PubMed Health⌕ Search

Biomedical subjects

P Sansonetti

Publications and source records attributed to P Sansonetti.

At least 37 records · Page 2Linked to original sources
PubMed1999

Myosin IB from Entamoeba histolytica is involved in phagocytosis of human erythrocytes.

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery in humans. The disease is prevalent worldwide. Infection with E. histolytica results in invasion of the intestine by the parasite, followed by tissue damage and inflammation. During this invasive process, parasites kill and phagocytose human epithelial cells, immune cells and erythrocytes. Expression of amoebic pathogenicity requires a dynamic cytoskeleton that allows movement, tissue penetration and changes in parasite morphology. Myosin IB is a member of the myosin I family of motor proteins. Studies conducted both with Dictyostelium discoideum, a non-pathogenic amoeba, and with the yeast Saccharomyces cerevisiae indicate the involvement of myosin IB in cellular processes including movement, phagocytosis and endocytosis. Recently, we isolated the gene encoding myosin IB from E. histolytica. Thus, we decided to analyze the role of myosin IB in pathogenesis of amoeba. Using a specific anti-myosin IB antibody, this protein was localized in cell regions including the pseudopod, vesicles and underneath the plasma membrane. When E. histolytica was activated for erythrophagocytosis, myosin IB was markedly recruited to both the phagocytic cup and around internalized phagosomes. To analyze the role of myosin IB in phagocytosis, a strain overexpressing the myosin IB gene was constructed. This strain synthesizes threefold more myosin IB than the wild-type strain. Challenge of the transfected cell line with erythrocytes showed that these amoebae were deficient in erythrophagocytosis mainly in the uptake step, suggesting a role for myosin IB in the pathogenic activity of a human parasite.

3,3'-Diaminobenzidine↗
PubMed1998

Virulence and functions of myosin II are inhibited by overexpression of light meromyosin in Entamoeba histolytica.

Several changes in cell morphology take place during the capping of surface receptors in Entamoeba histolytica. The amoebae develop the uroid, an appendage formed by membrane invaginations, which accumulates ligand-receptor complexes resulting from the capping process. Membrane shedding is particularly active in the uroid region and leads to the elimination of accumulated ligands. This appendage has been postulated to participate in parasitic defense mechanisms against the host immune response, because it eliminates complement and specific antibodies bound to the amoeba surface. The involvement of myosin II in the capping process of surface receptors has been suggested by experiments showing that drugs that affect myosin II heavy-chain phosphorylation prevent this activity. To understand the role of this mechanoenzyme in surface receptor capping, a myosin II dominant negative strain was constructed. This mutant is the first genetically engineered cytoskeleton-deficient strain of E. histolytica. It was obtained by overexpressing the light meromyosin domain, which is essential for myosin II filament formation. E. histolytica overexpressing light meromyosin domain displayed a myosin II null phenotype characterized by abnormal movement, failure to form the uroid, and failure to undergo the capping process after treatment with concanavalin A. In addition, the amoebic cytotoxic capacities of the transfectants on human colon cells was dramatically reduced, indicating a role for cytoskeleton in parasite pathogenicity.

Animals↗
PubMed1998

Alteration of HLA-B27 peptide presentation after infection of transfected murine L cells by Shigella flexneri.

Shigella flexneri is a triggering agent for reactive arthritis in HLA-B27-susceptible individuals. Considering the intracellular multiplication of bacteria, it seems likely that bacterial peptides may be presented by the major histocompatibility complex (MHC) class I pathway. To examine this hypothesis, we infected HLA-B*2705- and/or human beta2-microglobulin-transfected murine L-cell lines with M90T, an invasive strain of S. flexneri. Bacterial infection induced no detectable modifications in the biosynthesis and expression level of HLA-B27, as assessed by immunoprecipitation, Northern blot analysis, and flow cytometry. Using confocal microscopy, we observed that bacterial infection induced a clustering of HLA-B27 molecules during macropinocytosis and before bacterial dissemination from cell to cell. Peptides naturally bound to HLA-B27 molecules were acid eluted from infected cells and separated by high-performance liquid chromatography. Major differences were observed in high-performance liquid chromatography profiles and in the nature of peptides presented following bacterial infection. Although most of the antigens presented were not accessed by Edman degradation, we obtained two sequences partially homologous to bacterial proteins. These peptides lacked the major HLA-B27 peptide anchor (Arg) at position 2, and one had an unusual length of 14 amino acids. These data suggest that alterations in the peptide presentation by HLA-B27 occur during infection, which could be relevant to the pathogenesis of HLA-B27-related arthritis.

Animals↗
PubMed1998

The small GTP-binding protein RacG regulates uroid formation in the protozoan parasite Entamoeba histolytica.

Entamoeba histolytica is a protozoan parasite that invades human intestine leading to ulceration and destruction of tissue. Amoebic movement and phagocytosis of human cells is accompanied by characteristic changes in cell morphology. Amoebae become polarized, developing a frontal pseudopod and a well-defined rear zone of membrane accumulation designated the uroid. In motile eukaryotic cells, a phenomenon that contributes to movement is the capping of receptors at the cell surface. During the capping process, E. histolytica concentrates ligand-receptor complexes in the uroid. Interestingly, some of these surface receptors are involved in the survival of the parasite. While looking for regulators of capping and uroid formation, we identified RacG, an E. histolytica protein that is homologous to human Rac1. This protein belongs to the Rac subfamily of small GTPases implicated in interactions between the actin cytoskeleton and the membrane of mammalian cells. Cloning of the EhracG gene and analysis of the protein activity either in murine fibroblasts or in E. histolytica revealed that EhRacG induces a characteristic Rac phenotype. When expressed in amoebae, an EhRacG-V12 mutant protein not only deregulated cell polarity, but also caused a defect in cytokinesis. Analysis of the cytoskeleton in amoebae bearing this mutant revealed that F-actin concentrated at the periphery of the cell. In addition, the number and localization of uroids were modified. These results suggest a role for EhRacG in amoebic morphogenesis and cytokinesis.

3T3 Cells↗
PubMed1997

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy.

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.

Biopsy↗
PubMed1997

Functional analysis of the Shigella flexneri IpaC invasin by insertional mutagenesis.

The ability of Shigella to enter epithelial cells, to escape from the phagocytic vacuole, and to induce apoptosis in macrophages requires the IpaB, IpaC, and IpaD proteins. An extracellular complex containing IpaB and IpaC can promote the uptake of inert particles by epithelial cells. To determine whether the function of IpaC is to act as an extracellular chaperone for IpaB in the Ipa complex or as an effector of entry involved in a direct interaction with the cell surface, we have constructed eight IpaC recombinant proteins by inserting the coding sequence for a 12- to 14-amino-acid fragment into restriction sites scattered within the ipaC gene. We have investigated the ability of recombinant proteins to bind IpgC in the bacterial cytoplasm and IpaB in the extracellular medium and to complement an ipaC null mutant for entry into HeLa cells, lysis of erythrocytes, and escape from the phagocytic vacuole in infected macrophages. Most recombinant proteins were produced and secreted at a level similar to that of wild-type IpaC and did not exhibit altered susceptibility to proteolysis by trypsin, and all were able to bind IpgC and IpaB. Some recombinant proteins did not complement the ipaC mutant for entry into HeLa cells, lysis of erythrocytes, or escape from the phagocytic vacuole, which indicates that IpaC plays an active role in these processes and does not act solely as a chaperone for IpaB. In addition, some insertions which were located outside of the hydrophobic region of IpaC differentially affected the abilities of Shigella to enter epithelial cells and to lyse cell membranes.

Antibodies, Monoclonal↗
PubMed1997

Molecular characterization of myosin IB from the lower eukaryote Entamoeba histolytica, a human parasite.

The complete amino acid sequence of the Entamoeba histolytica unconventional myosin IB (Eh-myosin IB) is reported. Sequencing of overlapping cDNA fragments reveals a single open reading frame which predicts a 130 kDa protein of 1049 aa. Eh-myosin IB presents the three characteristics domains of myosins I subclass 1. This protein presents homology with myosins IB from other amoebae, but striking homologies with vertebrate unconventional myosins were also observed. The predicted actin and ATP-binding sites are located in the head domain. The heavy chain phosphorylation region is homologous to metazoan myosins I with an acidic residue present at the phosphorylation site. In the neck domain, an IQ motif indicates potential binding of calmodulin to the myosin I heavy chain. In the tail of Eh-myosin IB the three characteristic regions of myosin I are found. A putative membrane binding domain a very short domain rich in alanine and proline we demonstrate to be functional for actin binding, and the src-homology 3 domain. The Entamoeba histolytica myosin IB is the first unconventional myosin so far described in a lower eukaryote.

Actins↗
PubMed1996

Rho-dependent membrane folding causes Shigella entry into epithelial cells.

The small GTPase rho is functionally involved in the formation of cytoskeletal structures like stress fibers or focal adhesion plaques. Shigella entry into HeLa cells induces a blossom-like membrane structure at the bacterial entry site. We show here that this membrane-folding process is rho-dependent. The three rho isoforms were recruited into bacterial entry sites with differential localization relative to the membrane structure. A rho-specific inhibitor abolished Shigella-induced membrane folding and impaired bacterial entry accordingly. S1-myosin labeling indicated that rho was involved in Shigella-induced actin polymerization but not actin nucleation in the bacterial invasion site. This provides a major link in the signalization cascade allowing entry of a bacterial pathogen into a eukaryotic cell.

Amino Acid Sequence↗
PubMed1996

The secreted Ipa complex of Shigella flexneri promotes entry into mammalian cells.

The bacterial pathogen Shigella flexneri causes bacillary dysentery in humans by invading coloncytes. Upon contact with epithelial cells, S. flexneri elicits localized plasma membrane projections sustained by long actin filaments which engulf the microorganism. The products necessary for Shigella entry include three secretory proteins: IpaB, IpaC, and IpaD. Extracellular IpaB and IpaC associate in a soluble complex, the Ipa complex. We have immunopurified this Ipa complex on latex beads and found that they were efficiently internalized into HeLa cells. Like S. flexneri entry, uptake of the beads bearing the Ipa complex was associated with membrane projections and polymerization of actin at the site of cell-bead interaction and was dependent on small Rho GTPases. These results indicate that a secreted factor can promote S. flexneri entry into epithelial cells.

Actins↗
PubMed1996

Identification and cellular localization of the actin-binding protein ABP-120 from Entamoeba histolytica.

Several actin-binding proteins participate in the morphological changes that occur during amoeboid movement. The gene encoding one of these proteins, the gelation factor ABP-120, was identified and characterized from trophozoites of Entamoeba histolytica. The sequence contains 2574 nucleotides, with an open reading frame of 858 amino acids, giving a protein of 93 kDa belonging to the spectrin family. The N-terminal domain of ABP-120 from E. histolytica revealed a consensus site for actin binding homologous to the actin-binding sites of ABP-120 of Dictyostelium discoideum, alpha-actinin and spectrin. Analysis of the central domain revealed the presence of four repeats of a 73-amino-acid motif constituting 31% of the protein. In addition, a stretch of 105 amino acids was highly divergent when compared with the C-terminal domain of D. discoideum ABP-120. This sequence showed short motifs that are homologous to microtubule-binding domains. We found that ABP-120 from E. histolytica binds to F-actin. In addition, upon motility of the parasite, this protein localized in the pseudopod and the uroid region, implying a role for ABP-120 in movement and capping of surface receptors in E. histolytica.

Actins↗
PubMed1996

Induction of a local anti-IpaC antibody response in mice by use of a Shigella flexneri 2a vaccine candidate: implications for use of IpaC as a protein carrier.

The capacity of Shigella flexneri to act as a delivery system for stimulation of local immunity was investigated by use of an S. Flexneri 2a vaccine candidate (SC602). This vaccine strain was constructed by deletion of virulence genes responsible for dissemination (icsA) and survival (iuc:iut) of bacteria within the colonic mucosa. Among the most immunogenic S. flexneri 2a proteins inducing a local immunoglobulin A antibody response, the IpaC invasion was selected as a potential protein carrier. Overexpression of IpaC from a plasmid in trans within SC602 (SC602/pIpaC) was shown to be required for the induction of optimal anti-IpaC antibody response in the murine pulmonary infection model. A weak anti-IpaC antibody response was obtained after intranasal inoculations with SC602/pIpaC live bacteria. This response was enhanced by combining systemic priming with SC602/pIpaC killed bacteria and local boosting with SC602/pIpaC live bacteria. These results suggest that naive B cells primed systemically could account for local antibody production. This immunization protocol will allow further evaluation of the immunogenicity of IpaC hybrid proteins expresses in SC602.

Animals↗
PubMed1996

Analysis of epithelial cell stress response during infection by Shigella flexneri.

Shigella flexneri-infected macrophage cells undergo an apoptotic-like death as early as one hour after infection (A. Zychlinsky, M. C. Prévost, and P. J. Sansonetti, Nature [London] 358:167-168, 1992). To determine the fate of infected epithelial cells, we characterized the viability, morphology, and several metabolic activities of HeLa cells after treatment with M90T, an invoffve isolate of S. flexneri serotype 5, or BS176, a noninvasive isolate cured of the 220-kb virulence plasmid. Using standard assays, we found that for at least 4 h after infection with M90T, HeLa cells remained viable and did not detach or lyse. The ultrastructural morphology of HeLa cells heavily infected with M90T was free of hallmarks associated with cells undergoing apoptosis. Consistent with the idea that intracellular bacterial growth is metabolically stressful to the host cell, we observed that, compared with BS176 treated-HeLa cells, M90T-treated HeLa cells showed (i) a significant decrease in the total pool size of nucleoside triphosphates, (ii) a reduced ability to incorporate extracellular radiolabeled methionine into the soluble and insoluble cell fractions, and (iii) a stimulation of glucose uptake. However, there was no detectable increase in expression of the stress-inducible hsp70 gene in M90T-infected HeLa cells or activation of the anaerobic metabolic pathway as determined by measuring total lactate levels. These results demonstrate clearly that the fate of S.flexneri-infected cells can vary dramatically between cell types and agree with the hypothesis that the destruction of epithelial cells observed in experimental models of shigellosis is due to the host inflammatory response and probably not bacterial intracellular multiplication per se.

Base Sequence↗
PubMed1995

Monoclonal immunoglobulin A antibody directed against serotype-specific epitope of Shigella flexneri lipopolysaccharide protects against murine experimental shigellosis.

To determine the role of humoral mucosal immune response in protection against shigellosis, we have obtained a monoclonal dimeric immunoglobulin A (IgA) antibody specific for Shigella flexneri serotype 5a lipopolysaccharide (mIgA) and used a murine pulmonary infection model that mimics the lesions occurring in natural intestinal infection. Adult BALB/c mice challenged with 10(7) S. flexneri organisms developed a rapid inflammatory response characterized by polymorphonuclear cell infiltration around and within the bronchi and strong systemic interleukin 6 response. Implantation of hybridoma cells in the back of mice, resulting in the development of a myeloma tumor producing mIgA in the serum and subsequently secretory mIgA in local secretions, or direct intranasal administration of these antibodies, protected the animals against subsequent intranasal challenge with S. flexneri serotype 5a. Absence of histopathological lesion and significant decrease in bacterial load of the lungs and of systemic interleukin 6 response were the three major criteria of protection. This protection was shown to be serotype-specific and dependent on local concentration of mIgA. These data demonstrate that mucosal antibodies directed against a single polysaccharidic surface epitope of Shigella can protect against the disease.

Administration, Intranasal↗
PubMed1995

Live attenuated Shigella flexneri mutants as vaccine candidates against shigellosis and vectors for antigen delivery.

Up to now, no effective vaccine is available against shigellosis, a dysenteric syndrome caused by Shigella, a Gram-negative bacterium which invades the human colonic mucosa. About 40 years of research in the field have led to the conclusion that orally administered live Shigella vaccine strains are more effective in eliciting protection than killed bacteria given parenterally. Recently, the construction of promising new live attenuated vaccines has come with a better understanding of the fundamental determinants governing pathogenesis. As the development of new vaccine strategies requires knowledge of both pathogenesis and the immune response against infection, the current view of the pathogenic process of Shigella infection and the anti-Shigella immune responses elicited by the host are presented. The attempts in Shigella vaccine design are reviewed and, the future of these vaccines discussed.

Animals↗