Role of lipid rafts in E-cadherin– and HGF-R/Met–mediated entry of Listeria monocytogenes into host cells

Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-β-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity

A Natural System of Chromosome Transfer in Yersinia pseudotuberculosis

The High Pathogenicity Island of Yersinia pseudotuberculosis IP32637 was previously shown to be horizontally transferable as part of a large chromosomal segment. We demonstrate here that at low temperature other chromosomal loci, as well as a non-mobilizable plasmid (pUC4K), are also transferable. This transfer, designated GDT4 (Generalized DNA Transfer at 4°C), required the presence of an IP32637 endogenous plasmid (pGDT4) that carries several mobile genetic elements and a conjugation machinery. We established that cure of this plasmid or inactivation of its sex pilus fully abrogates this process. Analysis of the mobilized pUC4K recovered from transconjugants revealed the insertion of one of the pGDT4–borne ISs, designated ISYps1, at different sites on the transferred plasmid molecules. This IS belongs to the IS6 family, which moves by replicative transposition, and thus could drive the formation of cointegrates between pGDT4 and the host chromosome and could mediate the transfer of chromosomal regions in an Hfr-like manner. In support of this model, we show that a suicide plasmid carrying ISYps1 is able to integrate itself, flanked by ISYps1 copies, at multiple locations into the Escherichia coli chromosome. Furthermore, we demonstrate the formation of RecA-independent cointegrates between the ISYps1-harboring plasmid and an ISYps1-free replicon, leading to the passive transfer of the non-conjugative plasmid. We thus demonstrate here a natural mechanism of horizontal gene exchange, which is less constrained and more powerful than the classical Hfr mechanism, as it only requires the presence of an IS6-type element on a conjugative replicon to drive the horizontal transfer of any large block of plasmid or chromosomal DNA. This natural mechanism of chromosome transfer, which occurs under conditions mimicking those found in the environment, may thus play a significant role in bacterial evolution, pathogenesis, and adaptation to new ecological niches

Apical invasion of intestinal epithelial cells by salmonella typhimurium requires villin to remodel the brush border actin cytoskeleton

Funding Information: We thank R. Friedman, C. Mulet and T. Pedron for technical help. We thank T. Marlovits for antibodies, H.D. Hardt and J. Galan for Salmonella strains, and D. Zhou and V. Koronakis for plasmids. We acknowledge France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04-01, «Investments for the future»). This work was supported by the ERC (P.S. Advanced Grant HOMEOEPITH, number 232798). P.J.S. is an HHMI senior foreign scholar. The authors declare no conflict of interest. Publisher Copyright: © 2015 Elsevier Inc.Salmonella invasion of intestinal epithelial cells requires extensive, though transient, actin modifications at the site of bacterial entry. The actin-modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin-severing activity. We investigated a possible role for villin in Salmonella invasion. The absence of villin, which is normally located at the bacterial entry site, leads to a decrease in Salmonella invasion. Villin is necessary for early membrane-associated processes and for optimal ruffle assembly by balancing the steady-state level of actin. The severing activity of villin is important for Salmonella invasion in vivo. The bacterial phosphatase SptP tightly regulates villin phosphorylation, while the actin-binding effector SipA protects F-actin and counterbalances villin-severing activity. Thus, villin plays an important role in establishing the balance between actin polymerization and actin severing to facilitate the initial steps of Salmonella entry.publishersversionpublishe

Apical invasion of intestinal epithelial cells by salmonella typhimurium requires villin to remodel the brush border actin cytoskeleton

Funding Information: We thank R. Friedman, C. Mulet and T. Pedron for technical help. We thank T. Marlovits for antibodies, H.D. Hardt and J. Galan for Salmonella strains, and D. Zhou and V. Koronakis for plasmids. We acknowledge France-BioImaging infrastructure supported by the French National Research Agency (ANR-10-INSB-04-01, «Investments for the future»). This work was supported by the ERC (P.S. Advanced Grant HOMEOEPITH, number 232798). P.J.S. is an HHMI senior foreign scholar. The authors declare no conflict of interest. Publisher Copyright: © 2015 Elsevier Inc.Salmonella invasion of intestinal epithelial cells requires extensive, though transient, actin modifications at the site of bacterial entry. The actin-modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin-severing activity. We investigated a possible role for villin in Salmonella invasion. The absence of villin, which is normally located at the bacterial entry site, leads to a decrease in Salmonella invasion. Villin is necessary for early membrane-associated processes and for optimal ruffle assembly by balancing the steady-state level of actin. The severing activity of villin is important for Salmonella invasion in vivo. The bacterial phosphatase SptP tightly regulates villin phosphorylation, while the actin-binding effector SipA protects F-actin and counterbalances villin-severing activity. Thus, villin plays an important role in establishing the balance between actin polymerization and actin severing to facilitate the initial steps of Salmonella entry.publishersversionpublishe

Extracellular Bacterial Pathogen Induces Host Cell Surface Reorganization to Resist Shear Stress

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow–generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream

Role of AmiA in the Morphological Transition of Helicobacter pylori and in Immune Escape

The human gastric pathogen Helicobacter pylori is responsible for peptic ulcers and neoplasia. Both in vitro and in the human stomach it can be found in two forms, the bacillary and coccoid forms. The molecular mechanisms of the morphological transition between these two forms and the role of coccoids remain largely unknown. The peptidoglycan (PG) layer is a major determinant of bacterial cell shape, and therefore we studied H. pylori PG structure during the morphological transition. The transition correlated with an accumulation of the N-acetyl-D-glucosaminyl-β(1,4)-N-acetylmuramyl-L-Ala–D-Glu (GM-dipeptide) motif. We investigated the molecular mechanisms responsible for the GM-dipeptide motif accumulation, and studied the role of various putative PG hydrolases in this process. Interestingly, a mutant strain with a mutation in the amiA gene, encoding a putative PG hydrolase, was impaired in accumulating the GM-dipeptide motif and transforming into coccoids. We investigated the role of the morphological transition and the PG modification in the biology of H. pylori. PG modification and transformation of H. pylori was accompanied by an escape from detection by human Nod1 and the absence of NF-κB activation in epithelial cells. Accordingly, coccoids were unable to induce IL-8 secretion by AGS gastric epithelial cells. amiA is, to our knowledge, the first genetic determinant discovered to be required for this morphological transition into the coccoid forms, and therefore contributes to modulation of the host response and participates in the chronicity of H. pylori infection

Complete Chromosome Sequence of Carnobacterium maltaromaticum LMA 28

Within the lactic acid bacterium genus Carnobacterium, Carnobacterium maltaromaticum is one of the most frequently isolated species from natural environments and food. It potentially plays a major role in food product biopreservation. We report here on the 3.649-Mb chromosome sequence of C. maltaromaticum LMA 28, which was isolated from ripened soft cheese

Mycoplasma penetrans bacteremia and primary antiphospholipid syndrome.

Mycoplasma penetrans, a rare bacterium so far only found in HIV-infected persons, was isolated in the blood and throat of a non-HIV-infected patient with primary antiphospholipid syndrome (whose etiology and pathogenesis are unknown)

Two Distinct Pathways Leading to Nuclear Apoptosis

Apaf-1−/− or caspase-3−/− cells treated with a variety of apoptosis inducers manifest apoptosis-associated alterations including the translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, large scale DNA fragmentation, and initial chromatin condensation (stage I). However, when compared with normal control cells, Apaf-1−/− or caspase-3−/− cells fail to exhibit oligonucleosomal chromatin digestion and a more advanced pattern of chromatin condensation (stage II). Microinjection of such cells with recombinant AIF only causes peripheral chromatin condensation (stage I), whereas microinjection with activated caspase-3 or its downstream target caspase-activated DNAse (CAD) causes a more pronounced type of chromatin condensation (stage II). Similarly, when added to purified HeLa nuclei, AIF causes stage I chromatin condensation and large-scale DNA fragmentation, whereas CAD induces stage II chromatin condensation and oligonucleosomal DNA degradation. Furthermore, in a cell-free system, concomitant neutralization of AIF and CAD is required to suppress the nuclear DNA loss caused by cytoplasmic extracts from apoptotic wild-type cells. In contrast, AIF depletion alone suffices to suppress the nuclear DNA loss contained in extracts from apoptotic Apaf-1−/− or caspase-3−/− cells. As a result, at least two redundant parallel pathways may lead to chromatin processing during apoptosis. One of these pathways involves Apaf-1 and caspases, as well as CAD, and leads to oligonucleosomal DNA fragmentation and advanced chromatin condensation. The other pathway, which is caspase-independent, involves AIF and leads to large-scale DNA fragmentation and peripheral chromatin condensation

Le théâtre de Dumas père, entre héritage et renouvellement

Ce livre part d'un constat : malgré une abondante production, étalée sur près de cinquante ans, le théâtre de Dumas subit aujourd'hui une indéniable éclipse. Or, son auteur a joué un rôle de premier plan dans la « bataille » romantique ; il a continué sans interruption d'écrire pour la scène jusqu'à sa mort ; refusant l'exclusive, il a pratiqué tous les genres. Il laisse un corpus officiel de soixante-six pièces, en réalité plus de cent si l'on comptabilise les textes non signés mais où sa collaboration est attestée. Cette œuvre s'inscrit dans un héritage, revendiqué ou implicite, de l'Antiquité à la production contemporaine : tragédie classique et néo-classique, comédie d'intrigue et de caractère, scène historique, drame bourgeois, vaudeville, mélodrame, proverbe… Elle en fait un usage complexe, entre emprunts, variations et détournements, en jouant de la contamination entre modèles « nobles » et modèles « mineurs ». Parcouru par un double processus d'assimilation et de transformation, le théâtre de Dumas fonctionne comme un creuset dramatique ; il fait évoluer les genres, modernise les enjeux et renouvelle le spectaculaire, marquant ainsi un repère capital pour les générations suivantes. Travail collectif d'une équipe de chercheurs engagés dans la publication du Théâtre complet de Dumas, ce volume veut rendre hommage à la richesse et à la diversité d'une œuvre qui résume un demi-siècle de vie dramatique