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Microarray Strategies for Exploring Bacterial Surface Glycans and Their Interactions With Glycan-Binding Proteins
Publication . Campanero-Rhodes, María Asunción; Palma, Angelina Sá; Menéndez, Margarita; Solís, Dolores; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Frontiers Research Foundation
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.
Single human B cell-derived monoclonal anti-Candida antibodies enhance phagocytosis and protect against disseminated candidiasis
Publication . Rudkin, Fiona M.; Raziunaite, Ingrida; Workman, Hillary; Essono, Sosthene; Belmonte, Rodrigo; MacCallum, Donna M.; Johnson, Elizabeth M.; Silva, Lisete M.; Palma, Angelina S.; Feizi, Ten; Jensen, Allan; Erwig, Lars P.; Gow, Neil A.R.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Nature Portfolio
The high global burden of over one million annual lethal fungal infections reflects a lack of protective vaccines, late diagnosis and inadequate chemotherapy. Here, we have generated a unique set of fully human anti-Candida monoclonal antibodies (mAbs) with diagnostic and therapeutic potential by expressing recombinant antibodies from genes cloned from the B cells of patients suffering from candidiasis. Single class switched memory B cells isolated from donors serum-positive for anti-Candida IgG were differentiated in vitro and screened against recombinant Candida albicans Hyr1 cell wall protein and whole fungal cell wall preparations. Antibody genes from Candida-reactive B cell cultures were cloned and expressed in Expi293F human embryonic kidney cells to generate a panel of human recombinant anti-Candida mAbs that demonstrate morphology-specific, high avidity binding to the cell wall. The species-specific and pan-Candida mAbs generated through this technology display favourable properties for diagnostics, strong opsono-phagocytic activity of macrophages in vitro, and protection in a murine model of disseminated candidiasis.
Assignment of new roles for malectin-like domains to understand their divergent evolution
Publication . Lourenço, Frederico Miguel da Conceição; Pinheiro, Benedita
Malectin is a highly-conserved animal lectin from the endoplasmic reticulum (ER), with a quality control function in the N-Glycosylation process. It has a β-sandwich core with long loops connecting the β-sheets. Malectin binding-pocket is in the loops region. Several carbohydrate-binding modules (CBMs) discovered in other domains of life that shared sequence homology with the malectin, were classified and grouped as a novel CBM57 family by Carbohydrate-Active Enzymes (CAZy) database. The members of this family are expected to have a highly conserved β-sandwich core, but high variance in the binding-pocket residues.
To investigate if the specificity of these modules is the same as the malectin, a bioinformatic analysis was performed with 315 members of the CBM57 family found in CAZy database. Several programs were used to predict the protein architecture and to analyse the conservation of amino acids sequences, especially in the binding-pocket. Based on this analysis, we predict animal CBM57 modules to have the same specificity as malectin. However, bacterial CBM57 modules in bacteria domain are predicted, after highlighting the modules associated with glycoside hydrolases from family 2, to have various specificities, and thus different biological functions. For verifying these assumptions, a total of 7 CBMs (family 57 and homologous) associated with glycoside hydrolases from family 2 and belonging to the human gut microbiome – Bacteroides ovatus and Bacteroides thetaiotaomicron- were chosen for characterization studies.
A re-cloning was initially performed for the recombinant DNAs, changing the His-tag position. Afterwards, expression tests were realized, in which 2 CBMs of different bacteria were expressed in soluble form. The production of the proteins was then performed at a larger scale, followed by affinity chromatography purification. By the analysis of the gels, the eluted samples had high purity and were suitable for characterization studies.
Glycan microarrays were performed for determining the binding-specificities of the 2 CBM modules. The CBM module from B.thetaiotaomicron revealed high specificity for pectin polysaccharides, possible recognizing α 1-3 linked galacturonic acid and ramnose. For structural characterization by X-ray crystallography, several crystallization trials were performed. Crystals were obtained for the B.thetaiotaomicron CBM module, which diffracted to high resolution. The structure is, yet, to be solved.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/QUI-QUI/112537/2009