Biomedical subjects

P Gounon

Publications and source records attributed to P Gounon.

At least 19 recordsLinked to original sources

PubMed2005

Membrane curvature and the control of GTP hydrolysis in Arf1 during COPI vesicle formation.

The GTP switch of the small G-protein Arf1 (ADP-ribosylation factor 1) on lipid membranes promotes the polymerization of the COPI (coat protein complex I) coat, which acts as a membrane deforming shell to form transport vesicles. Real-time measurements for coat assembly on liposomes gives insights into how the GTPase cycle of Arf1 is coupled in time with the polymerization of the COPI coat and the resulting membrane deformation. One key parameter seems to be the membrane curvature. Arf-GAP1 (where GAP stands for GTPase-activating protein), which promotes GTP hydrolysis in the Arf1-COPI complex is highly sensitive to lipid packing. Its activity on Arf1-GTP increases by two orders of magnitude as the diameter of the liposomes approaches that of authentic transport vesicles (60 nm). This suggests that during membrane budding, Arf1-GTP molecules are progressively eliminated from the coated area where the membrane curvature is positive, but are protected from Arf-GAP1 at the bud neck due to the negative curvature of this region. As a result, the coat should be stable as long as the bud remains attached and should disassemble as soon as membrane fission occurs.

ADP-Ribosylation Factor 1↗

PubMed2001

A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier.

Listeria monocytogenes is responsible for severe food-borne infections, but the mechanisms by which bacteria cross the intestinal barrier are unknown. Listeria monocytogenes expresses a surface protein, internalin, that interacts with a host receptor, E-cadherin, to promote entry into human epithelial cells. Murine E-cadherin, in contrast to guinea pig E-cadherin, does not interact with internalin, excluding the mouse as a model for addressing internalin function in vivo. In guinea pigs and transgenic mice expressing human E-cadherin, internalin was found to mediate invasion of enterocytes and crossing of the intestinal barrier. These results illustrate how relevant animal models for human infections can be generated.

Animals↗

PubMed2001

A monoclonal antibody against the immunodominant epitope of the ribosomal P2beta protein of Trypanosoma cruzi interacts with the human beta 1-adrenergic receptor.

Monoclonal antibodies were raised against a recombinant ribosomal P2beta protein of Trypanosoma cruzi. One of these reacted with the C terminus of this protein (peptide R13, EEEDDDMGFGLFD) and epitope mapping confirmed that this epitope was the same as the one defined by the serum of immunized mice, and similar to the previously described chronic Chagas' heart disease (cChHD) anti-P epitope. Western blotting showed that the monoclonal antibody recognized the parasite ribosomal P proteins, as well as the human ribosomal P proteins. Electron microscopy showed that it stained different structures in parasite and human cells. Interestingly, surface plasmon resonance measurements indicated that the affinity for the parasite ribosomal P protein epitope (R13) was five times higher than for its human counterpart (peptide H13, EESDDDMGFGLFD). Since the human epitope contained an acidic region (EESDD) similar to the AESDE peptide recognized by cChHD patients in the second extra-cellular loop of the human beta1-adrenergic receptor, the biological activity of the antibody was assessed on neonatal rat cardiomyocytes in culture. The monoclonal antibody had an agonist-like effect. These results, together with the fact that the monoclonal reacted in Western blots with the different isoforms of the heart beta1-adrenergic receptor, confirm the possible pathogenic role of antibodies against the parasite ribosomal P protein based on their cross-reaction with the human beta1-adrenergic receptor.

Animals↗

PubMed2001

Structure and composition of the Shigella flexneri "needle complex", a part of its type III secreton.

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram-negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the "needle complex" (NC), produced an average image at 17 A resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2-3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N-terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.

Adhesins, Bacterial↗

PubMed2001

Identification of the Helicobacter pylori anti-sigma28 factor.

Flagellar motility is essential for colonization of the human gastric mucosa by Helicobacter pylori. The flagellar filament is composed of two subunits, FlaA and FlaB. Transcription of the genes encoding these proteins is controlled by the sigma28 and sigma54 factors of RNA polymerase respectively. The expression of flagellar genes is regulated, but no sigma28-specific effector was identified. It was also unclear whether H. pylori possessed a checkpoint for flagellar synthesis, and no gene encoding an anti-sigma28 factor, FlgM, could be identified by sequence similarity searches. To investigate the sigma28-dependent regulation, a new approach based on genomic data was used. Two-hybrid screening with the H. pylori proteins identified a protein of unknown function (HP1122) interacting with the sigma28 factor and defined the C-terminal part of HP1122 (residues 48-76) as the interaction domain. HP1122 interacts with region 4 of sigma28 and prevents its association with the beta-region of H. pylori RNA polymerase. Thus, HP1122 presented the characteristics of an anti-sigma28 factor. This was confirmed in H. pylori by RNA dot-blot hybridization and electron microscopy. The level of sigma28-dependent flaA transcription was higher in a HP1122-deficient strain and was decreased by the overproduction of HP1122. The overproduction of HP1122 also resulted in H. pylori cells with highly truncated flagella. These results demonstrate that HP1122 is the H. pylori anti-sigma28 factor, FlgM, a major regulator of flagellum assembly. Potential anti-sigma28 factors were identified in Campylobacter jejuni, Pseudomonas aeruginosa and Thermotoga maritima by sequence homology with the C-terminal region of HP1122.

Amino Acid Sequence↗

PubMed2001

Internalization of Bordetella pertussis adenylate cyclase-haemolysin into endocytic vesicles contributes to macrophage cytotoxicity.

Bordetella pertussis adenylate cyclase-haemolysin is a critical virulence factor in the murine model of intranasal infection, where it is required for several pathological effects, including macrophage apoptosis. Based on biochemical and immunological properties, it was proposed that the toxin was delivered directly to the cytoplasm of eukaryotic cells without trafficking through the endocytic pathway. In the present study, we analysed the cellular distribution of the adenylate cyclase-haemolysin during intoxication of macrophages. We showed that, shortly after its initial binding to the plasma membrane of macrophages, the toxin gains access to intracellular compartments that become progressively positive for the endosomal marker transferrin, but not for the lysosomal membrane protein CD107a/Lamp1. Importantly, the vesicular trafficking of the adenylate cyclase-haemolysin appears to be required for its ability to induce macrophage death. Inhibitors of actin polymerization and of macropinocytosis, as well as depletion of plasma membrane cholesterol and disruption of the Golgi network, reduce the toxin's ability to kill macrophages. Altogether, these results suggest that internalization of the adenylate cyclase-haemolysin into endocytic vesicles, at least partly through macropinocytosis, contributes to cytotoxicity.

Adenylate Cyclase Toxin↗

PubMed2001

Variations in biological features of West Nile viruses.

Pathological findings in humans, horses, and birds with West Nile (WN) encephalitis show neuronal degeneration and necrosis in the central nervous system (CNS), with diffuse inflammation. The mechanisms of WN viral penetration of the CNS and pathophysiology of the encephalitis remain largely unknown. Since 1996, several epizootics involving hundreds of humans, horses, and thousands of wild and domestic bird cases of encephalitis and mortality have been reported in Europe, North Africa, the Middle East, Russia, and the USA (see specific chapters in this issue). However, biological and molecular markers of virus virulence should be characterized to assess whether novel strains with increased virulence are responsible for this recent proliferation of outbreaks.

Aedes↗

PubMed2001

Characterization of AfaE adhesins produced by extraintestinal and intestinal human Escherichia coli isolates: PCR assays for detection of Afa adhesins that do or do not recognize Dr blood group antigens.

Operons of the afa family are expressed by pathogenic Escherichia coli strains associated with intestinal and extraintestinal infections in humans and animals. The recently demonstrated heterogeneity of these operons (L. Lalioui, M. Jouve, P. Gounon, and C. Le Bouguénec, Infect. Immun. 67:5048-5059, 1999) was used to develop a new PCR assay for detecting all the operons of the afa family with a single genetic tool. This PCR approach was validated by investigating three collections of human E. coli isolates originating from the stools of infants with diarrhea (88 strains), the urine of patients with pyelonephritis (97 strains), and the blood of cancer patients (115 strains). The results obtained with this single test and those previously obtained with several PCR assays were closely correlated. The AfaE adhesins encoded by the afa operons are variable, particularly with respect to the primary sequence encoded by the afaE gene. The receptor binding specificities have not been determined for all of these adhesins; some recognize the Dr blood group antigen (Afa/Dr(+) adhesins) on the human decay-accelerating factor (DAF) as a receptor, and others (Afa/Dr(-) adhesins) do not. Thus, the afa operons detected in this study were characterized by subtyping the afaE gene using specific PCRs. In addition, the DAF-binding capacities of as-yet-uncharacterized AfaE adhesins were tested by various cellular approaches. The afaE8 subtype (Afa/Dr(-) adhesin) was found to predominate in afa-positive isolates from sepsis patients (75%); it was frequent in afa-positive pyelonephritis E. coli (55.5%) and absent from diarrhea-associated strains. In contrast, Afa/Dr(+) strains (regardless of the afaE subtype) were associated with both diarrhea (100%) and extraintestinal infections (44 and 25% in afa-positive pyelonephritis and sepsis strains, respectively). These data suggest that there is an association between the subtype of AfaE adhesin and the physiological site of the infection caused by afa-positive strains.

Adhesins, Escherichia coli↗

PubMed2001

Nonenveloped nucleocapsids of hepatitis C virus in the serum of infected patients.

One of the characteristics of hepatitis C virus (HCV) is the high incidence of persistent infection. HCV core protein, in addition to forming the viral nucleocapsid, has multiple regulatory functions in host-cell transcription, apoptosis, cell transformation, and lipid metabolism and may play a role in suppressing host immune response. This protein is thought to be present in the bloodstream of the infected host as the nucleocapsid of infectious, enveloped virions. This study provides evidence that viral particles with the physicochemical, morphological, and antigenic properties of nonenveloped HCV nucleocapsids are present in the plasma of HCV-infected individuals. These particles have a buoyant density of 1.32 to 1.34 g/ml in CsCl, are heterogeneous in size (with predominance of particles 38 to 43 or 54 to 62 nm in diameter on electron microscopy), and express on their surface epitopes located in amino acids 24 to 68 of the core protein. Similar nucleocapsid-like particles are also produced in insect cells infected with recombinant baculovirus bearing cDNA for structural HCV proteins. HCV core particles isolated from plasma were used to generate anti-core monoclonal antibodies (MAbs). These MAbs stained HCV core in the cytoplasm of hepatocytes from experimentally infected chimpanzees in the acute phase of the infection. These chimpanzees had concomitantly HCV core antigen in serum. These findings suggest that overproduction of nonenveloped nucleocapsids and their release into the bloodstream are properties of HCV morphogenesis. The presence of circulating cores in serum and accumulation of the core protein in liver cells during the early phase of infection may contribute to the persistence of HCV and its many immunopathological effects in the infected host.

Amino Acid Sequence↗

PubMed2000

Characterization of the AfaD-like family of invasins encoded by pathogenic Escherichia coli associated with intestinal and extra-intestinal infections.

The afimbrial adhesive sheath, encoded by the afa-3 gene cluster, is composed of two proteins with different roles in bacterium-HeLa cell interactions. AfaE is required for adhesion and AfaD for internalization. In this study, we found that the AfaD invasin was structurally and functionally conserved among human afa-expressing strains, independently of AfaE subtype and clinical origin of the Escherichia coli isolate. The AggB protein from enteroaggregative E. coli was also found to be an AfaD-related invasin. These data suggest that AfaD is the prototype of a family of invasins encoded by adhesion-associated operons in pathogenic E. coli.

Adhesins, Bacterial↗

PubMed2000

Electron microscopic improvement in the study of diarrheagenic Escherichia coli.

In the versatile single species of Escherichia coli, the diarrheagenic group displays a remarkable array of virulence traits. These comprise microbial attachment, production of secretory endotoxins or cell-destroying cytotoxins, direct epithelial cell invasion, and localized effacement of the epithelium. The knowledge of how enteric E. coli induce disease has become increasingly important in the world, because of new pathogen emergence, increasing threats of drug resistance, and growing awareness of their importance in malnutrition and diarrhea. Numerous research programs have demonstrated various mechanisms of pathogenesis. We point out how some pathogens are able to develop intercourse with their host through subversion of its cytoskeleton and signaling processes without toxin secretion or heavy invasiveness. In that domain, the cellular biology of infected cells owes fundamental data to the electron microscopic approach. Combined with advances in microbiology and molecular biology, this approach may provide answers to many unanswered questions.

Animals↗

PubMed2000

Pilus formation and protein secretion by the same machinery in Escherichia coli.

The secreton (type II secretion) and type IV pilus biogenesis branches of the general secretory pathway in Gram-negative bacteria share many features that suggest a common evolutionary origin. Five components of the secreton, the pseudopilins, are similar to subunits of type IV pili. Here, we report that when the 15 genes encoding the pullulanase secreton of Klebsiella oxytoca were expressed on a high copy number plasmid in Escherichia coli, one pseudopilin, PulG, was assembled into pilus-like bundles. Assembly of the 'secreton pilus' required most but not all of the secreton components that are essential for pullulanase secretion, including some with no known homologues in type IV piliation machineries. Two other pseudopilins, pullulanase and two outer membrane-associated secreton components were not associated with pili. Thus, PulG is probably the major component of the pilus. Expression of a type IV pilin gene, the E.coli K-12 gene ppdD, led to secreton-dependent incorporation of PpdD pilin into pili without diminishing pullulanase secretion. This is the first demonstration that pseudopilins can be assembled into pilus-like structures.

Bacterial Proteins↗

PubMed2000

Immunocytochemistry of the AfaE adhesin and AfaD invasin produced by pathogenic Escherichia coli strains during interaction of the bacteria with HeLa cells by high-resolution scanning electron microscopy.

We used a recent scanning electron microscope equipped with field emission gun and highly sensitive detectors to develop a fast and simple protocol for double immunogold staining using 10- and 15-nm gold particles. We used this approach to analyse the afimbrial adhesive sheath produced by pathogenic Escherichia coli interacting with the surface of epithelial cells. We demonstrated that AfaE adhesin and AfaD invasin were exposed at the bacterial surface during the interaction. This method could be easily and widely extended to the study of the early invasion process of many bacterial and viral pathogens, by immunocytochemical probing.

Adhesins, Escherichia coli↗

PubMed2000

Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain guinea pig inclusion conjunctivitis.

The infectious cycle of phiCPG1, a bacteriophage that infects the obligate intracellular pathogen, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis, was observed using transmission electron microscopy of phage-hyperinfected, Chlamydia-infected HeLa cells. Phage attachment to extracellular, metabolically dormant, infectious elementary bodies and cointernalisation are demonstrated. Following entry, phage infection takes place as soon as elementary bodies differentiate into metabolically active reticulate bodies. Phage-infected bacteria follow an altered developmental path whereby cell division is inhibited, producing abnormally large reticulate bodies, termed maxi-reticulate bodies, which do not mature to elementary bodies. These forms eventually lyse late in the chlamydial developmental cycle, releasing abundant phage progeny in the inclusion and, upon lysis of the inclusion membrane, into the cytosol of the host cell. Structural integrity of the hyperinfected HeLa cell is markedly compromised at late stages. Released phage particles attach avidly to the outer leaflet of the outer membranes of lysed and unlysed Chlamydiae at different stages of development, suggesting the presence of specific phage receptors in the outer membrane uniformly during the chlamydial developmental cycle. A mechanism for phage infection is proposed, whereby phage gains access to replicating chlamydiae by attaching to the infectious elementary body, subsequently subverting the chlamydial developmental cycle to its own replicative needs. The implications of phage infection in the context of chlamydial infection and disease are discussed.

Animals↗

PubMed2000

The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins.

The Arp2/3 complex is an essential regulator of actin polymerization in response to signalling and generates a dendritic array of filaments in lamellipodia. Here we show that the activated Arp2/3 complex interacts with the barbed ends of filaments to initiate barbed-end branching. Barbed-end branching by Arp2/3 quantitatively accounts for polymerization kinetics and for the length correlation of the branches of filaments observed by electron microscopy. Filament branching is visualized at the surface of Listeria in a reconstituted motility assay. The functional antagonism between the Arp2/3 complex and capping proteins is essential in the maintenance of the steady state of actin assembly and actin-based motility.

Actin Cytoskeleton↗

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

CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells.

Shigella entry into epithelial cells is characterized by a transient reorganization of the host cell cytoskeleton at the site of bacterial interaction with the cell membrane, which leads to bacterial engulfment in a macropinocytic process. Using affinity chromatography on HeLa cell extracts, we show here that the hyaluronan receptor CD44 associates with IpaB, a Shigella protein that is secreted upon cell contact. Overlay and solid-phase assays indicated that IpaB binds directly to the extracellular domain of CD44; binding is saturable and inhibitable, with a half-maximal inhibitory concentration of 175 nM. Immunoprecipitation experiments showed that IpaB associates with CD44 during Shigella entry. CD44 is recruited at bacterial entry sites and localizes at the plasma membrane of cellular extensions induced by Shigella. Pretreatment of cells with an anti-CD44 monoclonal antibody resulted in inhibition of Shigella-induced cytoskeletal reorganization, as well as inhibition of bacterial entry, whereas transfection of CD44 in cells that are deficient for CD44 results in increased bacterial binding to cells and internalization. The IpaB-CD44 interaction appears to be required for Shigella invasion by initiating the early steps of the entry process.

Antibodies, Monoclonal↗

PubMed2000

Proteolysis of microtubule associated protein 2 and sensitivity of pancreatic tumours to docetaxel.

We have studied the state of microtubule associated protein 2 (MAP2) in the pancreatic ductal adenocarcinomas P03 and P02 (sensitive and refractory to docetaxel respectively) since they express the corresponding mRNA and MAP2-related peptides. Immunohistochemical localization showed that in tumour P03 the MAP2-related peptides are highly expressed and confined to the epithelial malignant cells while in P02 the Intensity of the immunostaining is lower. However, anti alpha-tubulin staining followed a similar pattern suggesting that the net amount of macromolecular structures in the sensitive tumour is higher than in the refractory one. This may explain its higher sensitivity to docetaxel, because tubulin assembled into microtubules is the target of the drug. We found that protein extracts from both tumours differed in their proteolytic activity on rat brain MAP2. Since the proteolysis pattern obtained was similar to the one produced by Cathepsin D, we studied the effect of MAP2 proteolysed by this enzyme on microtubule formation in vitro. Proteolysis was found to increase the tendency of tubulin to assemble into macromolecular structures (microtubules and aggregates) in the presence of docetaxel. This suggests that in vivo proteolysis of MAP2 might increase microtubule alterations and potentiate the antitumour effect of docetaxel.

Amino Acid Sequence↗