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

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

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

Development and Evaluation of a Microarray Platform for Detection of Serum Antibodies against Streptococcus pneumoniae Capsular Polysaccharides

7 pags., 1 fig., 2 tabs.Streptococcus pneumoniae is responsible for severe infections, causing millions of deaths yearly. Immunoglobulin G (IgG) antibodies against the capsular polysaccharide (CPS) offer S. pneumoniae serotype-specific protection. In this work, we examined the applicability of the microarray technology to detect CPS type-specific IgGs in serum, using a collection of 22 microarray-printed S. pneumoniae CPSs. First, printing of five CPSs onto nitrocellulose-coated glass slides was tested. Successful printing was only achieved for certain CPS types and concentrations. This behavior was tentatively related with diverse viscosities of the CPS solutions. Measurement of dynamic viscosities fully supported this assumption and helped to establish suitable CPS type-and concentration-dependent printing conditions. Next, the potential of CPS microarrays for detecting recognition by anti-CPS IgGs was examined using well-defined rabbit pneumococcal antisera. In all cases, the expected antiserum-CPS binding signals were detected, prompting a proof-of-concept analysis of human serum samples. Clearly distinct serum-and CPS-specific binding patterns and intensities were observed, evidencing selective detection of CPS type-specific IgGs. Compared to the ELISA assay commonly used to quantitate CPS type-specific IgGs in serum, the newly developed S. pneumoniae CPS microarrays offer the advantage of enabling the simultaneous analysis of multiple CPS-serum interactions using minute CPS amounts and significantly reduced serum volumes. Therefore, the approach could be particularly valuable for gauging the presence of CPS type-specific IgGs in human serum when sample volumes are limited and/or numerous serum samples are being examined.We gratefully acknowledge financial support from the Spanish Ministry of Economy, Industry, and Competitiveness (Grants BFU2015-70052-R and SAF2017-88664-R), the Spanish Ministry of Science, Innovation, and Universities, the Spanish State Research Agency, and the European Regional Development Fund (Grant RTI2018-099985-B-I00, MCIU/AEI/ FEDER, UE), and the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII)

The role of collectins and galectins in lung innate immune defense

10 pags, 2 figsDifferent families of endogenous lectins use complementary defense strategies against pathogens. They may recognize non-self glycans typically found on pathogens and/or host glycans. The collectin and galectin families are prominent examples of these two lectin categories. Collectins are C-type lectins that contain a carbohydrate recognition domain and a collagen-like domain. Members of this group include surfactant protein A (SP-A) and D (SP-D), secreted by the alveolar epithelium to the alveolar fluid. Lung collectins bind to several microorganisms, which results in pathogen aggregation and/or killing, and enhances phagocytosis of pathogens by alveolar macrophages. Moreover, SP-A and SP-D influence macrophage responses, contributing to resolution of inflammation, and SP-A is essential for tissue-repair functions of macrophages. Galectins also function by interacting directly with pathogens or by modulating the immune system in response to the infection. Direct binding may result in enhanced or impaired infection of target cells, or can have microbicidal effects. Immunomodulatory effects of galectins include recruitment of immune cells to the site of infection, promotion of neutrophil function, and stimulation of the bactericidal activity of infected macrophages. Moreover, intracellular galectins can serve as danger receptors, promoting autophagy of the invading pathogen. This review will focus on the role of collectins and galectins in pathogen clearance and immune response activation in infectious diseases of the respiratory system.This study was supported by the Spanish Ministerio de Economía y Competitividad through Grants SAF2015-65307-R (to CC) and BFU2015-70052-R (to DS) and Instituto de Salud Carlos III (CIBERES-CB06/06/0002 to CC and CB06/06/1102 to DS)

Development of a new microarray set-up for high through-put screening of exosome glycosylation

11as Jornadas formación CIBERES, Jornadas conjuntas con CIBERONC. Aula Magna Gustavo Pitaluga. Escuela Nacional de Sanidad Instituto de Salud Carlos, del 15-16 de Noviembre de 2018 (Madrid, España).- http:///C:/Users/biblioteca3/Downloads/programa-cienti-fico-jornadas-formacio-n-ciberes-ciberonc-2018(7).pdfExtracellular vesicles are membrane-enclosed vesicles released from cells, whose composition may change under different physiological and pathological conditions. They are involved in intercellular communication and regulation of cellular functions. An elevated concentration of extracellular vesicles in blood and altered composition can be a sign of a pathological state. Host cell-derived vesicles include apoptotic bodies, microvesicles, and exosomes, which vary in size, composition, and biosynthesis. Exosomes, the smallest vesicles (30-100 nm), contain host-derived proteins, carbohydrates, lipids, and nucleic acids. Numerous studies have examined the lipid, protein and RNA/DNA content of exosomes. However, very scarce information on their carbohydrate composition is available. Exosomes are expected to share glycosylation patterns with their parental cell, which could change in response to different pathologies. In particular, evidence for a correlation between surface glycosylation and properties of tumour cells, as e.g. tumour-immune escape, is emerging. Recently, a cell surface proteoglycan, glypican-1 (GPC1) has been identified as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer, as it is specifically enriched on cancer cell-derived exosomes. Thus, isolation and characterization of exosomes in body fluids could enable the identification of specific markers that distinguish cancer exosomes from normal exosomes, aiding in the diagnosis and management of cancer

Combined Bacteria Microarray and Quartz Crystal Microbalance Approach for Exploring Glycosignatures of Nontypeable Haemophilus influenzae and Recognition by Host Lectins

Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria-host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface.We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (Grants BFU2012-36825, BFU2015-70052-R, SAF2012-31166, and SAF2015-66520-R), the Department of Health of the Navarra Government (ref 359/2012), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Marie Curie Initial Training Networks DYNANO (Grant PITN-GA-2011-289033), GLYCOPHARM (Grant PITN-GA-2012-317297), and WntsApp (GA-No. 608180, FP7-PEOPLE-2013). I.K. and D.P. were funded by Marie Curie contracts from the European Commission.Peer Reviewe

Exploring Glycosignatures of Pathogenic Bacteria and Cell-Derived Extracellular Vesicles as Prospective Biomarkers

Póster presentado en la National Biotechnology Conference de la American Association of Pharmaceutical Scientists (AAPS), en San Diego, California, del 19 al 21 de mayo de 2014Purpose Glycans are becoming attractive targets in the search for novel biomarkers in many biomedically relevant processes. As significant example, specific carbohydrate determinants on the surface of pathogens are recognized by host receptors for triggering defense responses. Analogously, evidence for a correlation between surface glycosylation and properties of tumor cells, as e.g. tumor-immune escape, is emerging. The purpose of this work was to establish a proof-of-concept on the use of designer¿s microarrays for the analysis of surface glycosylation of whole cells, as pathogenic bacteria and cell-derived extracellular vesicles (EVs), the latter regarded as promising tumor biomarkers. Methods Fixed bacteria and EVs were immobilized in microarrays. Non typable (NT)- Haemophilus influenzae, a non-capsulated opportunistic pathogen, and EVs derived from THP-1 cells, an acute monocytic leukemia cell line, were selected as probes. Printing quality, immobilization efficiency and stability of the arrays were controlled using probe-specific protocols. As pattern-reading tool, a panel of plant lectins with known oligosaccharide-binding specificities was used. The selected system for monitoring lectin binding was biotin/AlexaFluor-647-streptavidin. Results Evidence for probe- and lectin-specific binding patterns was obtained. Results for NT-H. influenzae demonstrated extensive galactoside decoration of the bacterial surface and inhibition assays with lectin-specific haptens confirmed carbohydratedependent recognition. In the case of EVs, galactosides also appeared as prominent markers; furthermore, specific glycosylation motifs among vesicle types (exosomes and microvesicles), as eg. mannosylation, sialylation or fucosylation, were unveiled. Conclusion Glycosylation patterns of the surface of bacteria and cell-derived EVs have been explored by using designer¿s microarrays. Our method allows the evaluation of glycans¿ accessibility on the cell surface, a factor possibly having a significant impact on their biomarker potential. Furthermore, any operative synergetic contribution of other molecules will not be overlooked. Of general importance, the approach is applicable to the screening of any type of biomarker on the cell surface, beyond glycans.Peer Reviewe

A combined Bacteria Microarray and Quartz Crystal Microbalance approach for exploring lectin recognition of nontypeable Haemophilus influenzae

Trabajo presentado en el III Biennal Meeting of the Chemical Biology Meeting / XII Carbohydrate Symposium, celebrado en Madrid (España), del 14 al 16 de marzo de 2016Bacterial glycosignatures are intimately associated with virulence and regularly used for strain typification. Carbohydrate structures anchored on the bacterial surface are targeted by endogenous lectins for triggering defence responses. Elucidation of the recognition traditionally requires laborious protocols for the purification of both pathogen and host target molecules. Recently, we employed bacteria microarrays to study glycan-mediated interactions taking place at the bacterial surface, using Klebsiella pneumoniae as model[1]. In contrast to Klebsiella species, other Gram-negative bacteria are nonencapsulated or present lipooligosaccharides (LOS) instead of complex lipopolysaccharides. This is the case for nontypeable Haemophilus influenzae (NTHi), an important opportunistic pathogen responsible for respiratory and even systematic infections. In this work, we combined NTHi microarrays with the quartz crystal microbalance (QCM) technology for detection of carbohydrate epitopes and analysis of their recognition by lectins. A panel of mutant strains of the clinical isolate NTHi375 presenting a sequentially truncated LOS was examined, along with clinical isolates of different pathological origin. Reference lectins with well-defined binding specificities were used as pattern-reading tool, competition assays with lectin-specific haptens confirming carbohydrate-dependent recognition. Furthermore, the microarray-based approach was shown to be efficient for detecting strain-selective binding of three lectins of the innate immune system, namely surfactant protein D, human galectin-8 and Siglec-14. Selected bacteria-lectin pairs were analysed by QCM using novel bacteria chips, granting additional information on binding kinetics and affinity. The parameters obtained were strain- and lectin-specific, providing further insights into the interactions occurring at the bacterial surface and aiding in the establishment of functional correlations. Acknowledgements: This work was supported by the Marie Curie Initial Training Networks DYNANO (PITN-GA-2011-289033) and GLYCOPHARM (PITN-GA-2012-317297), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Spanish Ministry of Economy and Competitiveness (grants BFU2012-36825 and SAF2012-31166). I.K. was funded by a Marie Curie contract from the European Commission. References [1] Campanero-Rhodes, M. A.; Llobet, E.; Bengoechea, J. A.; Solis, D. RSC Adv. 2015, 5 (10), 7173.This work was supported by the Marie Curie Initial Training Networks DYNANO (PITN-GA-2011-289033) and GLYCOPHARM (PITN-GA-2012-317297), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Spanish Ministry of Economy and Competitiveness (grants BFU2012-36825 and SAF2012-31166). I.K. was funded by a Marie Curie contract from the European CommissionPeer reviewe