection. The unrelated pathogens Shigella and Rickettsia use a similar process of actin-based motility to disseminate in infected tissues. This review focuses on the bacterial and cellular factors involved in the actin-based motility of L. monocytogenes.

Actin Cytoskeleton↗
PubMed1994

Five Listeria monocytogenes genes preferentially expressed in infected mammalian cells: plcA, purH, purD, pyrE and an arginine ABC transporter gene, arpJ.

Listeria monocytogenes is a bacterial pathogen that multiplies within the cytosol of eukaryotic cells. To identify Listeria genes with preferentially intracellular expression (pic genes), a library of Tn917-lac insertion mutants was screened for transcriptional fusions to lacZ with higher expression inside a macrophage-like cell line than in a rich broth medium. Five pic genes with up to 100-fold induction inside cells were identified. Three of them (purH, purD and pyrE) were involved in nucleotide biosynthesis. One was part of an operon encoding an ABC (ATP-binding cassette) transporter for arginine. The corresponding mutants were not affected in intracellular growth, cell-to-cell spread or virulence, except for the transporter mutant, whose LD50 after intravenous infection of mice was twofold higher than the wild-type. The fifth gene was plcA, a previously identified virulence gene that encodes a phosphatidylinositol-phospholipase C, and is cotranscribed with prfA, a gene encoding a pleiotropic transcriptional activator of known virulence genes. Although plcA expression is known to depend on PrfA, a prfA promoter-lacZ fusion was highly expressed both inside and outside cells. Furthermore, in the presence of cellobiose, a disaccharide recently shown to repress plcA and hly expression, plcA and hly mRNA levels were dramatically reduced without any decrease in the monocistronic prfA mRNA levels. These results demonstrate that virulence gene activation does not depend only on prfA transcript accumulation.

ATP-Binding Cassette Transporters↗
PubMed1994

The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.

Most known Listeria monocytogenes virulence genes cluster within a 9.6-kb chromosomal region. This region is flanked on one end by two uncharacterized open reading frames (ORF A and ORF B) and ldh, an ORF presumably encoding the L. monocytogenes lactate dehydrogenase (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). We report here that the other end is flanked by prs, and ORF homologous to phosphoribosyl PPi synthetase genes. ORF B and prs were detected in all Listeria species and thus delimit the virulence region. This virulence gene cluster was detected exclusively in hemolytic Listeria species, Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species.

Animals↗
PubMed1994

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are similar to cadA and cadC of Staphylococcus aureus and are induced by cadmium.

pLm74 is the smallest known plasmid in Listeria monocytogenes. It confers resistance to the toxic divalent cation cadmium. It contains a 3.1-kb EcoRI fragment which hybridizes with the cadAC genes of plasmid pI258 of Staphylococcus aureus. When introduced into cadmium-sensitive L. monocytogenes or Bacillus subtilis strains, this fragment conferred cadmium resistance. The DNA sequence of the 3.1-kb EcoRI fragment contains two open reading frames, cadA and cadC. The deduced amino acid sequences are similar to those of the cad operon of plasmid pI258 of S. aureus, known to prevent accumulation of Cd2+ in the bacteria by an ATPase efflux mechanism. The cadmium resistance determinant of L. monocytogenes does not confer zinc resistance, in contrast to the cadAC determinant of S. aureus, suggesting that the two resistance mechanisms are slightly different. Slot blot DNA-RNA hybridization analysis showed cadmium-inducible synthesis of L. monocytogenes cadAC RNA.

Adenosine Triphosphatases↗
PubMed1994

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are present on Tn5422, a novel transposon closely related to Tn917.

The complete (6,449-bp) nucleotide sequence of the first-described natural transposon of Listeria monocytogenes, designated Tn5422, was determined. Tn5422 is a transposon of the Tn3 family delineated by imperfect inverted repeats (IRs) of 40 bp. It contains two genes which confer cadmium resistance (M. Lebrun, A. Audurier, and P. Cossart, J. Bacteriol. 176:3040-3048, 1994) and two open reading frames that encode a transposase (TnpA) and a resolvase (TnpR) of 971 and 184 amino acids, respectively. The cadmium resistance genes and the transposition genes are transcribed in opposite directions and are separated by a putative recombination site (res). The structural elements presumed to be involved in transposition of Tn5422 (IRs, transposase, resolvase, and res) are very similar to those of Tn917, suggesting a common origin. The transposition genes were not induced by cadmium. Analysis of sequences surrounding Tn5422 in nine different plasmids of L. monocytogenes indicated that Tn5422 is a functional transposon, capable of intramolecular replicative transposition, generating deletions. This transposition process is probably the reason for the size diversity of the L. monocytogenes plasmids. Restriction analysis and Southern hybridization revealed the presence of Tn5422 in all the plasmid-mediated cadmium-resistant L. monocytogenes strains tested but not in strains encoding cadmium resistance on the chromosome.

Adenosine Triphosphatases↗
PubMed1993

Internalin-mediated invasion of epithelial cells by Listeria monocytogenes is regulated by the bacterial growth state, temperature and the pleiotropic activator prfA.

Entry of Listeria monocytogenes into epithelial cells requires expression of inIA, the first gene of an operon comprising two genes: inIA, which encodes internalin, a 800-amino-acid protein, and inIB, which encodes a 630-amino-acid protein. We report here that the inI locus is transcribed on two transcripts in constant relative ratio: a 5 kb transcript spanning inIA and inIB, and a 2.9 kb transcript that covers only inIA. The promoter is located 397 bp from the GTG initiator of inIA and displays in its -35 region a palindrome similar to that found in promoters controlled by the pleiotropic activator prfA. Transcription of the inI locus is, as are several other L. monocytogenes virulence genes, activated by prfA and regulated by temperature--with higher expression at 37 degrees C versus 25 degrees C--and bacterial growth state. It is maximal during exponential growth and correlates with maximal invasivity of the bacteria in the human epithelial cell line Caco-2. It also correlates with maximum amounts of internalin present on the bacterial surface. Internalin is also detected in substantial amounts in culture supernatants. Taken together, these data suggest that surface-bound internalin plays an important role in bacterial entry but do not exclude a role for the released form.

Bacterial Proteins↗
PubMed1993

Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor type 1.

Cytotoxic necrotizing factor type 1 (CNF1) from strains of pathogenic Escherichia coli induces in human epithelial HEp-2 cells, a profound reorganization of the actin cytoskeleton into prominent stress fibres and membrane ruffles. We report here that this process is associated with induction of phagocytic-like activity. CNF1-treated cells acquired the ability to ingest latex beads as well as non-invasive bacteria such as Listeria innocua, which were taken as a model system. Uptake of bacteria was similar to pathogen-induced phagocytosis, since L. innocua transformed with DNA coding for the pore-forming toxin listeriolysin O behaved, with respect to intracellular growth, like the invasive, pathogenic species L. monocytogenes. Our results raise the possibility that, in vivo, pathogenic CNF1-producing E. coli may invade epithelia by this novel induced phagocytic-like mechanism.

Actin Cytoskeleton↗