A PHAGE DISPLAY LIBRARY APPROACH TO ISOLATE FACTORS THAT PROMOTE ADHESION OF BRUCELLA TO HOST TISSUES

POSADAS, D. M.; RUIZ, V.; MARTÍN, F A; ZORREGUIETA, A

Buenos Aires

Congreso; IV Congreso Argentino de Microbiología General SAMIGE; 2007

SAMIGE

P33: A PHAGE DISPLAY LIBRARY APPROACH TO ISOLATE FACTORS THAT PROMOTE ADHESION OF BRUCELLA TO HOST TISSUES Diana M. Posadas; Verónica Ruiz; Fernando A. Martín; Angeles Zorreguieta. Fundación Instituto Leloir, IIBBA CONICET y FCEyN, Universidad de Buenos Aires. Patricias Argentinas 435, (1405) Bs As, Argentina. e-mail: dposadas@leloir.org.ar Brucella is an intracellular pathogen responsible of the zoonotic disease called brucellosis. A key feature of Brucella virulence is its ability to invade and proliferate inside professional and non-professional phagocytic cells, were it establishes.  It is well known that attachment of bacteria to host cells is one of the earliest steps in many infectious processes. Firm adhesion of Brucella to the tissues of their hosts allows a close contact between bacterial and eukaryotic cells, a prerequisite for the progress of the infection. Attachment appears to be a complex multistep process consisting of several, both specific and unspecific, interactions. It has been shown that brucellae are able to adhere to the surface of cultured epithelial cells and that sialic acid residues were involved in the interaction of Brucella with erythrocytes, macrophages and epithelial cells, indicating the presence of bacterial lectins participating in the recognition of cellular receptors. Brucella also was able to bind to several extracellular matrix proteins, in particular fibronectin and vitronectin, as other pathogenic organisms. However, we still know very little about the adhesion mechanism used by different Brucella and what physiological changes are induced in the bacteria upon direct physical contact with host cells.  We are interested in discover which are the factors that promote adhesion and invasion of the host tissues in Brucella. Shotgun phage display cloning is a useful tool for studying interactions between bacterial and host proteins. Libraries are constructed by cloning randomly fragmented prokaryotic DNA into phagemid vectors. Theoretically, these libraries consist of phages that together display all proteins encoded by the bacterial genome.  From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. This procedure can lead to the selection of clones that present a true binding ability.    We used this technique to identify bacterial adhesins, receptins and their minimal binding domains. A B. suis phage display library was constructed by cloning shotgun digested genomic DNA into the pG8SAET phagemid vector. The phages were panned several times against immobilized ligands; fibronectin and fetuin were used as ligands in different panning experiments. After enrichment for a certain binding capacity, a number of candidates were selected and the sequences of the inserted foreign DNA were determined. The interaction of these phage candidates with the ligand will be analyzed further in future experiments.