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P J Sansonetti

Publications and source records attributed to P J Sansonetti.

At least 19 recordsLinked to original sources
PubMed1997

Purification of IpaC, a protein involved in entry of Shigella flexneri into epithelial cells and characterization of its interaction with lipid membranes.

Entry of Shigella flexneri into epithelial cells and lysis of the phagosome involve the secreted IpaA-D proteins. A complex containing IpaC and IpaB is able to promote uptake of inert particles by epithelial cells. This suggested that Ipa proteins, either individually or as a complex, might interact with the cell membrane. We have purified IpaC and demonstrated its interaction with lipid vesicles. This interaction is modulated by the pH, which might be relevant to the dual role of Ipa proteins, in induction of membrane ruffles upon entry and lysis of the endosome membrane thereafter.

Antigens, Bacterial↗
PubMed1996

The nadB gene of Salmonella typhimurium complements the nicotinic acid auxotrophy of Shigella flexneri.

Shigella species are characteristically nicotinic acid (NA) auxotrophs. The invasive S. flexneri strain M90T, transformed with the multicopy plasmid pZT349 encoding the nadB gene of Salmonella typhimurium, can grow in minimal glucose medium without exogenous NA, whereas, M90T containing the control vector, pUC18 does not, suggesting that this species lacks L-aspartic acid oxidase, the first enzyme in the de novo NAD biosynthetic pathway. The estimated growth rate of strain M90T (pZT349) in HeLa cells was identical to that of M90T (pUC18), indicating the available intracellular concentration of NA is not limiting for bacterial growth.

Humans↗
PubMed1996

Bacterial entry into epithelial cells: the paradigm of Shigella.

Shigella flexneri is a model for the entry of bacterial pathogens into nonphagocytic epithelial cells. On contact with the epithelial cell surface, the Ipa proteins are secreted from the bacterium. The Ipa complex then triggers a reorganization of the host-cell cytoskeleton leading to the formation of membrane ruffles, which engulf the bacterium.

Bacterial Adhesion↗
PubMed1996

In vivo apoptosis in Shigella flexneri infections.

Shigella flexneri, an etiological agent of bacillary dysentery, causes apoptosis in vitro. Here we show that it also induces apoptosis in vivo. We were able to quantify the number of apoptotic cells in rabbit Peyer's patches infected with S. flexneri by detecting cells with fragmented DNA. Infection with virulent S. flexneri results in massive numbers of apoptotic cells within the lymphoid follicles. In contrast, neither an avirulent strain nor an avirulent strain capable of colonizing Peyer's patches increases the background level of apoptotic cells. Macrophages, T cells, and B cells are shown to undergo apoptosis in vivo. These results indicate that apoptosis may play a crucial role in the pathogenesis of shigellosis.

Animals↗
PubMed1996

Lack of cleavage of IcsA in Shigella flexneri causes aberrant movement and allows demonstration of a cross-reactive eukaryotic protein.

Once in the cytoplasm of mammalian cells, Shigella flexneri expresses a motile phenotype caused by polar directional assembly of actin. This process depends on accumulation of IcsA (VirG), a 120-kDa protein with ATPase activity, at the pole of the bacterium opposite to that at which ongoing septation occurs. IcsA is also secreted into the bacterial supernatant as a 95-kDa species, after cleavage at an SSRRASS sequence which, when mutagenized, blocks processing. MAbF15, an anti-IcsA monoclonal antibody, recognizes an epitope located within repeated Gly-rich boxes in the N-terminal half of the protein. We used this monoclonal antibody to visualize the location of a noncleavable 120-kDa IcsA mutant protein expressed in S. flexneri. We found that this noncleavable IcsA protein no longer localized exclusively to the pole of the bacterium but also could be detected circumferentially. Whereas the monoclonal antibody detected the wild-type cleavable form of IcsA in only 40% of the cells expressing this protein, the noncleavable was easily detectable in all the cells carrying the icsA mutant allele. Similar aberrant localization of the IcsA mutant protein on bacteria growing within the cytoplasm of HeLa cells was observed. The strains expressing the noncleavable IcsA protein expressed abnormal intracellular movement and were often observed moving in a direction perpendicular to their longitudinal axis. The putative protease which processes IcsA may therefore play a role in achieving polar expression of this protein and providing maximum asymmetry essential to directional movement. In addition, MAbF15 allowed us to identify a 70-kDa eukaryotic protein cross-reacting with IcsA. This protein accumulated in the actin tails of motile bacteria and in membrane ruffles of the cells.

Actins↗
PubMed1996

Induction of adherence and degranulation of polymorphonuclear leukocytes: a new expression of the invasive phenotype of Shigella flexneri.

In the present study, the ability of Shigella flexneri to activate polymorphonuclear neutrophils (PMN) was examined. The invasive serotype 5 strain M90T induced strong PMN adherence, which was dependent on both the multiplicity of infection and the duration of incubation. When tested under the same experimental conditions, the noninvasive strain BS176 (cured of the 220-kb virulence plasmid) was less efficient. Indeed, incubation of PMN for 2 h with either M90T or BS176 (multiplicity of infection, 100) induced 51.8% +/- 10.5% and 15.2% +/- 4.2% adherence, respectively (n = 3; P < 0.05). Stronger PMN activation by M90T was confirmed by evaluating PMN degranulation induced by the two strains. Whereas M90T triggered significant PMN secretion, BS176 did not. M90T strains with mutations in ipa genes were then analyzed. When PMN were incubated with these mutants, their activation was of the same intensity as that obtained with BS176. These data provide the first evidence for PMN activation induced by S. flexneri, a process which appears to be mediated by Ipa invasins.

Adhesins, Bacterial↗
PubMed1996

Infection of rabbit Peyer's patches by Shigella flexneri: effect of adhesive or invasive bacterial phenotypes on follicle-associated epithelium.

In order to invade the colonic mucosa, the bacterial pathogen Shigella flexneri must find a site of entry. Experiments with the rabbit ligated intestinal loop model described here confirm that M cells of the follicle-associated epithelium (FAE) that covers lymphoid structures of the Peyer's patches represent a major site of entry for invasive microorganisms. In addition, in an isogenic Shigella background, expression of an adhesive phenotype, or of an invasive phenotype, is required for bacteria to efficiently colonize the FAE. A nonadhesive, noninvasive mutant barely interacted with FAE. Adhesive and invasive strains induced dramatic but different alterations on FAE. Invasive strain M90T caused major inflammation-mediated tissue destruction after 8 h of infection. Adhesive strain BS15 caused limited inflammation, but major architectural changes, characterized by an increase in the size of M cells that became stretched over large pockets containing an increased number of mononuclear cells, were observed. M cells progressively occupied large surface areas of the FAE at the expense of enterocytes. This contributed to enterocytes losing contact with the lumen. These experiments demonstrate that various remodeling patterns may occur in Peyer's patches in response to bacterial pathogens, depending on the virulence phenotype expressed by the pathogenic strain.

Animals↗
PubMed1995

[Prevention of diarrheal diseases and oral vaccination].

Vaccination against diarrhoeal diseases has been set up as a primary goal for developing countries. Recent progress in understanding the molecular and cellular bases of these infections and the function of the mucosal immune system opens the way to new strategies of vaccination. Mucosal surfaces, particularly the intestine, appear as suitable targets for a new generation of vaccines among which live attenuated vectors are promising. However, we are still a long way from mastering all the relevant parameters of such approaches.

Administration, Oral↗
PubMed1995

Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-src-mediated signalling pathway.

Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon. Cell invasion occurs via bacterium-directed phagocytosis, a process requiring polymerization of actin at the site of bacterial entry. We show that invasion of HeLa cells by S.flexneri induces tyrosine phosphorylation of cortactin, a host cell protein previously identified as a cytoskeleton-associated protein tyrosine kinase (PTK) substrate for the proto-oncoprotein pp60c-src. Immunolocalization experiments indicate that cortactin is recruited to submembranous actin filaments formed during bacterial entry. In particular, cortactin is highly enriched in membrane ruffles of the entry structure, which engulf entering bacteria, and also in the periphery of the phagosome early after bacterial internalization. The proto-oncoprotein pp60c-src appears to mediate tyrosine phosphorylation of cortactin, since overexpression of this PTK in HeLa cells specifically increases the level of cortactin tyrosine phosphorylation induced during bacterial entry. Immunolocalization studies in pp60c-src-overexpressing HeLa cells indicate that pp60c-src is recruited to the entry structure and to the periphery of the phagosome, where pp60c-src appears to accumulate in association with the membrane. Our results suggest that epithelial cell invasion by S.flexneri involves recruitment and kinase activation of pp60c-src. Signalling by the proto-oncoprotein pp60c-src may play a role in cytoskeletal changes that facilitate S.flexneri uptake into epithelial cells, since transient overexpression of pp60c-src in HeLa cells can provoke membrane ruffling and appears also to stimulate bacterial uptake of a non-invasive S.flexneri strain.

Actins↗
PubMed1995

Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigella flexneri into HeLa cells.

Shigella flexneri is an enteroinvasive bacterium which causes bacillary dysentery in humans. A major feature of its pathogenic potential is the capacity to invade epithelial cells. Shigella entry into epithelial cells is considered a parasite-induced internalization process requiring polymerization of actin. Here we describe the cytoskeletal rearrangements during S. flexneri invasion of HeLa cells. After an initial contact of the bacterium with the cell surface, distinct nucleation zones of heavy chain actin polymerization appear in close proximity to the contact site underneath the parasite with long filaments being polymerized. These structures then push cellular protrusions that rise beside the entering bacterium, being sustained by tightly bundled long actin filaments organized in parallel orientation with their positive ends pointing to the cytoplasmic membrane. Finally, the cellular projections coalesce above the bacterial body, leading to its internalization. In addition, we found the actin-bundling protein plastin to be concentrated in these protrusions. Since plastin is known to bundle actin filaments in parallel orientation, colocalization of parallel actin filaments and plastin in the cellular protrusions strongly suggested a functional role of this protein in the architecture of parasite-induced cellular projections. Using transfection experiments, we show the differential recruitment of the two plastin isoforms (T- and L-) into Shigella entry zones. By transient expression of a truncated T-plastin which is deprived of one of its actin-binding sites, we also demonstrate the functional role of T-plastin in Shigella entry into HeLa cells.

Actins↗
PubMed1995

SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion.

In addition to Ipa proteins and IcsA, which are involved in entry into epithelial cells and intercellular spread, respectively, Shigella secretes a 110 kDa protein, designated SepA. We report the identification, cloning, and nucleotide sequence determination of the sepA gene, analysis of SepA secretion, and construction and characterization of a sepA mutant. The sepA gene is carried by the virulence plasmid and codes for a 150 kDa precursor. Upon secretion, which does not involve accessory proteins encoded by the virulence plasmid, the precursor is converted to a mature protein of 110 kDa by two cleavages removing an N-terminal signal sequence and a C-terminal fragment. Extensive similarities were detected between the sequence of the first 500 residues of mature SepA and the N-terminal region of IgA1 proteases from Neisseria gonorrhoeae and Haemophilus influenzae, the Tsh haemagglutinin of an avian pathogenic Escherichia coli, and the Hap protein involved in adhesion and penetration of H. influenzae. The C-terminal domain of the SepA precursor, which is not present in the secreted protein, exhibits sequence similarity with pertactin of Bordetella pertussis and the ring-forming protein of Helicobacter mustelae. Construction and phenotypic characterization of a sepA mutant indicated that SepA is required neither for entry into cultured epithelial cells nor for intercellular dissemination. However, in the rabbit ligated ileal loop model, the sepA mutant exhibited an attenuated virulence, which suggests that SepA might play a role in tissue invasion.

Amino Acid Sequence↗
PubMed1995

MxiG, a membrane protein required for secretion of Shigella spp. Ipa invasins: involvement in entry into epithelial cells and in intercellular dissemination.

Entry of Shigella flexneri into epithelial cells involves secretory proteins, the Ipa proteins, and their dedicated secretion apparatus, the Mxi-Spa translocon, which is encoded by the mxi and spa operons. We have characterized the mxiG gene that is located at the proximal part of the mxi operon. Inactivation of mxiG abolished lpa secretion, which indicates that MxiG is an essential component of the Mxi-Spa translocon. Immunoblotting analysis of membrane fractions suggests that the 42 kDa MxiG protein is associated with both the inner and outer membranes. Taking advantage of the complementation of the mxiG mutant by a plasmid carrying a wild-type copy of mxiG (which restored Ipa secretion, entry into HeLa cells, and cell-to-cell spread) we mutagenized the mxiG gene carried by the complementing plasmid to replace the RGD motif of MxiG by RAD. This mutation (mxiG*), which had no effect on the stability of the protein, did not affect Ipa secretion in vitro or entry into HeLa cells, but impaired intercellular dissemination. Therefore, MxiG and possibly proteins secreted by the Mxi-Spa translocation are involved not only in entry but also in spread of Shigella between epithelial cells.

Adhesins, Bacterial↗
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

Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures.

Genes required for entry of Shigella flexneri into epithelial cells in vitro are clustered in two adjacent loci, one of which encodes secretory proteins, the IpaA-D proteins, and the other their dedicated secretion apparatus, the Mxi-Spa translocon. Ipa secretion, which is induced upon contact of bacteria with epithelial cells, is prevented during growth in vitro. Here, we show that ipaB and ipaD mutations lead to enhanced secretion of a set of about 15 proteins. These extracellular proteins and some Ipas associate in organized structures consisting of extended sheets. Growth of the wild-type strain in the presence of Congo red is shown to induce protein secretion through the Mxi-Spa translocon. Cultures grown to stationary phase in the presence of Congo red contain extracellular filaments whose composition and morphology are similar to those produced by the hypersecreting ipaB and ipaD mutants.

Antigens, Bacterial↗
PubMed1995

Role of interleukin-1 in the pathogenesis of experimental shigellosis.

The effect of human recombinant interleukin-1 receptor antagonist on intestinal inflammation, tissue destruction, and bacterial invasion during experimental shigellosis caused by Shigella flexneri was studied in the rabbit-ligated loop infection model. Intravenous infusion of the inhibitor at a dose of 2 mg/kg per h, was initiated 30 min before intestinal loops were ligated and infected, and continued during the 8-h period of infection. The animals treated with IL-1 receptor antagonist showed a striking decrease in inflammation, destruction, and bacterial invasion of their tissues, both at the level of the villous intestine and Peyer's patches. This is conclusive evidence that interleukin-1 plays a critical role in the pathogenesis of shigellosis. This proinflammatory cytokine is here proposed as a major trigger of the inflammatory reaction which is characteristic of this invasive disease of the intestine, due to the particular interaction existing between S. flexneri and macrophages.

Animals↗
PubMed1994

Acute inflammation causes epithelial invasion and mucosal destruction in experimental shigellosis.

The gram-negative pathogen Shigella flexneri causes bacillary dysentery, an invasive disease of the human colonic mucosa. A major characteristic of the infectious process is the occurrence of an acute inflammatory reaction of mucosal tissues which is generally consequence of primary invasion and destruction of colonic epithelial cells by the pathogen. Confirming in vitro demonstration that S. flexneri is unable to invade the apical pole of colonic cells and that polymorphonuclear (PMN) cells may assist them in reaching the basal side of epithelial cells where they can invade, we have provided here in vivo evidence that S. flexneri enters the epithelial barrier essentially through the dome of lymphoid follicles at the early stage of infection and that subsequent invasion and destruction of the epithelium is primarily due to immigration of leukocytes, particularly PMN that destroy cohesion of the epithelial barrier. These conclusions are based on experiments carried out in infected rabbit ligated intestinal loops, with some animals treated by an anti-CD18 monoclonal antibody that blocked immigration of leukocytes into infected tissues.

Acute Disease↗
PubMed1994

Molecular and cellular mechanisms of tissue invasion by Shigella flexneri.

Shigella flexneri, a member of the family of enterobacteriaceae, causes bacillary dysentery by invading the human colonic mucosa and provoking a very intense inflammation. Recent in vitro data allow us to integrate different phenomena into a model of the infectious process during shigellosis. In vivo, bacteria appear to enter the submucosa via the M cells, specialized cells that cover the follicular structures of the intestinal mucosa. Once inside the submucosa, shigellae encounter resident tissue macrophages, which are infected, and apoptosis is rapidly induced. During programmed cell death the inflammatory cytokine interleukin-1 (IL-1) is released. Interleukin-1 triggers an inflammatory reaction characterized by extravasation of polymorphonuclear (PMN) cells. The inflammation is probably potentiated by the production of other cytokines by epithelial, endothelial, and PMN cells. Polymorphonuclear cells migrate through the epithelium into the lumen of the colon, destabilizing the integrity of the epithelial barrier. The damaged epithelium allows massive entry of bacteria into the submucosa. Further colonization of the epithelium aggravates inflammation, which in turn causes extensive tissue destruction. Both the in vitro and in vivo results that support this model are discussed.

Apoptosis↗