Search PubMed⌕ Search

Biomedical subjects

P J Sansonetti

Publications and source records attributed to P J Sansonetti.

At least 19 recordsLinked to original sources
PubMed2000

Invasive Shigella flexneri activates NF-kappa B through a lipopolysaccharide-dependent innate intracellular response and leads to IL-8 expression in epithelial cells.

The pathogenesis of Shigella flexneri infection centers on the ability of this organism to invade epithelial cells and initiate an intense inflammatory reaction. Because NF-kappa B is an important transcriptional regulator of genes involved in inflammation, we investigated the role of this transcription factor during S. flexneri infection of epithelial cells. Infection of HeLa cells with invasive S. flexneri induced NF-kappa B DNA-binding activity; noninvasive S. flexneri strains did not lead to this activation. The pathway leading to NF-kappa B activation by invasive S. flexneri involved the kinases, NF-kappa B-inducing kinase, I kappa B kinase-1, and I kappa B kinase-2. NF-kappa B activation was linked to inflammation, because invasive S. flexneri activated an IL-8 promoter-driven reporter gene, and the kappa B site within this promoter was indispensable for its induction. Microinjection of bacterial culture supernatants into HeLa cells suggested that LPS is responsible for NF-kappa B activation by S. flexneri infection. In conclusion, the eukaryotic transcription factor NF-kappa B was activated during S. flexneri infection of epithelial cells, which suggests a role for this transcriptional regulator in modulating the immune response during infection in vivo.

Antigens, Bacterial↗
PubMed2000

The pathogenesis of Shigella flexneri infection: lessons from in vitro and in vivo studies.

Shigella flexneri is a Gram-negative facultatively intracellular pathogen responsible for bacillary dysentery in humans. More than one million deaths occur yearly due to infections with Shigella spp. and the victims are mostly children of the developing world. The pathogenesis of Shigella centres on the ability of this organism to invade the colonic epithelium where it induces severe mucosal inflammation. Much information that we have gained concerning the pathogenesis of Shigella has been derived from the study of in vitro models of infection. Using these techniques, a number of the molecular mechanisms by which Shigella invades epithelial cells and macrophages have been identified. In vivo models of shigellosis have been hampered since humans are the only natural hosts of Shigella. However, experimental infection of macaques as well as the murine lung and rabbit ligated ileal loop models have been important in defining some of the immune and inflammatory components of the disease. In particular, the murine lung model has shed light on the development of systemic and local immune protection against Shigella infection. It would be naive to believe that any one model of Shigella infection could adequately represent the complexity of the disease in humans, and more sophisticated in vivo models are now necessary. These models require the use of human cells and tissue, but at present such models remain in the developmental stage. Ultimately, however, it is with such studies that novel treatments and vaccine candidates for the treatment and prevention of shigellosis will be designed.

Adaptation, Physiological↗
PubMed2000

Production of IFN-gamma and IL-10 to Shigella invasins by mononuclear cells from volunteers orally inoculated with a Shiga toxin-deleted Shigella dysenteriae type 1 strain.

Volunteers were orally administered invasive, non-Shiga toxin-producing Shigella dysenteriae 1 to establish a challenge model to assess vaccine efficacy. In stepwise fashion, four separate groups were given 3 x 10(2), 7 x 10(3), 5 x 10(4), or 7 x 10(5) CFU. Using PBMC, proliferative responses and cytokine production were measured to S. dysenteriae whole-cell preparations and to purified recombinant invasion plasmid Ags (Ipa) C and IpaD. Anti-LPS and anti-Ipa Abs and Ab-secreting cells were also evaluated. Preinoculation PBMC produced considerable quantities of IL-10 and IFN-gamma, probably secreted by monocytes and NK cells, respectively, of the innate immune system. Following inoculation, PBMC from 95 and 87% of volunteers exhibited an increased production of IFN-gamma and IL-10, respectively, in response to Shigella Ags. These increases included responses to IpaC and IpaD among those volunteers receiving the lowest inoculum. No IL-4 or IL-5 responses were detected. Whereas there were no Ab or Ab-secreting cell responses in volunteers receiving the lowest inoculum, other dose groups had moderate to strong anti-LPS and anti-Ipa responses. These results suggest that in humans, type 1 responses play an important role in mucosal and systemic immunity to S. dysentariae 1.

Adhesins, Bacterial↗
PubMed2000

Caspase-1 activation of IL-1beta and IL-18 are essential for Shigella flexneri-induced inflammation.

Caspases are intracellular proteases that mediate mammalian cell apoptosis. Caspase-1 (Casp-1) is a unique caspase because it activates the proinflammatory cytokines interleukin (IL)-1beta and IL-18. Shigella flexneri, the etiological agent of bacillary dysentery, induces macrophage apoptosis, which requires Casp-1 and results in the release of mature IL-1beta and IL-18. Here we show that casp-1(-/-) mice infected with S. flexneri do not develop the acute inflammation characteristic of shigellosis and are unable to resolve the bacterial infection. Using casp-1(-/-) mice supplemented with recombinant cytokines and experiments with IL-1beta(-/-) and IL-18(-/-) mice, we show that IL-1beta and IL-18 are both required to mediate inflammation in S. flexneri infections. Together, these data demonstrate the importance of Casp-1 in acute inflammation and show the different roles of its substrates, IL-1beta and IL-18, in this response.

Animals↗
PubMed2000

Diversion of cytoskeletal processes by Shigella during invasion of epithelial cells.

Shigella, the causative agent of bacillar dysentery, invades colonic epithelial cells and moves intracellularly to spread from cell to cell. The processes of Shigella entry, determined by the Ipa proteins, and of actin-based motility, dependent on the IcsA/VirG protein, represent different levels of bacterial manipulation of the cell cytoskeleton.

Actins↗
PubMed2000

IpgD, a protein secreted by the type III secretion machinery of Shigella flexneri, is chaperoned by IpgE and implicated in entry focus formation.

Invasion of epithelial cells by Shigella flexneri involves entry and intercellular dissemination. Entry of bacteria into non-phagocytic cells requires the IpaA-D proteins that are secreted by the Mxi-Spa type III secretion machinery. Type III secretion systems are found in several Gram-negative pathogens and serve to inject bacterial effector proteins directly into the cytoplasm of host cells. In this study, we have analysed the IpgD protein of S. flexneri, the gene of which is located on the virulence plasmid at the 5' end of the mxi-spa locus. We have shown that IpgD (i) is stored in the bacterial cytoplasm in association with a specific chaperone, IpgE; (ii) is secreted by the Mxi-Spa type III secretion system in amounts similar to those of the IpaA-D proteins; (iii) is associated with IpaA in the extracellular medium; and (iv) is involved in the modulation of the host cell response after contact of the bacterium with epithelial cells. This suggests that IpgD is an effector that might be injected into host cells to manipulate cellular processes during infection.

Bacterial Proteins↗
PubMed2000

Induction of necrosis in human neutrophils by Shigella flexneri requires type III secretion, IpaB and IpaC invasins, and actin polymerization.

Infection by Shigella flexneri is characterized by infiltration of neutrophils in the intestinal mucosa and by a strong inflammatory reaction. Although neutrophils are constitutively programmed to die by apoptosis, we show that isolated human neutrophils undergo necrosis 2 h after infection with virulent S. flexneri strain M90T but not with the virulence plasmid-cured strain BS176. This was demonstrated by the release of azurophil granule proteins concomitant with the release of lactate dehydrogenase (LDH), disruption of the plasma membrane, and absence of DNA fragmentation. Mutants with the mxiD1 gene, coding for an essential component of the secretion type III machinery, or the genes coding for IpaB or IpaC invasins deleted were not cytotoxic. Neutrophil necrosis occurred independently of the bacterial ability to leave phagosomes, and it involved actin polymerization, as the addition of cytochalasin D after phagocytosis of Shigella inhibited the release of LDH. In conclusion, Shigella kills neutrophils by necrosis, a process characterized by the release of tissue-injurious granular proteins. This probably contributes to disruption of the epithelial barrier, leading to the dysentery observed in shigellosis and allowing Shigella to enter its host cells.

Actins↗
PubMed1999

IkappaBepsilon-deficient mice: reduction of one T cell precursor subspecies and enhanced Ig isotype switching and cytokine synthesis.

Three major inhibitors of the NF-kappaB/Rel family of transcription factors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, have been described. To examine the in vivo role of the most recently discovered member of the IkappaB family, IkappaBepsilon, we generated a null allele of the murine IkappaBepsilon gene by replacement of all coding sequences with nlslacZ. Unlike IkappaBalpha nullizygous mice, mice lacking IkappaBepsilon are viable, fertile, and indistinguishable from wild-type animals in appearance and histology. Analysis of beta-galactosidase expression pattern revealed that IkappaBepsilon is mainly expressed in T cells in the thymus, spleen, and lymph nodes. Flow cytometric analysis of immune cell populations from the bone marrow, thymus, spleen, and lymph nodes did not show any specific differences between the wild-type and the mutant mice, with the exception of a reproducible 50% reduction of the CD44-CD25+ T cell subspecies. The IkappaBepsilon-null mice present constitutive up-regulation of IgM and IgG1 Ig isotypes together with a further increased synthesis of these two isotypes after immunization against T cell-dependent or independent Ags. The failure of observable augmentation of constitutive nuclear NF-kappaB/Rel-binding activity is probably due to compensatory mechanisms involving IkappaBalpha and IkappaBbeta, which are up-regulated in several organs. RNase-mapping analysis indicated that IL-1alpha, IL-1beta, IL-1Ra, and IL-6 mRNA levels are constitutively elevated in thioglycolate-elicited IkappaBepsilon-null macrophages in contrast to GM-CSF, G-CSF, and IFN-gamma, which remain undetectable.

Animals↗
PubMed1999

Binding of the Shigella protein IpaA to vinculin induces F-actin depolymerization.

Shigella flexneri, the causative agent of bacillary dysentery, enters into epithelial cells by a macropinocytic process. IpaA, a Shigella protein secreted upon cell contact, binds to the focal adhesion protein vinculin and is required for efficient bacterial uptake. IpaA was shown here to bind with high affinity to the N-terminal residues 1-265 of vinculin. Using co-sedimentation and solid-phase assays, we demonstrated that binding of IpaA to vinculin strongly increases the association of vinculin with F-actin. We also characterized a depolymerizing activity on actin filaments associated with the vinculin-IpaA complex both in vitro and in microinjected cells. We propose that the conformational change of vinculin induced by IpaA binding allows interaction of the vinculin-IpaA complex with F-actin and subsequent depolymerization of actin filaments.

Actin Cytoskeleton↗
PubMed1999

Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility.

To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH(2)-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.

Actin-Related Protein 2↗
PubMed1999

IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells.

Shigella proteins that are targeted to host cells by a type III secretion apparatus are essential for reorganization of the cytoskeleton during cell invasion. We have developed a semi-permeabilized cell assay that tests the effects of bacterial proteins on the actin cytoskeleton. The Shigella IpaC protein was found to induce the formation of filopodial and lamellipodial extensions in these semi-permeabilized cells. Microinjection of IpaC into cells, or cellular expression of IpaC also led to the formation of filopodial structures. Monoclonal antibodies (mAbs) directed against the C-terminus of IpaC inhibited the IpaC-induced extensions, whereas an anti-N-terminal IpaC mAb stimulated extensive lamellae formation. Shigella induced foci of actin polymerization in the permeabilized cells and these were inhibited by anti-C-terminal IpaC mAbs. Consistent with a role for IpaC in Shigella-induced cytoskeletal rearrangements during entry, stable transfectants expressing IpaC challenged with Shigella showed increased bacterial internalization. IpaC-induced extensions were inhibited by a dominant-interfering form of Cdc42 or the Cdc42-binding domain of WASP, whereas a dominant-interfering form of Rac resulted in inhibition of lamellae formation. We conclude that IpaC leads to activation of Cdc42 which in turn, causes activation of Rac, both GTPases being required for Shigella entry.

3T3 Cells↗
PubMed1999

Trafficking of Shigella lipopolysaccharide in polarized intestinal epithelial cells.

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395-4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

Biomarkers↗
PubMed1999

M cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process.

M cells are key sites of antigen sampling for the mucosal-associated lymphoid system (MALT) and consequently are essential components of the structures serving as inductive sites for mucosal immunity. In addition, they have recently been recognized as major sites of adherence and major ports of entry for enteric pathogens. In the case of enteroinvasive pathogens, such as Salmonella, Yersinia and Shigella, few clinical evidences, but lots of experimental data indicate that, at least at the early stage of infection, M cells of the follicular associated epithelium transport the pathogens. This has significantly altered our view on the pathogenesis of enteroinvasive infections. Crossing the epithelial barrier seems an achievable task for these bacteria which express adherence and invasion mechanisms which have often been well characterized in epithelial cell lines. These systems seem to be also used for entering and crossing M cells, although reproducible in vitro assays for M cell infection are now required. Having crossed the epithelial lining, the bacteria face phagocytic cells, particularly the macrophages that are present in the follicle dome. Depending on the capacity to survive in the presence of macrophages, and how this survival is achieved by a given invasive species, the outcome of infection can be dramatically affected. In consequence, M cells can be considered as pathogen translocators toward immunocompetent areas of the gut, thus opening the possibility to harness this property in order to design new mucosal vaccines or vaccine vectors.

Antigens↗
PubMed1999

Mechanism of Shigella entry into epithelial cells.

Shigella, the causative agent of bacillary dysentery, invades epithelial cells by reorganizing the cell cytoskeleton during bacterial entry. This entry process requires the Shigella Ipa proteins that are secreted by a type III secretion apparatus and that act in concert to fine tune cell responses. Actin polymerization at the site of entry is dependent on the IpaB and IpaC proteins, whereas IpaA further modulates cytoskeletal rearrangements by binding to vinculin.

Animals↗