A Label-Free Optical Detection of Pathogens in Isopropanol as a First Step towards Real-Time Infection Prevention.

The detection of pathogens is a major public health issue. Every year, thousands of people die because of nosocomial infections. It is therefore important to be able to detect possible outbreaks as early as possible, especially in the hospital environment. Various pathogen detection techniques have already been demonstrated. However, most of them require expensive and specific equipment, and/or complex protocols, which, most of the time, involve biochemical reaction and labelling steps. In this paper, a new method that combines microscopic imaging and machine learning is described. The main benefits of this approach are to be low-cost, label-free and easy to integrate in any suitable medical device, such as hand hygiene dispensers. The suitability of this pathogen detection method is validated using four bacteria, both in PBS (Phosphate Buffered Saline) and in isopropanol. In particular, we demonstrated an efficient pathogenic detection that is sensible to changes in the composition of a mixture of pathogens, even in alcohol-based solutions.

Pseudomonas aeruginosa lectin LecB impairs keratinocyte fitness by abrogating growth factor signalling.

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.

Carbohydrate-dependent B cell activation by fucose-binding bacterial lectins.

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 co-receptor CD19, as well as on spleen tyrosine kinase (Syk) activity. The resulting release of intracellular Ca2+ was followed by an increase in the cell surface abundance of the activation marker CD86, augmented 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.

Dynamic Cooperative Glycan Assembly Blocks the Binding of Bacterial Lectins to Epithelial Cells.

Pathogens frequently rely on lectins for adhesion and cellular entry into the host. Since these interactions typically result from multimeric binding of lectins to cell-surface glycans, novel therapeutic strategies are being developed with the use of glycomimetics as competitors of such interactions. Herein we study the benefit of nucleic acid based oligomeric assemblies with PNA-fucose conjugates. We demonstrate that the interactions of a lectin with epithelial cells can be inhibited with conjugates that do not form stable assemblies in solution but benefit from cooperativity between ligand-protein interactions and PNA hybridization to achieve high affinity. A dynamic dimeric assembly fully blocked the binding of the fucose-binding lectin BambL of Burkholderia ambifaria, a pathogenic bacterium, to epithelial cells with an efficiency of more than 700-fold compared to l-fucose.

Uptake of Marasmius oreades agglutinin disrupts integrin-dependent cell adhesion.

BACKGROUND: Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a lectin from the fairy ring mushroom with specificity for Galα1-3Gal containing carbohydrates. This lectin is composed of an N-terminal carbohydrate-binding domain and a C-terminal dimerization domain. The dimerization domain of MOA shows in addition calcium-dependent cysteine protease activity, similar to the calpain family. METHODS: Cell detachment assay, cell viability assay, immunofluorescence, live cell imaging and Western blot using MDCKII cell line. RESULTS: In this study, we demonstrate in MDCKII cells that after internalization, MOA protease activity induces profound physiological cellular responses, like cytoskeleton rearrangement, cell detachment and cell death. These changes are preceded by a decrease in FAK phosphorylation and an internalization and degradation of ß1-integrin, consistent with a disruption of integrin-dependent cell adhesion signaling. Once internalized, MOA accumulates in late endosomal compartments. CONCLUSION: Our results suggest a possible toxic mechanism of MOA, which consists of disturbing the cell adhesion and the cell viability. GENERAL SIGNIFICANCE: After being ingested by a predator, MOA might exert a protective role by diminishing host cell integrity.

Plasma membrane reorganization: A glycolipid gateway for microbes.

Ligand-receptor interactions, which represent the core for cell signaling and internalization processes are largely affected by the spatial configuration of host cell receptors. There is a growing piece of evidence that receptors are not homogeneously distributed within the plasma membrane, but are rather pre-clustered in nanodomains, or clusters are formed upon ligand binding. Pathogens have evolved many strategies to evade the host immune system and to ensure their survival by hijacking plasma membrane receptors that are most often associated with lipid rafts. In this review, we discuss the early stage molecular and physiological events that occur following ligand binding to host cell glycolipids. The ability of various biological ligands (e.g. toxins, lectins, viruses or bacteria) that bind to glycolipids to induce their own uptake into mammalian cells by creating negative membrane curvature and membrane invaginations is explored. We highlight recent trends in understanding nanoscale plasma membrane (re-)organization and present the benefits of using synthetic membrane systems. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.

Membrane deformation by neolectins with engineered glycolipid binding sites.

Lectins are glycan-binding proteins that are involved in the recognition of glycoconjugates at the cell surface. When binding to glycolipids, multivalent lectins can affect their distribution and alter membrane shapes. Neolectins have now been designed with controlled number and position of binding sites to decipher the role of multivalency on avidity to a glycosylated surface and on membrane dynamics of glycolipids. A monomeric hexavalent neolectin has been first engineered from a trimeric hexavalent bacterial lectin, From this neolectin template, 13 different neolectins with a valency ranging from 0 to 6 were designed, produced, and analyzed for their ability to bind fucose in solution, to attach to a glycosylated surface and to invaginate glycolipid-containing giant liposomes. Whereas the avidity only depends on the presence of at least two binding sites, the ability to bend and invaginate membranes critically depends on the distance between two adjacent binding sites.

Reduction of lectin valency drastically changes glycolipid dynamics in membranes but not surface avidity.

Multivalency is proposed to play a role in the strong avidity of lectins for glycosylated cell surfaces and also in their ability to affect membrane dynamics by clustering glycosphingolipids. Lectins with modified valency were designed from the ß-propeller fold of Ralstonia solanacearum lectin (RSL) that presents six fucose binding sites. After identification of key amino acids by molecular dynamics calculations, two mutants with reduced valency were produced. Isothermal titration calorimetry confirmed the loss of three high affinity binding sites for both mutants. Crystal structures indicated that residual low affinity binding occurred in W76A but not in R17A. The trivalent R17A mutant presented unchanged avidity toward fucosylated surfaces, when compared to hexavalent RSL. However, R17A is not able anymore to induce formation of membrane invaginations on giant unilamellar vesicules, indicating the crucial role of number of binding sites for clustering of glycolipids. In the human lung epithelial cell line H1299, wt-RSL is internalized within seconds whereas the kinetics of R17A uptake is largely delayed. Neolectins with tailored valency are promising tools to study membrane dynamics.

Fucose-binding lectin from opportunistic pathogen Burkholderia ambifaria binds to both plant and human oligosaccharidic epitopes.

Burkholderia ambifaria is generally associated with the rhizosphere of plants where it has biocontrol effects on other microorganisms. It is also a member of the Burkholderia cepacia complex, a group of closely related bacteria that cause lung infections in immunocompromised patients as well as in patients with granulomatous disease or cystic fibrosis. Our previous work indicated that fucose on human epithelia is a frequent target for lectins and adhesins of lung pathogens (Sulák, O., Cioci, G., Lameignère, E., Balloy, V., Round, A., Gutsche, I., Malinovská, L., Chignard, M., Kosma, P., Aubert, D. F., Marolda, C. L., Valvano, M. A., Wimmerová, M., and Imberty, A. (2011) PLoS Pathog. 7, e1002238). Analysis of the B. ambifaria genome identified BambL as a putative fucose-binding lectin. The 87-amino acid protein was produced recombinantly and demonstrated to bind to fucosylated oligosaccharides with a preference for αFuc1-2Gal epitopes. Crystal structures revealed that it associates as a trimer with two fucose-binding sites per monomer. The overall fold is a six-bladed ß-propeller formed by oligomerization as in the Ralstonia solanacearum lectin and not by sequential domains like the fungal fucose lectin from Aleuria aurantia. The affinity of BambL for small fucosylated glycans is very high as demonstrated by microcalorimetry (K(D) < 1 µM). Plant cell wall oligosaccharides and human histo-blood group oligosaccharides H-type 2 and Lewis Y are bound with equivalent efficiency. Binding to artificial glycosphingolipid-containing vesicles, human saliva, and lung tissues confirmed that BambL could recognize a wide spectrum of fucosylated epitopes, albeit with a lower affinity for biological material from nonsecretor individuals.

Palmitoylation of interferon-alpha (IFN-alpha) receptor subunit IFNAR1 is required for the activation of Stat1 and Stat2 by IFN-alpha.

Type I interferons (IFNs) bind IFNAR receptors and activate Jak kinases and Stat transcription factors to stimulate the transcription of genes downstream from IFN-stimulated response elements. In this study, we analyze the role of protein palmitoylation, a reversible post-translational lipid modification, in the functional properties of IFNAR. We report that pharmacological inhibition of protein palmitoylation results in severe defects of IFN receptor endocytosis and signaling. We generated mutants of the IFNAR1 subunit of the type I IFN receptor, in which each or both of the two cysteines present in the cytoplasmic domain are replaced by alanines. We show that cysteine 463 of IFNAR1, the more proximal of the two cytoplasmic cysteines, is palmitoylated. A thorough microscopic and biochemical analysis of the palmitoylation-deficient IFNAR1 mutant revealed that IFNAR1 palmitoylation is not required for receptor endocytosis, intracellular distribution, or stability at the cell surface. However, the lack of IFNAR1 palmitoylation affects selectively the activation of Stat2, which results in a lack of efficient Stat1 activation and nuclear translocation and IFN-alpha-activated gene transcription. Thus, receptor palmitoylation is a previously undescribed mechanism of regulating signaling activity by type I IFNs in the Jak/Stat pathway.

Interfering with interferon receptor sorting and trafficking: impact on signaling.

Interferons (IFNs) and their receptors (IFN-Rs) play fundamental roles in a multitude of biological functions. Many articles and reviews emphasize that the JAK/STAT machinery is obligatory for relay of the information transmitted by IFNs after binding to their cognate receptors at the plasma membrane. In contrast, very few studies have addressed the endocytosis and the intracellular trafficking of IFN-Rs, the immediate step following IFN binding. However, recent findings have shed light on the importance of IFN-R sorting and trafficking in the control of IFN signaling. Thus, IFN-Rs can be included in the growing family of signaling receptors for which regulation of biological activity critically involves endocytosis and trafficking.

New natural intergenotypic (2/5) recombinant of hepatitis C virus.

A 9.2-kb sequence from a hepatitis C virus (HCV) strain found in southwest France was compared to sequences from reference strains in HCV sequence databases. We found a recombinant virus with genotype 2 at the 5' end and genotype 5 at the 3' end. The crossover point was located between genes NS2 and NS3. Recombination between HCV genotypes must now be considered in studies on HCV epidemiology and evolution and in predictions of the virus response to antiviral therapy. Knowing the location of the recombination point may also be useful for constructing infectious chimeric viruses.