استكشف المجموعة الواسعة من العناوين المتاحة.

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane-derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including beta-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP-mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche.

Key Points On infection, bacterial pathogens interact with host membranes to trigger various cellular processes through different mechanisms. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways that are involved in vesicle trafficking. Many bacterial effectors manipulate phosphoinositide (PI) homeostasis at the plasma membrane to destabilize actin dynamics and alter the morphology of the membrane. This facilitates the entry of pathogens or, in other cases, damages the cells by disrupting membrane integrity and eventually leading to rapid cell lysis in the later stage of infection. Some pathogens use bacterial phosphatases or PI adaptor proteins to form intracellular vacuoles that are derived from host membranes in order to establish a replicative niche. Altered PI levels at the surfaces of these vacuoles as a result of the activity of bacterial phosphatases can block phagosomal maturation to avoid lysosomal fusion. The GTPase signalling pathway is often targeted by bacterial pathogens to manipulate the actin cytoskeleton and endosomal trafficking. RAB GTPases , which have an important role in vesicular trafficking pathways, are recruited to bacterium-containing vacuoles, where their active state can be differentially regulated by effectors. Bacterial effectors mimic GTPase-activating protein (GAP) or guanine nucleotide exchange factor (GEF) activity to target RHO-family GTPases that are key regulators of actin dynamics. This results in loss of cell shape, motility and ability to phagocytose pathogens. Autophagy is one of the cellular defence mechanisms against the invasion of pathogenic bacteria. However, some pathogens have evolved strategies to subvert autophagy to their own advantage by establishing autophagic vesicles as their replicative niche. This allows them to survive inside host cells and avoid lysosomal degradation. Some bacterial effectors are speculated to induce autophagy during infection. This may not only protect the bacteria from degradative enzymes and immune responses, but also provide nutrients from cellular debris. For extracellular pathogens, inducing autophagy helps prevent phagocytosis. Bacterial pathogens secrete a range of effector proteins to target the signalling pathways that regulate host cell membranes. Here, Orth and colleagues describe the bacterial effectors that target phosphoinositide signalling, GTPase signalling and autophagy, and discuss how targeting these pathways can alter host membrane dynamics. Bacterial pathogens interact with host membranes to trigger a wide range of cellular processes during the course of infection. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways involved in vesicle trafficking. Such changes facilitate the entry and replication of the pathogen, and prevent its phagocytosis and degradation. In this Review, we describe the manipulation of host membranes by numerous bacterial effectors that target phosphoinositide metabolism, GTPase signalling and autophagy.

Organisms undergoing open mitosis disassemble their nuclei during cell division. Upon returning to interphase, the nuclear en-velope (NE) and nuclear pore complexes (NPCs) must reassemble to reestablish cell compartmentalization and nucleocytoplasmic icles, ER-embedded membrane proteins, membrane fusion, and NPC assembly from soluble nucleoporin complexes. This complex process involves numerous players that need to coordinate in space and time, and is so far little understood.To explore NE and NPC assembly mechanisms and the roles ofransmembrane Protein 209), we generated target-specific nanobodies (Nbs). Some Nbs allowed protein visualization via confocal and super-resolution microscopy, while others disrupted essential protein-protein interactions, hindering functional nuclei assembly. Nbs targeting the GTPase and ER integral membrane protein Atlastin impeded import-competent nuclei assembly in vitro, illustrating the Nbs' utility in disrupting membrane protein functions. Conversely, Nbs against the Lamin B Receptor facilitated the tracking of the proteins' recruitment during NE assembly via immunofluorescence microscopy.Orthogonal Nbs, targeting the Transmembrane Protein 209 (TMEM209), allowed us to follow its recruitment during NE assembly, and to pinpoint its localization by super-resolution microscopy. We showed that TMEM209 co-localizes with NPC proteins and established its relative position in the NPC with regard to some of the NUPs, and thus identified TMEM209 as a novel transmembrane NUP.

The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER)-derived replicative organelle named the \"Brucella-containing vacuole\" (BCV). Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D) gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC) and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER) and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

  First identified to be cargo-selective transport carriers during yolk uptake and storage within oocytes of blood-fed mosquitoes [2], clathrin-coated vesicles are now known to support many vital cellular processes, ranging from nutrient uptake, cellular locomotion, and transcriptional regulation and proliferation to complex developmental morphogenetic events. Perturbing PtdIns(4,5)P2 production in cultured cells leads to an almost immediate dissolution of preexisting clathrin-coated structures at the cell surface [8],[10],[11]. Because transmission electron microscope (EM) images typically reveal isolated, invaginating coated buds all along the cell surface, and clathrin immunolabeling often shows a profusion of small, separated puncta apparently randomly scattered over the surface membrane (Figure 2), it seems reasonable to suspect that clathrin-coated vesicles might form de novo for each internalization cycle.

Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium Bacillus subtilis to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron scattering spectra, we confirmed that the B. subtilis cell membrane is lamellar and determined that its average hydrophobic thickness is 24.3 ± 0.9 Ångstroms (Å). Furthermore, by creating neutron contrast within the plane of the membrane using a mixture of H- and D-fatty acids, we detected lateral features smaller than 40 nm that are consistent with the notion of lipid rafts. These experiments-performed under biologically relevant conditions-answer long-standing questions in membrane biology and illustrate a fundamentally new approach for systematic in vivo investigations of cell membrane structure.

Both gram-negative and gram-positive bacteria release extracellular vesicles (EVs) that contain components from their mother cells. Bacterial EVs are similar in size to mammalian-derived EVs and are thought to mediate bacteria–host communications by transporting diverse bioactive molecules including proteins, nucleic acids, lipids, and metabolites. Bacterial EVs have been implicated in bacteria–bacteria and bacteria–host interactions, promoting health or causing various pathologies. Although the science of bacterial EVs is less developed than that of eukaryotic EVs, the number of studies on bacterial EVs is continuously increasing. This review highlights the current state of knowledge in the rapidly evolving field of bacterial EV science, focusing on their discovery, isolation, biogenesis, and more specifically on their role in microbiota–host communications. Knowledge of these mechanisms may be translated into new therapeutics and diagnostics based on bacterial EVs.

Most proteins fold into 3D structures that determine how they function and orchestrate the biological processes of the cell. Recent developments in computational methods for protein structure predictions have reached the accuracy of experimentally determined models. Although this has been independently verified, the implementation of these methods across structural-biology applications remains to be tested. Here, we evaluate the use of AlphaFold2 (AF2) predictions in the study of characteristic structural elements; the impact of missense variants; function and ligand binding site predictions; modeling of interactions; and modeling of experimental structural data. For 11 proteomes, an average of 25% additional residues can be confidently modeled when compared with homology modeling, identifying structural features rarely seen in the Protein Data Bank. AF2-based predictions of protein disorder and complexes surpass dedicated tools, and AF2 models can be used across diverse applications equally well compared with experimentally determined structures, when the confidence metrics are critically considered. In summary, we find that these advances are likely to have a transformative impact in structural biology and broader life-science research. Here, the authors evaluate the performance of AlphaFold2 and its predicted structures on common structural biological applications, including missense variants, function and ligand binding site prediction, modeling of interactions and modeling of experimental structural data.