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25 pags., 6 figs., 3 tabs. 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. This work was financed by the Spanish Ministry of Economy and Competitiveness (Grant BFU2015-70052-R), the 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), the Fundação para a Ciência e a Tecnologia (FCT-MCTES), Portugal (Grants PTDC/QUI-QUI/112537/2009, PTDC/BIA-MIB/31730/2017, and IF/00023/2012), and the Applied Molecular Biosciences Unit (UCIBIO) (Financed by FCT-MCTES, UID/Multi/04378/2013/2019)

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).pdf, Extracellular 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.

29th International Carbohydrate Symposium - ICS 14-19 of July (2018), Lisboa, Portugal .-- http://www.ics2018.eventos.chemistry.pt/, Bacterial adhesion is a key step for initiating infection that frequently involves specific recognition of host glycans by microbial adhesins. This is the case for uropathogenic Escherichia coli (UPEC), which exploits FimH, a prevalent mannose-specific adhesin, for infecting the human urothelium. Different monovalent and multivalent compounds have been synthesized and evaluated as microbial antiadhesives. We here report on the development of a versatile microarray approach for evaluation of bacterial adhesion and efficiency of FimH-targeted compounds. A straightforward set-up was established, allowing to directly monitor the binding of fluorescently labeled E. coli UTI89 (UPEC clinical isolate) to custom-designed mannan microarrays. Three novel fullerene hexakis-adducts bearing 12 peripheral mannose residues grafted onto the C60 core by different spacers were tested in competition assays as representative compounds. The IC50 values obtained for the fullerenes evidenced a clear multivalency effect, with up to 18-fold enhancement of the relative inhibitory potential per mannose residue compared to methyl ¿-D-mannopyranoside, in line with previous studies. Differences among fullerenes were detected, pointing to a noticeable impact on compound activity of the precise structure and length of the spacer used for linking mannose to the fullerene core. Comparison of array-derived results obtained for the archetypal mannose-binding lectin concanavalin A with data obtained by isothermal titration calorimetry analyses of ConA-mannofullerene interactions fully validated the utility of the arrays for evaluating the inhibitory potential of anti-adhesive compounds., This work was supported by the Marie Curie Initial Training Network DYNANO (PITN-GA-2011-289033), 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, BFU2015-70052-R). I.K. and M.A.F. were funded by Marie Curie contracts from the European Commission.

Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria¿host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment., We gratefully acknowledge financial support from the Spanish Ministry of Economy andCompetitiveness (Grants BFU2012-36825, BFU2015-70052-R, SAF2012-31166, andSAF2015-66520-R), the Department of Health of the Navarra Government (ref.359/2012), the CIBER of Respiratory Diseases (CIBERES), an initiative from the SpanishInstitute of Health Carlos III (ISCIII), and the Marie Curie Initial Training NetworksDYNANO (Grant PITN-GA-2011-289033), GLYCOPHARM (Grant PITN-GA-2012-317297), and WntsApp (GA-No. 608180, FP7-PEOPLE-2013). I.K. and D.P. werebeneficiaries of Marie Curie contracts from the European Commissio

Trabajo presentado en el 68th Congress of the Hellenic Society of Biochemistry and Molecular Biology, celebrado en Atenas (Grecia), del 10 al 12 de noviembre de 2017, Bacterial virulence often correlates with the carbohydrate structures displayed on the pathogen surface and their recognition, or lack of recognition, by lectins of the innate immune system. Using bacteria-based microarrays and a panel of lectins of known specificity as pattern-reading tolos, the presence on nontypeable Haemophilus influenzae (NTHi) of sugar epitopes that could be recognised by galectins was detected. The carbohydrate-recognition domain (CRD) of this lectin family is highly conserved and shares ß-galactoside-binding specificity, although small variations in the architecture of the binding site lead to distinct fine oligosaccharide-binding selectivities. Moreover, their structural organisation defines three groups, i.e. proto, chimera, and tandem-repeat type, with dissimilar capacities to bind ligand clusters and/or act as bridges. In this work, we explored whether human galectins (hGal) other than hGal-8 are capable to bind Gal-bearing NTHi carbohydrate structures. To this end, the binding of selected human galectins, including members of the three structural subgroups, to microarray-printed NTHi375 was examined. SDS-PAGE and fluorescence microscopy analyses of NTHi375 suspensions incubated with galectins confirmed direct galectin binding to live bacteria and cell crosslinking, respectively. Dissection of the recognized epitopes was facilitated by the availability of isogenic mutant strains lacking enzymes involved in lipooligosaccharide (LOS) biosynthesis, also enabling LOS-based microarray binding assays. The results unveiled striking differences in NTHi recognition, even for the two CRDs of the tandem-repeat galectins tested. In particular, LOS truncation had detrimental effects on the binding to NTHi of hGal-4 and -8, pointing to the LOS serving as ligand. However, the driving forces for binding were found to be different, hinting at different epitopes recognized in each case.

Trabajo presentado en el 19th European Carbohydrate Symposium EuroCarb, celebrado en Barcelona (España), del 2 al 6 de julio de 2017, Bacterial glycocalyx components are intimately associated with virulence and commonly used for strain typification. Up until recently, analysis of their recognition by lectins and antibodies was commonly performed following isolation of selected constituents from the pathogen surface. Of note, in these ligand-targeted approaches, the natural presentation and accessibility of glycans along with possible cooperativity of neighbour molecules are not considered, an aspect particularly relevant in the case of weak binders. We recently developed a combined approach of static microarrays and QCM biosensor assays under flow, based on the use of entire bacteria, and demonstrated its usefulness for detection and real-time monitoring of lectin recognition processes [1]. We benefitted from the availability of a panel of nontypeable Heamophilus influenzae (strain NTHi375) mutants, defective for selected lipooligosaccharide (LOS)-specific enzymes, and focused our studies on the effect of LOS truncation in lectin binding to the bacteria. Intriguingly, LOS truncation gave rise to distinct binding profiles for Viscum album and Ricinus communis agglutinins, pointing to the recognition of different Gal-bearing targets. Besides the LOS molecule, a variety of glycoconjugates can be present at the bacterial surface, also encompassing glycoproteins of great significance in pathogenesis. In particular, in NTHi375, hmw loci code for two heavily glycosylated adhesins that are absent in other NTHi strains, as e.g. Rd KW20, a noninvasive laboratory strain. In this work, we pursued the dissection of bacterial surface ligands recognised by these two lectins. To this aim, we comparatively examined their binding to NTHi375, an NTHi375Δomp5 mutant suspected to overexpress LOS, Rd KW20, and an isogenic Rd KW20 mutant expressing HMW1, using our combined bacteria-based microarray and QCM approach. In parallel, recognition of the isolated NTHi375 and Rd KW20 LOSs, whose structures are known to be different, was also examined using LOS-based microarrays. Furthermore, saturation transfer difference NMR experiments allowed the identification of the sugar residues involved in the recognition. Altogether, the results revealed strain- and lectin-specific binding patterns, from the level of LOS molecules to the intact bacterial surface., This work was supported by the Marie Curie Initial Training Networks DYNANO (PITN-GA-2011-289033), GLYCOPHARM (PITN-GA-2012-317297) and WntsApp (PITN-GA-2013- 608180), 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 CTQ2015-64597-C2- 2-P, BFU2012-36825, BFU2015-70052-R, SAF2012-31166 and SAF2015-66520-R). I.K. was funded by a Marie Curie contract from the European Commission.

Trabajo presentado en el Ciclo quincenal de seminarios del IdAB (2016-2017), celebrado en Mutilva (España), el 29 de marzo de 2016, Recognition of carbohydrates by proteins is a crucial step in a myriad of processes relevant to health and disease, from fertilization and embryonic development to cell differentiation and malignancy. Carbohydrates also serve as self/non-self recognition signals. The ABO blood group system and the Gal xenoantigens are prominent examples. Pathogens¿ surfaces are also coated with distinct carbohydrate structures that are targeted by host receptors, including antibodies and lectins of the innate immune system, for triggering defence responses. Conversely, bacterial adhesion to host cells is frequently mediated by pathogens¿ glycan-binding proteins that use carbohydrates expressed on the surface of host cells as docking points for initiating infection. In order to investigate these host¿pathogen interactions, we have developed novel microarray and quartz crystal microbalance (QCM) strategies based on the use of entire bacteria. Bacteria microarrays have proved very useful for exploring the presence of carbohydrate epitopes on the bacterial surface and their recognition by host counter-receptors. In addition, QCM bacteria-chips have served to analyse lectin-binding kinetics and affinity, providing further insights into the interactions occurring at the bacterial surface. The functionality of glycan-binding adhesins has also been explored using tailored microarray strategies, including analysis of the binding of live bacteria to case-designed microarrays. Illustrative examples of the development, validation and application of these strategies will be presented, with main focus on the human pathogens Haemophilus influenzae and uropathogenic Escherichia coli.

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 2016, Bacterial 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 Commission