Bacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world’s population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.

Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.

SummaryIn the embryonic zebrafish, skeletal muscle fibres are formed from muscle progenitors in the paraxial mesoderm. The embryonic myotome is mostly constituted of fast-twitch-specific fibres, which are formed from a fast-specific progenitor cell pool. The most lateral fraction of the fast domain in the myotome of zebrafish embryos derives from the Pax7-positive dermomyotome-like cells. In this study, we show that two genes, belonging to the sine oculus class 1 (six1) genes (six1a and six1b), are both essential for the regulation of Pax7 + cell proliferation and, consequently, in their differentiation during the establishment of the zebrafish dermomyotome. In both six1a and six1b morphant embryos, Pax7 + cells are initially formed but fail to proliferate, as detected by reduced levels of the proliferation marker phosphohistone3 and reduced brdU incorporation. In congruence, overexpression of six1a or six1b leads to increased Pax7 + cell number and reduced or alternatively delayed fibre cell differentiation. Bone morphogenetic protein signalling has previously been suggested to inhibit differentiation of Pax7 + cells in the dermomyotome. Here we show that the remaining Pax7 + cells in six1a and six1b morphant embryos also have significantly reduced pSmad1/5/8 levels and propose that this leads to a reduced proliferative activity, which may result in a premature differentiation of Pax7 + cells in the zebrafish dermomyotome. In summary, we show a mechanism for Six1a and Six1b in establishing the Pax7 + cell derived part of the fast muscle and suggest new important roles for Six1 in the regulation of the Pax7 + muscle cell population through pSmad1/5/8 signalling.

Epidemic keratoconjunctivitis (EKC) is a severe, contagious ocular disease that affects 20 to 40 million individuals worldwide every year. EKC is mainly caused by six types of human adenovirus (HAdV): HAdV-8, -19, -37, -53, -54, and -56. Of these, HAdV-8, -19, and -37 use sialic acid-containing glycans as cellular receptors. αVβ3, αVβ5, and a few additional integrins facilitate entry and endosomal release of other HAdVs. With the exception of a few biochemical analyses indicating that HAdV-37 can interact physically with αVβ5, little is known about the integrins used by EKC-causing HAdVs. Here, we investigated the overall integrin expression on human corneal cells and found expression of α2, α3, α6, αV, β1, and β4 subunits in human corneal in situ epithelium and/or in a human corneal epithelial (HCE) cell line but no or less accessible expression of α4, α5, β3, or β5. We also identified the integrins used by HAdV-37 through a series of binding and infection competition experiments and different biochemical approaches. Together, our data suggest that HAdV-37 uses αVβ1 and α3β1 integrins for infection of human corneal epithelial cells. Furthermore, to confirm the relevance of these integrins in the HAdV-37 life cycle, we developed a corneal multilayer tissue system and found that HAdV-37 infection correlated well with the patterns of αV, α3, and β1 integrin expression. These results provide further insight into the tropism and pathogenesis of EKC-causing HAdVs and may be of importance for future development of new antiviral drugs.IMPORTANCE Keratitis is a hallmark of EKC, which is caused by six HAdV types (HAdV-8, -19, -37, -53, -54, and -56). HAdV-37 and some other HAdV types interact with integrin αVβ5 in order to enter nonocular human cells. In this study, we found that αVβ5 is not expressed on human corneal epithelial cells, thus proposing other host factors mediate corneal infection. Here, we first characterized integrin expression patterns on corneal tissue and corneal cells. Among the integrins identified, competition binding and infection experiments and biochemical assays pointed out αVβ1 and α3β1 to be of importance for HAdV-37 infection of corneal tissue. In the absence of a good animal model for EKC-causing HAdVs, we also developed an in vitro system with multilayer HCE cells and confirmed the relevance of the suggested integrins during HAdV-37 infection.

Endocytic mechanisms have been suggested to be important for plasma membrane repair in response to pore-forming toxins such as listeriolysin O (LLO), which form membrane pores that disrupt cellular homeostasis. Yet, little is known about the specific role of distinct endocytic machineries in this process. Here, we have addressed the importance of key endocytic pathways and developed reporter systems for real-time imaging of the endocytic response to LLO pore formation. We found that loss of clathrin-independent endocytic pathways negatively influenced the efficiency of membrane repair. However, we did not detect any increased activity of these pathways, or co-localisation with the toxin or markers of membrane repair, suggesting that they were not directly involved in removal of LLO pores from the plasma membrane. In fact, markers of clathrin-independent carriers (CLICs) were rapidly disassembled in the acute phase of membrane damage due to Ca2+ influx, followed by a reassembly about 2 min after pore formation. We propose that these endocytic mechanisms might influence membrane repair by regulating the plasma membrane composition and tension, but not via direct internalisation of LLO pores.