Microarray Strategies for Exploring Bacterial Surface Glycans and Their Interactions With Glycan-Binding Proteins

Spanish Ministry of Economy and Competitiveness (Grant BFU2015-70052-R). Spanish Ministry of Science, Innovation, and Universities, the Spanish State Research Agency, the European Regional Development Fund (RTI2018-099985-B-I00, MCIU/AEI/FEDER, UE), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII). PTDC/BIA-MIB/31730/2017. IF/00023/2012. Applied Molecular Biosciences Unit (UCIBIO) (Financed by FCT-MCTES, UID/Multi/04378/2013/2019).Bacterial surfaces are decorated with distinct carbohydrate structures that may substantially differ among species and strains. These structures can be recognized by a variety of glycan-binding proteins, playing an important role in the bacteria cross-talk with the host and invading bacteriophages, and also in the formation of bacterial microcolonies and biofilms. In recent years, different microarray approaches for exploring bacterial surface glycans and their recognition by proteins have been developed. A main advantage of the microarray format is the inherent miniaturization of the method, which allows sensitive and high-throughput analyses with very small amounts of sample. Antibody and lectin microarrays have been used for examining bacterial glycosignatures, enabling bacteria identification and differentiation among strains. In addition, microarrays incorporating bacterial carbohydrate structures have served to evaluate their recognition by diverse host/phage/bacterial glycan-binding proteins, such as lectins, effectors of the immune system, or bacterial and phagic cell wall lysins, and to identify antigenic determinants for vaccine development. The list of samples printed in the arrays includes polysaccharides, lipopoly/lipooligosaccharides, (lipo)teichoic acids, and peptidoglycans, as well as sequence-defined oligosaccharide fragments. Moreover, microarrays of cell wall fragments and entire bacterial cells have been developed, which also allow to study bacterial glycosylation patterns. In this review, examples of the different microarray platforms and applications are presented with a view to give the current state-of-the-art and future prospects in this field.publishersversionpublishe

The C-type lectin receptor CLECSF8 (CLEC4D) is expressed by myeloid cells and triggers cellular activation through syk kinase

11 pags, 7 figsCLECSF8 is a poorly characterized member of the "Dectin-2 cluster" of C-type lectin receptors and was originally thought to be expressed exclusively by macrophages. We show here that CLECSF8 is primarily expressed by peripheral blood neutrophils and monocytes and weakly by several subsets of peripheral blood dendritic cells. However, expression of this receptor is lost upon in vitro differentiation of monocytes into dendritic cells or macrophages. Like the other members of the Dectin-2 family, which require association of their transmembrane domains with signaling adaptors for surface expression, CLECSF8 is retained intracellularly when expressed in non-myeloid cells. However, we demonstrate that CLECSF8 does not associate with any known signaling adaptor molecule, including DAP10, DAP12, or the FcRγ chain, and we found that the C-type lectin domain of CLECSF8 was responsible for its intracellular retention. Although CLECSF8 does not contain a signaling motif in its cytoplasmic domain, we show that this receptor is capable of inducing signaling via Syk kinase in myeloid cells and that it can induce phagocytosis, proinflammatory cytokine production, and the respiratory burst. These data therefore indicate that CLECSF8 functions as an activation receptor on myeloid cells and associates with a novel adaptor molecule. Characterization of the CLECSF8-deficient mice and screening for ligands using oligosaccharide microarrays did not provide further insights into the physiological function of this receptor. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.This work was funded by the Wellcome Trust, the National Research Foundation, the Deutscher Akademischer Austauschdienst, the University of Cape Town, the UK Research Council Basic Technology Initiative “Glycoar-rays” (GRS/79268), and the UK Medical Research Council. A. S. P is a fellowof the Fundação para a Ciência e Tecnologia (SFRH/BPD/26515/2006, Portugal) and M. A. C. of the Consejo Superior de Investigaciones Cientificas, Programe “Junta para la Ampliación de Estudios” (JaeDoc/098/2011) cofinanced by the Fondo Social Europeo

Atomic resolution insight into host cell recognition by Toxoplasma gondii.

Accepted versio

Lipopolysaccharide O-antigen molecular and supramolecular modifications of plant root microbiota are pivotal for host recognition

11 pags., 5 figs.Lipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis or bacterial virulence, depending on the bacterial lifestyle. Plant microbiota shows great complexity, promotes plant health and growth and assures protection from pathogens. How plants perceive LPS from plant-associated bacteria and discriminate between beneficial and pathogenic microbes is an open and urgent question. Here, we report on the structure, conformation, membrane properties and immune recognition of LPS isolated from the Arabidopsis thaliana root microbiota member Herbaspirillum sp. Root189. The LPS consists of an O-methylated and variously acetylated D-rhamnose containing polysaccharide with a rather hydrophobic surface. Plant immunology studies in A. thaliana demonstrate that the native acetylated O-antigen shields the LPS from immune recognition whereas the O-deacylated one does not. These findings highlight the role of Herbaspirillum LPS within plant-microbial crosstalk, and how O-antigen modifications influence membrane properties and modulate LPS host recognition.This study was supported by PRIN 2017 "Glytunes" (2017XZ2ZBK, 2019-2022) to AS; by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme under grant agreement No 851356 to RM. Neutron Reflectivity (NR) measurements were performed at the INTER instrument at ISIS Pulsed Neutron and Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Didcot, UK. The authors thank the ISIS facility for provision of beam time. MACR and DS gratefully acknowl- edge financial support from the Spanish Ministry of Science, Innovation, and Universities (RTI2018-099985-B-I00), and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII). AZ and LM acknowledge support from the Cluster of Excellence on Plant Sciences (CEPLAS) funded by the Deutsche For- schungsgemeinschaft (DFG, German Research Foundation) under Ger- many’s Excellence Strategy-EXC 2048/1-Project ID: 390686111 and project ZU 263/11-1 (SPP DECRyPT)Peer reviewe

The Role of Sialyl Glycan Recognition in Host Tissue Tropism of the Avian Parasite Eimeria tenella

Eimeria spp. are a highly successful group of intracellular protozoan parasites that develop within intestinal epithelial cells of poultry, causing coccidiosis. As a result of resistance against anticoccidial drugs and the expense of manufacturing live vaccines, it is necessary to understand the relationship between Eimeria and its host more deeply, with a view to developing recombinant vaccines. Eimeria possesses a family of microneme lectins (MICs) that contain microneme adhesive repeat regions (MARR). We show that the major MARR protein from Eimeria tenella, EtMIC3, is deployed at the parasite-host interface during the early stages of invasion. EtMIC3 consists of seven tandem MAR1-type domains, which possess a high specificity for sialylated glycans as shown by cell-based assays and carbohydrate microarray analyses. The restricted tissue staining pattern observed for EtMIC3 in the chicken caecal epithelium indicates that EtMIC3 contributes to guiding the parasite to the site of invasion in the chicken gut. The microarray analyses also reveal a lack of recognition of glycan sequences terminating in the N-glycolyl form of sialic acid by EtMIC3. Thus the parasite is well adapted to the avian host which lacks N-glycolyl neuraminic acid. We provide new structural insight into the MAR1 family of domains and reveal the atomic resolution basis for the sialic acid-based carbohydrate recognition. Finally, a preliminary chicken immunization trial provides evidence that recombinant EtMIC3 protein and EtMIC3 DNA are effective vaccine candidates

Sialyllactose in Viral Membrane Gangliosides Is a Novel Molecular Recognition Pattern for Mature Dendritic Cell Capture of HIV-1

An accessible sialyllactose moiety on viral membrane gangliosides is shown to be essential for HIV-1 uptake into mature dendritic cells, thereby promoting viral transfer and infection of bystander CD4+ T lymphocytes

Virus Movements on the Plasma Membrane Support Infection and Transmission between Cells

How viruses are transmitted across the mucosal epithelia of the respiratory, digestive, or excretory tracts, and how they spread from cell to cell and cause systemic infections, is incompletely understood. Recent advances from single virus tracking experiments have revealed conserved patterns of virus movements on the plasma membrane, including diffusive motions, drifting motions depending on retrograde flow of actin filaments or actin tail formation by polymerization, and confinement to submicrometer areas. Here, we discuss how viruses take advantage of cellular mechanisms that normally drive the movements of proteins and lipids on the cell surface. A concept emerges where short periods of fast diffusive motions allow viruses to rapidly move over several micrometers. Coupling to actin flow supports directional transport of virus particles during entry and cell-cell transmission, and local confinement coincides with either nonproductive stalling or infectious endocytic uptake. These conserved features of virus–host interactions upstream of infectious entry offer new perspectives for anti-viral interference

Bacterial microarrays for examining bacterial glycosignatures and recognition by host lectins

14 pags., 2 figs., 1 tab.The surface of bacteria displays diverse carbohydrate structures that may significantly differ among bacteria with the same cell wall architecture and even among strains of a given bacterial species. These structures are often recognized by lectins of the innate immune system for triggering defense responses, although some bacterial pathogens exploit recognition by host lectins for favoring infection. Bacterial microarrays are a useful tool for profiling accessible bacterial surface glycans and for exploring their recognition by innate immune lectins. The use of array-printed bacterial cells enables evaluation of the recognition of the glycan epitopes in their natural presentation, i.e., preserving their real density and accessibility. Glycosylation patterns of bacterial surfaces can be examined by testing the binding to the bacterial arrays of a panel of lectins with known carbohydrate-binding preferences, and the recognition of surface glycans by innate immune lectins can easily be assessed using similar binding assays.We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (Grant BFU2015- 70052-R), the Ministry of Science, Innovation, and Universities (RTI2018-099985-B-I00), and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIIIPeer reviewe