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The opportunistic bacterium Pseudomonas aeruginosa produces the fucose-specific lectin LecB, which has been identified as virulence factor. LecB has a tetrameric structure with four opposing binding sites and has been shown to act as crosslinker. Here, we demonstrate that LecB strongly binds to the glycosylated moieties of β1-integrins on the basolateral plasma membrane of epithelial cells and caused rapid integrin endocytosis. Whereas internalized integrins were degraded via a lysosomal pathway, washout of LecB restored integrin cell surface localization, thus indicating a specific and direct action of LecB on integrins to bring about their endocytosis. Interestingly, LecB was able to trigger uptake of active and inactive β1-integrins and also of complete α3β1-integrin - laminin complexes. We provide a mechanistic explanation for this unique endocytic process by showing that LecB has the additional ability to recognize fucose-bearing glycosphingolipids and caused the formation of membrane invaginations on giant unilamellar vesicles. In cells, LecB recruited integrins to these invaginations by crosslinking integrins and glycosphingolipids. In epithelial wound healing assays, LecB specifically cleared integrins from the surface of cells located at the wound edge and blocked cell migration and wound healing in a dose-dependent manner. Moreover, the wild type P. aeruginosa strain PAO1 was able to loosen cell-substrate adhesion in order to crawl underneath exposed cells, whereas knockout of LecB significantly reduced crawling events. Based on these results we suggest that LecB has a role in disseminating bacteria along the cell - basement membrane interface.ImportancePseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the leading causes for nosocomial infections. P. aeruginosa is able to switch between planktonic, intracellular, and biofilm-based lifestyles, which allows it to evade the immune system as well as antibiotic treatment. Hence, alternatives to antibiotic treatment are urgently required to combat P. aeruginosa infections. Lectins, like the fucose-specific LecB, are promising targets, because removal of LecB resulted in decreased virulence in mouse models. Currently, several research groups are developing LecB inhibitors. However, the role of LecB in host-pathogen interaction is not well understood. The significance of our research is in identifying cellular mechanisms how LecB facilitates P. aeruginosa infection: We introduce LecB as new member to the list of bacterial molecules that bind integrins and show that P. aeruginosa can efficiently move forward underneath attached epithelial cells by loosening cell - basement membrane attachment in a LecB-dependent manner.

This study provides a novel function of the bacterial lectin LecB, which dampens cell proliferation and survival by targeting the insulin-like growth factor-1 for degradation, without receptor activation., Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria use lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa, which has developed diverse strategies to impair tissue repair processes and promote infection. Here, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, which extend beyond its role as an adhesion molecule. We found that LecB associates with insulin-like growth factor-1 receptor and dampens its signalling, leading to the arrest of cell cycle. In addition, we describe a novel LecB-triggered mechanism to down-regulate host cell receptors by showing that LecB leads to insulin-like growth factor-1 receptor internalisation and subsequent missorting towards intracellular endosomal compartments, without receptor activation. Overall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival.

International audience; Bacterial lectins are typically multivalent and bind noncovalently to specific carbohydrates on host tissues to facilitate bacterial adhesion. Here, we analyzed the effects of two fucose-binding lectins, BambL from Burkholderia ambifaria and LecB from Pseudomonas aeruginosa, on specific signaling pathways in B cells. We found that these bacterial lectins induced B cell activation, which, in vitro, was dependent on the cell surface expression of the B cell antigen receptor (BCR) and its coreceptor CD19, as well as on spleen tyrosine kinase (Syk) activity. The resulting release of intracellular Ca 2+ was followed by an increase in the cell surface abundance of the activation marker CD8, increased cytokine secretion, and subsequent cell death, replicating all of the events that are observed in vitro upon canonical and antigen-mediated B cell activation. Moreover, injection of BambL in mice resulted in a substantial, BCR-independent loss of B cells in the bone marrow with simultaneous, transient enlargement of the spleen (splenomegaly), as well as an increase in the numbers of splenic B cells and myeloid cells. Together, these data suggest that bacterial lectins can initiate polyclonal activation of B cells through their sole capacity to bind to fucose.

SummarySeveral pattern-recognition receptors sense HIV-1 replication products and induce type I interferon (IFN-I) production under specific experimental conditions. However, it is thought that viral sensing and IFN induction are virtually absent in the main target cells of HIV-1 in vivo. Here, we show that activated CD4+ T cells sense HIV-1 infection through the cytosolic DNA sensor cGAS and mount a bioactive IFN-I response. Efficient induction of IFN-I by HIV-1 infection requires proviral integration and is regulated by newly expressed viral accessory proteins: Vpr potentiates, while Vpu suppresses cGAS-dependent IFN-I induction. Furthermore, Vpr also amplifies innate sensing of HIV-1 infection in Vpx-treated dendritic cells. Our results identify cGAS as mediator of an IFN-I response to HIV-1 infection in CD4+ T cells and demonstrate that this response is modulated by the viral accessory proteins Vpr and Vpu. Thus, viral innate immune evasion is incomplete in the main target cells of HIV-1.

The HIV-1, HIV-2 and SIV Nef protein are known to modulate the expression of several cell surface receptors and molecules to escape the immune system, to alter T cell activation, to enhance viral replication, infectivity and transmission and overall to ensure the optimal environment for infection outcome. Consistent and continuous efforts have been made over the years to characterize the modulation of expression of each of these molecules, in the hope that a better understanding of these processes essential for HIV infection and/or pathogenesis will eventually highlight new therapeutic targets. In this article we provide an extensive review of the knowledge gained so far on this important and evolving topic.

The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting.

Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. In contrast, measurement of reverse transcriptase (RT) activity is a generic method which can be adapted for higher sensitivity using real-time PCR quantification (qPCR-based product-enhanced RT (PERT) assay). We present an evaluation of a modified SYBR Green I-based PERT assay (SG-PERT), using commercially available reagents such as MS2 RNA and ready-to-use qPCR mixes. This assay has a dynamic range of 7 logs, a sensitivity of 10 nU HIV-1 RT and outperforms p24 ELISA for HIV titer determination by lower inter-run variation, lower cost and higher linear range. The SG-PERT values correlate with transducing and infectious units in HIV-based viral vector and replication-competent HIV-1 preparations respectively. This assay can furthermore quantify Moloney Murine Leukemia Virus-derived vectors and can be performed on different instruments, such as Roche Lightcycler® 480 and Applied Biosystems ABI 7300. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories.

Infection by Human Immunodeficiency Virus is difficult to treat thanks to its persistent viral reservoir and to its high rate of mutation that allows appearance of resistance to the available treatment. An approach to discover drugable targets is the identification of cellular partner proteins interacting with the virus during its life cycle. We used RNAi technology to identify new HIV partners in the host cytoskeleton since it has been shown that cytoskeleton components and the irregulators have a role during several steps of HIV life cycle. By transducingseveral Tcell lines such as Jurkat CD4 CCR5, Jurkat E6-1 and SupT1 with lentiviral vectors expressings hRNA sequences, we silenced different target genes, members of pathways involved inactinrearrangement. By infection with HIV-NL4.3-eGFP reporter virus we evaluated HIV replication ratesin transducedcells. Surprisingly, the infection rate affected by the specific knock-down was dependent on the cell line used. lndeed, ashRNAtransduced in one cell line affected infection differently to what it did in another. Moreover, we observed that transduction on itself with a control vector expressinga scrambled shRNA sequence affected HIV infection rate in some but not all cell lines. Therefore, to obtain relevant results in screening co-factors for HIV infection, we turned toprimary cells, the naturaltargets of the virus in vivo. We optimized combinedlentiviral transduction and HIV infection in cultured peripheral blood CD4+ lymphocytes. In this setting, transduction with scrambled shRNA expressing lentivirus did not affect HIV replication, providing us a platform to assay gene-knockdown likely to generate the most relevant information for natural HIV infection invivo.

Down-modulation of the CD4 receptor expressed on T cells is one of the hallmarks of Human Immunodeficiency Virus (HIV) infection and it is believed to confer to the virus a selective replicative advantage in vivo. HIV has evolved redundant mechanisms to remove the receptor from the cell surface and accelerate its degradation. This process is mainly mediated by three viral proteins: Vpu, Env and Nef. Up to date, the mechanisms that lead to CD4 depletion from the surface of CD4+ T lymphocytes, the natural targets of HIV, are still poorly understood and only partially characterized. We are interested in the discovery of pathways and human proteins involved in the process, in order to eventually find potential new drug targets. To pursue our aim, we first performed a functional screening on HeLa CD4+ cells expressing Nef, using a shRNA lentiviral interference delivery system targeting the whole human genome compatible with an Affymetrix GeneChip Microarray. The read out was the rescue of the CD4 high phenotype, despite Nef expression. The results were analyzed with Affymetrix Expression Console 1.1 software and the web-based gene ontology resource DAVID. After four different screens we obtained a final list of 75 genes enriched in the cells sorted for high surface CD4. These genes appear to code for proteins involved in endocytic trafficking, trans-Golgi trafficking and lysosomal degradation pathways. We decided to validate our hits in primary blood CD4+ T cells, the most relevant for this kind of studies, and we set up a protocol for the transduction of these cells with lentiviral and retroviral vectors, necessary respectively for the expression of the shRN As and the viral proteins Vpu, Nef and Env. The actual down-regulation of the gene expression will be validated via quantitative Real Time PCR and Western Blot and the experiments will be eventually repeated in the context of HIV-1 infection.