Interaction of Candida Species with the Skin

The human skin is commonly colonized by diverse fungal species. Some Candida species, especially C. albicans, do not only reside on the skin surface as commensals, but also cause infections by growing into the colonized tissue. However, defense mechanisms at the skin barrier level are very efficient, involving residential non-immune and immune cells as well as immune cells specifically recruited to the site of infection. Therefore, the skin is an effective barrier against fungal infection. While most studies about commensal and pathogenic interaction of Candida species with host epithelia focus on the interaction with mucosal surfaces such as the vaginal and gastrointestinal epithelia, less is known about the mechanisms underlying Candida interaction with the skin. In this review, we focus on the ecology and molecular pathogenesis of Candida species on the skin and give an overview of defense mechanisms against C. albicans in this context. We also discuss new research avenues in dermal infection, including the involvement of neurons, fibroblasts, and commensal bacteria in both mouse and human model systems

Entry of Listeria monocytogenes in Mammalian Epithelial Cells: An Updated View

International audienceListeria monocytogenes is a bacterial pathogen that promotes its internalization into host epithelial cells. Interaction between the bacterial surface molecules InlA and InlB and their cellular receptors E-cadherin and Met, respectively, triggers the recruitment of endocytic effectors, the subversion of the phosphoinositide metabolism, and the remodeling of the actin cytoskeleton that lead to bacterial engulfment. Additional bacterial surface and secreted virulence factors also contribute to entry, albeit to a lesser extent. Here we review the increasing number of signaling effectors that are reported as being subverted by L. monocytogenes during invasion of cultured cell lines. We also update the current knowledge of the early steps of in vivo cellular infection, which, as shown recently, challenges previous concepts generated from in vitro data

Genome-Wide siRNA Screen Identifies Complementary Signaling Pathways Involved in Listeria Infection and Reveals Different Actin Nucleation Mechanisms during Listeria Cell Invasion and Actin Comet Tail Formation

Listeria monocytogenes enters nonphagocytic cells by a receptor-mediated mechanism that is dependent on a clathrin-based molecular machinery and actin rearrangements. Bacterial intra- and intercellular movements are also actin dependent and rely on the actin nucleating Arp2/3 complex, which is activated by host-derived nucleation-promoting factors downstream of the cell receptor Met during entry and by the bacterial nucleation-promoting factor ActA during comet tail formation. By genome-wide small interfering RNA (siRNA) screening for host factors involved in bacterial infection, we identified diverse cellular signaling networks and protein complexes that support or limit these processes. In addition, we could precise previously described molecular pathways involved in Listeria invasion. In particular our results show that the requirements for actin nucleators during Listeria entry and actin comet tail formation are different. Knockdown of several actin nucleators, including SPIRE2, reduced bacterial invasion while not affecting the generation of comet tails. Most interestingly, we observed that in contrast to our expectations, not all of the seven subunits of the Arp2/3 complex are required for Listeria entry into cells or actin tail formation and that the subunit requirements for each of these processes differ, highlighting a previously unsuspected versatility in Arp2/3 complex composition and function. IMPORTANCE: Listeria is a bacterial pathogen that induces its internalization within the cytoplasm of human cells and has been used for decades as a major molecular tool to manipulate cells in order to explore and discover cellular functions. We have inactivated individually, for the first time in epithelial cells, all the genes of the human genome to investigate whether each gene modifies positively or negatively the Listeria infectious process. We identified novel signaling cascades that have never been associated with Listeria infection. We have also revisited the role of the molecular complex Arp2/3 involved in the polymerization of the actin cytoskeleton, which was shown previously to be required for Listeria entry and movement inside host cells, and we demonstrate that contrary to the general dogma, some subunits of the complex are dispensable for both Listeria entry and bacterial movement

Rol de la Tropomiosina y del Adaptador NEDD9 durante la invasión celular de Listeria Mnocytogenes

Listeria monocytogenes es un patógeno de animales y humanos que logra invadir el espacio intracelular gracias a la interacción entre proteínas bacterianas de superficie y receptores en células hospedero, lo que permite activar cascadas de señalización que promueven la internalización de esta bacteria. El silenciamiento de la expresión génica en células de mamífero gracias a la técnica de transfección de ARNs pequeños de interferencia (siARN) ha permitido recientemente asociar nuevos efectores moleculares al proceso de internalización de distintos patógenos intracelulares en células eucariotas. Esta investigación hace uso de esta técnica para determinar la posible contribución de la tropomiosina (TPM) y de la proteína adaptadora NEDD9 a la invasión celular por parte de L. monocytogenes, así como de Salmonella typhimurium y de una cepa de Escherichia coli que expresa la invasina de Yersinia pseudotuberculosis. Utilizando ensayos de invasión se demuestra que el silenciamiento de la expresión de TPM, pero no de NEDD9, reduce significativamente la entrada de los tres patógenos intracelulares estudiados. Mediante microscopía de fluorescencia se observa que el silenciamiento de TPM y NEDD9 afecta en forma diferente la morfología celular y la distribución de los filamentos de actina. Estos resultados sugieren que TPM puede modular la entrada de patógenos bacterianos mediante una modificación de las propiedades de reorganización de la membrana plasmática dependientes del citoesqueleto de actina, propiedades que difieren de aquellas afectadas por NEDD9. Listeria monocytogenes is an animal and human pathogen that it is able to invade the intracellular space due to the interaction between bacterial surface proteins and host cell receptors, activating signaling cascades that promote pathogen internalization. Gene expression silencing in mammalian cells by transfection of small interfering RNAs (siRNA) has recently allowed the implication of novel molecular effectors to the internalization process of different intracellular pathogens in eukaryotic cells. The present work takes advantage of this technique to determine to potential contribution of tropomyosin (TPM) and the adaptor protein NEDD9 to cell invasion by L. monocytogenes, as well as Salmonella typhimurium and an Escherichia coli strain that expresses the invasin from Yersinia pseudotuberculosis. Using gentamicin invasion assays, it is shown that only TPM expression silencing reduces significantly the entry in HeLa cells of the three investigated bacterial pathogens. Fluorescence microscopy demonstrates that TPM and NEDD9 silencing affects differently HeLa cell morphology and the distribution of actin filaments. These results suggest that TPM may modulate the entry of bacterial pathogens by modifying the reorganization properties of the plasma membrane which are dependent on the actin cytoskeleton, and that these properties differ from those affected by NEDD9

Imaging InlC secretion to investigate cellular infection by the bacterial pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells

DC-SIGN as a receptor for phleboviruses

SummaryDuring natural transmission, bunyaviruses are introduced into the skin through arthropod bites, and dermal dendritic cells (DCs) are the first to encounter incoming viruses. DC-SIGN is a C-type lectin highly expressed on the surface of dermal DCs. We found that several arthropod-borne phleboviruses (Bunyaviridae), including Rift Valley fever and Uukuniemi viruses, exploit DC-SIGN to infect DCs and other DC-SIGN-expressing cells. DC-SIGN binds the virus directly via interactions with high-mannose N-glycans on the viral glycoproteins and is required for virus internalization and infection. In live cells, virus-induced clustering of cell surface DC-SIGN could be visualized. An endocytosis-defective mutant of DC-SIGN was unable to mediate virus uptake, indicating that DC-SIGN is an authentic receptor required for both attachment and endocytosis. After internalization, viruses separated from DC-SIGN and underwent trafficking to late endosomes. Our study provides real-time visualization of virus-receptor interactions on the cell surface and establishes DC-SIGN as a phlebovirus entry receptor

A Common Clathrin-Mediated Machinery Co-ordinates Cell–Cell Adhesion and Bacterial Internalization

Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E-cadherin, hijack the host adherens junction (AJ) machinery and invade non-phagocytic cells by a clathrin-dependent mechanism. Here, we investigate a potential role for clathrin in cell-cell adhesion. We observed that the initial steps of AJ formation trigger the phosphorylation of clathrin, and its transient localization at forming cell-cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of AJs. Using an InlA/E-cadherin chimera, we show that adherent cells expressing the chimera form AJs with cells expressing E-cadherin. We demonstrate that non-adherent cells expressing the InlA chimera, as bacteria, can be internalized by E-cadherin-expressing adherent cells. Together these results reveal that a common clathrin-mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes.Institut Pasteur, Inserm, INRA, ERC. Grant Number: Advanced Grant 233348; NIH grant. Grant Number: GM038093Peer Reviewe

A Common Clathrin-Mediated Machinery Co-ordinates Cell-Cell Adhesion and Bacterial Internalization

International audienceInvasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E-cadherin, hijack the host adherens junction (AJ) machinery and invade non- phagocytic cells by a clathrin-dependent mechanism. Here, we investigate a potential role for clathrin in cell–cell adhesion. We observed that the initial steps of AJ formation trigger the phosphorylation of clathrin, and its transient localization at forming cell – cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of AJs. Using an InlA/E-cadherin chimera, we show that adherent cells expressing the chimera form AJs with cells expressing E-cadherin. We demonstrate that non-adherent cells expressing the InlA chimera, as bacteria, can be internalized by E-cadherin-expressing adherent cells. Together these results reveal that a common clathrin-mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes

Computationally designed bispecific MD2/CD14 binding peptides show TLR4 agonist activity

Toll-like receptor 4 plays an important role in the regulation of the innate and adaptive immune response. The majority of TLR4 activators currently in clinical use are derivatives of its prototypic ligand LPS. The discovery of innovative TLR4 activators has the potential of providing new therapeutic immunomodulators and adjuvants. We used computational design methods to predict and optimize a total of 53 cyclic and linear peptides targeting myeloid differentiation 2 (MD2) and cluster of differentiation 14 (CD14), both coreceptors of human TLR4. Activity of the designed peptides was first assessed using NF-κB reporter cell lines expressing either TLR4/MD2 or TLR4/CD14 receptors, then binding to CD14 and MD2 confirmed and quantified using MicroScale Thermophoresis. Finally, we incubated select peptides in human whole blood and observed their ability to induce cytokine production, either alone or in synergy with LPS. Our data demonstrate the advantage of computational design for the discovery of new TLR4 peptide activators with little structural resemblance to known ligands and indicate an efficient strategy with which to identify TLR4 targeting peptides that could be used as easy-to-produce alternatives to LPS-derived molecules in a variety of settings