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In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes. Dengue virus (DENV) is an enveloped, positive-strand RNA virus belonging to the family Flaviviridae, which also includes tick-borne encephalitis virus, yellow fever virus, and West Nile virus. Flavivirus virions contain three structural proteins: the C (capsid) protein, the M (membrane) protein, and the E (envelope) protein (12, 26). Multiple copies of the C protein associate with the viral RNA to form the nucleocapsid (26). The nucleocapsid is surrounded by a lipid bilayer in which the M and E glycoproteins are inserted. In the infected cell, the M protein is produced as a precursor protein called prM, which is believed to function as a chaperone during the folding

The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many Proteobacteria to target effectors/toxins into both eukaryotic and prokaryotic cells. We report that Agrobacterium tumefaciens, a soil bacterium that triggers tumorigenesis in plants, produces a family of type VI DNase effectors (Tde) that are distinct from previously known polymorphic toxins and nucleases. Tde exhibits an antibacterial DNase activity that relies on a conserved HxxD motif and can be counteracted by a cognate immunity protein, Tdi. In vitro, A. tumefaciens T6SS could kill Escherichia coli but triggered a lethal counterattack by Pseudomonas aeruginosa upon injection of the Tde toxins. However, in an in planta coinfection assay, A. tumefaciens used Tde effectors to attack both siblings cells and P. aeruginosa to ultimately gain a competitive advantage. Such acquired T6SS-dependent fitness in vivo and conservation of Tde-Tdi couples in bacteria highlights a widespread antibacterial weapon beneficial for niche colonization.

Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules., Natural Killer (NK) cells are innate immune cells that secrete lytic granules to directly kill virus-infected or transformed cells across an immune synapse. However, a major gap in understanding this process is in establishing how lytic granules pass through the mesh of cortical actin known to underlie the NK cell membrane. Research has been hampered by the resolution of conventional light microscopy, which is too low to resolve cortical actin during lytic granule secretion. Here we use two high-resolution imaging techniques to probe the synaptic organisation of NK cell receptors and filamentous (F)-actin. A combination of optical tweezers and live cell confocal microscopy reveals that microclusters of NKG2D assemble into a ring-shaped structure at the centre of intercellular synapses, where Vav1 and Grb2 also accumulate. Within this ring-shaped organisation of NK cell proteins, lytic granules accumulate for secretion. Using 3D-structured illumination microscopy (3D-SIM) to gain super-resolution of ∼100 nm, cortical actin was detected in a central region of the NK cell synapse irrespective of whether activating or inhibitory signals dominate. Strikingly, the periodicity of the cortical actin mesh increased in specific domains at the synapse when the NK cell was activated. Two-colour super-resolution imaging revealed that lytic granules docked precisely in these domains which were also proximal to where the microtubule-organising centre (MTOC) polarised. Together, these data demonstrate that remodelling of the cortical actin mesh occurs at the central region of the cytolytic NK cell immune synapse. This is likely to occur for other types of cell secretion and also emphasises the importance of emerging super-resolution imaging technology for revealing new biology., Author Summary Natural Killer (NK) cells are immune cells that can recognise and kill virus-infected and cancerous cells. This killing requires an intercellular contact —termed an immune synapse—between the NK cell and its target cell through which molecules can be delivered to trigger lysis. Reorganisation of the NK cell cytoskeleton is essential for the delivery and release at the synapse of granules containing the cytolytic molecules. Understanding precisely how the cytoskeleton is involved in these cytolytic events has been hampered by our inability to resolve cytoskeletal structure at immune synapses by conventional light microscopy. Very recent advances in imaging technology have now provided the resolving power to see previously undetectable cellular structures. Here, we have used 3D super-resolution imaging to observe the structure of the actin cytoskeleton at the NK immune synapse. We found that a dense mesh of actin underlies the immune synapse and that it is remodelled upon NK cell activation. Domains within the actin meshwork open up specifying where the lytic granules dock and also where the microtubule-organising centre moves towards. Thus, actin remodelling occurs at the immune synapse during secretion and this may be important for the regulation of lytic granule secretion.

Natural killer (NK) cell activity is conserved throughout vertebrate development, but characterization of non-mammalian NK-cells has been hampered by the absence of specific mAbs for these cells.Monoclonal antibodies were generated against in vitro IL-2 expanded sorted CD3-CD8 alpha+ peripheral blood lymphocytes, previously described to contain chicken NK-cells. Screening of embryonic and adult splenocytes with hybridoma supernatants resulted in five candidate NK markers.Activation of chicken NK-cells with PMA/Ionomycin or with the NK target cell-line LSCC-RP9 resulted in increased expression of CD107 (LAMP-1) and a newly developed flow cytometry based cytotoxicity assay showed that NK-cells were able to kill target cells. Combining NK markers with functional assays indicated that marker positive cells showed NK-cell function.In conclusion, we generated new monoclonal antibodies and developed two functional assays which will enhance our understanding of the role of NK-cells in healthy and diseased chickens. (C) 2010 Elsevier Ltd. All rights reserved.

Studies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4+T cells, and CD8+T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected αβ T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of B1 cells suggested that B1 cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding. We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and leads to the production of high levels of RV-specific intestinal IgA. In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA. Cotransfer of mixtures of purified B1 cells and B1-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (B1-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA. To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4+T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4+T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4+T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4+T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.

It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co-localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor-ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions.