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Exposure of the vertebrate embryo to maternal hormones can have long-lasting effects on its phenotype, which has been studied extensively by experimentally manipulating maternal steroids, mostly androgens, in bird eggs. Yet, there is a severe lack of understanding of how and when these effects are actually mediated, hampering both underlying proximate and ultimate explanations. Here we report a novel finding that the embryo expresses androgen receptor (AR) and estrogen receptor (ERα) mRNA in its extraembryonic membranes (EMs) as early as before its own hormone production starts, suggesting a novel substrate for action of maternal hormones on the offspring. We also report the first experimental evidence for steroid receptor regulation in the avian embryo in response to yolk steroid levels: the level of AR is dependent on yolk androgen levels only in the EMs but not in body tissues, suggesting embryonic adaptation to maternal hormones. The results also solve the problem of uptake of lipophilic steroids from the yolk, why they affect multiple traits, and how they could mediate maternal effects without affecting embryonic sexual differentiation.

Dengue virus infects immune cells, including monocytes, macrophages and dendritic cells (DC). We compared virus infectivity in macrophages and DC, and found that the virus origin determined the cell tropism of progeny virus. The highest efficiency of re-infection was seen for macrophage-derived dengue virus. Furthermore, in the presence of enhancing antibodies, macrophage-derived virus gave greater enhancement of infection compared with immature DC derived virus. Taken together, our results highlight the importance of macrophages in dengue infection.

Ducks and chickens are hosts of avian influenza virus, each with distinctive responses to infection. To understand these differences, we characterized the innate immune response to low pathogenicity avian influenza virus H7N1 infection in chickens and ducks. Viral RNA was detected in the lungs of chickens from day 0.8 to 7, in ducks mainly at day 4. In both species viral RNA was detected in the bursa and gut. Infection in chickens resulted in up-regulation of interferon (IFN)-a and IFN-ß mRNA, while in the ducks IFN-¿ mRNA was strongly up-regulated in lung and bursa. In chickens and ducks all investigated pathogen recognition receptors (PRR) mRNA were up regulated, however, in the chicken lung Toll-like receptor (TLR)7 and melanoma differentiation-associated protein (MDA)-5 mRNA were strongly induced. TLR3, TLR7 and MDA-5 responses correlated with IFN-a and IFN-ß responses in chickens, but in ducks a correlation between IFN-a and TLR7, retinoic acid-inducible gene-I and MDA-5 was absent. We studied the responses of duck and chicken splenocytes to poly (I:C) and R848 analogues to analyze the regulation of PRRs without the interfering mechanisms of the influenza virus. This revealed IFN-a and IFN- ¿ responses in both species. MDA-5 was only strongly up-regulated in chicken splenocytes. In chickens splenocytes, time related PRR responses correlated with the IFN-a and IFN-ß response. In duck splenocytes this correlation was absent. In conclusion, chicken and duck differ in induction of MDA-5, TLR7 and IFN-a mRNA after an influenza virus infection in vivo and after in vitro stimulation with TLR antagonists.

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

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system. Technical and human support provided by SGIker (Analytical and High-Resolution Microscopy in Biomedicine Service of UPV/EHU) and Rocio Alonso for excellent technical assistance are gratefully acknowledged. We also thank Dr. N. Guiso from the Pasteur Institute (Paris, France) for the Bordetella pertussis strains BP18323 and non-virulent bgv negative BP18323H-. This study was supported by grants from the Spanish Ministerio de Ciencia y Tecnologia (Project BFU 2012-36241), and the Basque Government (Grupos Consolidados IT849-13 and ETORTEK Program). K.B.U and D.G.B. were recipients of a fellowship from the Bizkaia Biophysics Foundation. A.E.G. was a recipient of a research contract (INNPACTO 2010) and A.E.L. was a recipient of a fellowship from the University of the Basque Country (UPV/EH). A patent with reference N/Ref.: P1683USPC (2004) was approved.

The Type VI secretion system (T6SS) mediates toxin delivery into both eukaryotic and prokaryotic cells. It is composed of a cytoplasmic structure resembling the tail of contractile bacteriophages anchored to the cell envelope through a membrane complex composed of the TssL and TssM inner membrane proteins and of the TssJ outer membrane lipoprotein. The C-terminal domain of TssM is required for its interaction with TssJ, and for the function of the T6SS. In Citrobacter rodentium, the tssM1 gene does not encode the C-terminal domain. However, the stop codon is preceded by a run of 11 consecutive adenosines. In this study, we demonstrate that this poly-A tract is a transcriptional slippery site that induces the incorporation of additional adenosines, leading to frameshifting, and hence the production of two TssM1 variants, including a full-length canonical protein. We show that both forms of TssM1, and the ratio between these two forms, are required for the function of the T6SS in C. rodentium. Finally, we demonstrate that the tssM gene associated with the Yersinia pseudotuberculosis T6SS-3 gene cluster is also subjected to transcriptional frameshifting., Author Summary Nonstandard decoding mechanisms lead to the synthesis of different protein variants from a single DNA sequence. These mechanisms are particularly important when the genome length has to be limited such as viral genomes, limited by the available space in the capsid, or to synthesize two different polypeptides that have distinct functional properties. Here, we report that tssM, a gene encoded within the Citrobacter rodentium Type VI secretion (T6S) gene cluster, is interrupted by a premature stop codon; however, the stop codon is preceded by a slippery site constituted by 11 consecutive adenosines. Reiterative transcription leads to the incorporation of additional nucleotides in the mRNA and therefore restores the original framing. As a consequence, two different TssM variants are created by transcriptional frameshifting, including a full-length 130-kDa protein and an 88-kDa truncated variant. We further show that both forms, and the ratio between these two forms, are required for the function of the transport apparatus. Interestingly, a similar mechanism regulates the synthesis of two TssM variants in Yersinia pseudotuberculosis.

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.Cell Death and Differentiation advance online publication, 22 August 2014; doi:10.1038/cdd.2014.110.

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.

The bacterial type VI secretion system (T6SS) is a supra-molecular complex akin to bacteriophage tails, with VgrG proteins acting as a puncturing device. The Pseudomonas aeruginosa H1-T6SS has been extensively characterized. It is involved in bacterial killing and in the delivery of three toxins, Tse1–3. Here, we demonstrate the independent contribution of the three H1-T6SS co-regulated vgrG genes, vgrG1abc, to bacterial killing. A putative toxin is encoded in the vicinity of each vgrG gene, supporting the concept of specific VgrG/toxin couples. In this respect, VgrG1c is involved in the delivery of an Rhs protein, RhsP1. The RhsP1 C terminus carries a toxic activity, from which the producing bacterium is protected by a cognate immunity. Similarly, VgrG1a-dependent toxicity is associated with the PA0093 gene encoding a two-domain protein with a putative toxin domain (Toxin_61) at the C terminus. Finally, VgrG1b-dependent killing is detectable upon complementation of a triple vgrG1abc mutant. The VgrG1b-dependent killing is mediated by PA0099, which presents the characteristics of the superfamily nuclease 2 toxin members. Overall, these data develop the concept that VgrGs are indispensable components for the specific delivery of effectors. Several additional vgrG genes are encoded on the P. aeruginosa genome and are not linked genetically to other T6SS genes. A closer inspection of these clusters reveals that they also encode putative toxins. Overall, these associations further support the notion of an original form of secretion system, in which VgrG acts as the carrier.

The type VI secretion system (T6SS) of Gram-negative bacteria has been involved in various processes, notably bacterial competition and eukaryotic cell subversion. Most Pseudomonas aeruginosa strains possess three T6SS gene clusters, but only the function of the first T6SS (H1-T6SS) has been clearly elucidated. It is involved in the secretion of three toxins (Tse1 to -3) that target bacterial competitors. In the case of the H2- and H3-T6SS, no clear function has been assigned, and only one effector has been associated with these systems. Yet the H2-T6SS was proposed to promote P. aeruginosa internalization in nonphagocytic epithelial cells. Although the H2-T6SS genetic organization is conserved across P. aeruginosa isolates, one feature is the presence of an additional transcriptional unit in the PA14 strain H2-T6SS cluster, which is divergent from the core H2-T6SS genes. A specific set of four genes encodes an Hcp protein (Hcp2), a VgrG protein (VgrG14), an Rhs element (PA14_43100 or RhsP2), and a protein with no homologies with previously characterized proteins (PA14_43090). In this study, we engineered a P. aeruginosa PA14 strain carrying an arabinose-inducible H2-T6SS on the chromosome. We showed that arabinose induction readily promotes assembly of the H2-T6SS, as seen by monitoring Hcp2 secretion. We further studied the secretion fate of VgrG14 and RhsP2, but these were not detectable in the extracellular medium. We finally investigated whether activation of the PA14 H2-T6SS gene cluster could influence phenotypic traits such as internalization in eukaryotic cells, and we reported noteworthy differences compared to strain PAO1, which may be accounted for by the described genetic differences.

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 (YP T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of BI cells suggested that BL 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 Iii-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (BI-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.

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.