Junín virus induces autophagy in human A549 cells.

Autophagy, a highly regulated degradative process that promotes cellular homeostasis, is increasingly recognised as a fundamental component of the cellular response against viral infection. In this study, we investigated the role of autophagy during Junín virus (JUNV) multiplication using human A549 cells. We found that JUNV infection induces an increment of the LC3-II/LC3-I ratio, an accumulation of punctate pattern in RFP-LC3-transfected cells and the colocalisation of viral nucleoprotein and LC3 protein, suggesting autophagosome formation. JUNV infection also induced the degradation of the autophagy receptor p62, suggesting that complete autophagic flux was triggered. In addition, we showed that inhibition of autophagy with bafilomycin A1 or 3-methyladenine significantly reduces viral multiplication. Moreover, viral yield was increased when autophagy was induced using rapamycin. Furthermore, JUNV infection induced the colocalisation of p62, ATG16, RAB5, RAB7A and LAMP1 with the autophagosomal LC3 protein. That suggests that phagosomes undergo the maturation process during viral infection. Finally, we demonstrated that siRNA experiments targeting essential autophagy genes (ATG5, ATG7 and Beclin 1) reduce viral protein synthesis and viral yield. Overall, our results indicate that JUNV activates host autophagy machinery enhancing its multiplication.

A monoclonal antibody against the extracellular domain of mouse and human epithelial V-like antigen 1 reveals a restricted expression pattern among CD4- CD8- thymocytes.

Expression of transcripts for the homotypic adhesion protein epithelial V-like antigen 1 (EVA1), also known as myelin protein zero like-2 (Mpzl2), is known to be present in thymic stromal cells. However, protein expression within different thymic subsets, stromal and/or lymphoid, has not been characterized due a lack of specific reagents. To address this, we generated a hybridoma (G9P3-1) secreting a monoclonal antibody (G9P3-1Mab), reactive against both human and mouse EVA1. The G9P3-1Mab was generated by immunizing Mpzl2-deficient gene-targeted mice with the extracellular domain of EVA1, followed by a conventional hybridoma fusion protocol, illustrating the feasibility of using gene-targeted mice to generate monoclonal antibodies with multiple species cross-reactivity. We confirmed expression of EVA1 on cortical and medullary epithelial cell subsets and revealed a restricted pattern of expression on CD4- CD8- double negative (DN) cell subsets, with the highest level of expression on DN3 (CD44(low)CD25(+)) thymocytes. G9P3-1MAb is a valuable reagent to study thymic T cell development and is likely useful for the analysis of pathological conditions affecting thymopoiesis, such as thymic involution caused by stress or aging.

Multicomponent Moraxella catarrhalis outer membrane vesicles induce an inflammatory response and are internalized by human epithelial cells.

Moraxella catarrhalis is an emerging human respiratory pathogen in patients with chronic obstructive pulmonary disease (COPD) and in children with acute otitis media. The specific secretion machinery known as outer membrane vesicles (OMVs) is a mechanism by which Gram-negative pathogens interact with host cells during infection. We identified 57 proteins in M. catarrhalis OMVs using a proteomics approach combining two-dimensional SDS-PAGE and MALDI-TOF mass spectrometry analysis. The OMVs contained known surface proteins such as ubiquitous surface proteins (Usp) A1/A2, and Moraxella IgD-binding protein (MID). Most of the proteins are adhesins/virulence factors triggering the immune response, but also aid bacteria to evade the host defence. FITC-stained OMVs bound to lipid raft domains in alveolar epithelial cells and were internalized after interaction with Toll-like receptor 2 (TLR2), suggesting a delivery to the host tissue of a large and complex group of OMV-attributed proteins. Interestingly, OMVs modulated the pro-inflammatory response in epithelial cells, and UspA1-bearing OMVs were found to specifically downregulate the reaction. When mice were exposed to OMVs, a pulmonary inflammation was clearly seen. Our findings indicate that Moraxella OMVs are highly biologically active, transport main bacterial virulence factors and may modulate the epithelial pro-inflammatory response.

Superantigen- and TLR-dependent activation of tonsillar B cells after receptor-mediated endocytosis.

Classical B lymphocyte activation is dependent on BCR cross-linking in combination with physical interaction with Th cells. Other B cell molecules that contribute to the activation are complement, cytokine, and TLRs recognizing specific pathogen-associated molecular patterns. Moraxella (Branhamella) catarrhalis is a common Gram-negative respiratory pathogen that induces proliferation in human IgD-expressing B cells independently of T cell help. The activation is initiated by the B cell superantigen Moraxella IgD-binding protein (MID) through a nonimmune cross-linking of IgD. However, IgD cross-linking alone is not sufficient to induce proliferation. In this study, we characterized the significance of TLRs in superantigen-dependent B cell activation using whole bacteria or rMID in the presence or absence of TLR ligands. IgD cross-linking by MID sensitized B cells obtained from children with tonsillar hyperplasia for mainly TLR9, whereas TLRs 1, 2, 6, and 7 were less important. The Moraxella-induced activation was inhibited when a dominant-negative TLR9 ligand was added. Interestingly, BCR-mediated endocytosis of whole Moraxella and degradation of live bacteria in naive B cells were observed with fluorescence, confocal, and transmission electron microscopy. This unique observation proved the strong intracellular TLR9 response as well as highlighted the Ag-presenting function of B cells. In conclusion, our findings suggest an important role of TLRs in the adaptive immune response and reveal novel insights into the T cell-independent B cell activation induced by bacteria.

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils.

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-beta-cyclodextrin (MbetaCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.

Diseño de Vacuna Pertussis. Modulación fenotípica de Bordetella pertussis in vivo. Rol en la inmunoevasión y sobrevida dentro del hospedador

Debido a la divergencia encontrada entre las cepas vacunales y los aislamientos locales de B. pertussis en este estudio se incluyó un aislamiento local con fines comparativos. La cepa vacunal Tohama en fase virulenta (BpT), y en fase avirulenta (Bvg359) y un aislamiento nacional de B. pertussis en fase virulenta (Bp955) y en fase avirulenta obtenido por modulación fenotípica (Bp955 mod.) se cultivaron en condiciones de limitación y exceso de hierro, según se describe en Materiales y Métodos. La limitación de hierro determinó un retardo en la cinética de crecimiento de las bacterias y se confirmó por evaluación de producción de sideróforos según la técnica de Schwyn y Neilands (21). Las proteínas de membrana externa se obtuvieron a partir de fase exponencial tardía cultivadas en cada condición

Diseño de Vacuna Pertussis. Modulación fenotípica de Bordetella pertussis in vivo. Rol en la inmunoevasión y sobrevida dentro del hospedador

Debido a la divergencia encontrada entre las cepas vacunales y los aislamientos locales de B. pertussis en este estudio se incluyó un aislamiento local con fines comparativos. La cepa vacunal Tohama en fase virulenta (BpT), y en fase avirulenta (Bvg359) y un aislamiento nacional de B. pertussis en fase virulenta (Bp955) y en fase avirulenta obtenido por modulación fenotípica (Bp955 mod.) se cultivaron en condiciones de limitación y exceso de hierro, según se describe en Materiales y Métodos. La limitación de hierro determinó un retardo en la cinética de crecimiento de las bacterias y se confirmó por evaluación de producción de sideróforos según la técnica de Schwyn y Neilands (21). Las proteínas de membrana externa se obtuvieron a partir de fase exponencial tardía cultivadas en cada condición

Diseño de Vacuna Pertussis. Modulación fenotípica de Bordetella pertussis in vivo. Rol en la inmunoevasión y sobrevida dentro del hospedador

Debido a la divergencia encontrada entre las cepas vacunales y los aislamientos locales de B. pertussis en este estudio se incluyó un aislamiento local con fines comparativos. La cepa vacunal Tohama en fase virulenta (BpT), y en fase avirulenta (Bvg359) y un aislamiento nacional de B. pertussis en fase virulenta (Bp955) y en fase avirulenta obtenido por modulación fenotípica (Bp955 mod.) se cultivaron en condiciones de limitación y exceso de hierro, según se describe en Materiales y Métodos. La limitación de hierro determinó un retardo en la cinética de crecimiento de las bacterias y se confirmó por evaluación de producción de sideróforos según la técnica de Schwyn y Neilands (21). Las proteínas de membrana externa se obtuvieron a partir de fase exponencial tardía cultivadas en cada condición