Identification of novel adhesion molecules in Brucella suis

RUÍZ V; POSADAS DM; MARTÍN F.A.; ZORREGUIETA A.

Rosario, Santa Fé.

Congreso; 5ta Reunión de la Sociedad Argentina de Microbiología General SAMIGE; 2008

samige

Identification of novel adhesion molecules in Brucella suis Shotgun phage display cloning is a useful tool for studying interactions between bacteria and host proteins. Theoretically, these libraries consist of phages that together display all protein domains 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 with a true binding ability. We are interested in discovering the factors that promote adhesion and invasion of the host tissues in the intracellular pathogen Brucella suis. The ability of Brucella to invade and survive within cells is the cornerstone of its virulence, yet the mechanisms underlying both processes are not clear.It has been shown that Brucellae are able to adhere to cultured cells and that extracellular matrix components are involved in the interaction of Brucella with erythrocytes, macrophages, and epithelial cells. To identify bacterial adhesins, invasins, 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 (an extracellular matrix protein), fetuin (a sialic-rich protein) and hyaluronic acid (a carbohydrate polymer that is a major component of the extracellular matrix in connective, epithelial, and neural tissues) were used as ligands in different panning experiments. After enrichment, the sequences of the inserted foreign DNA were obtained for ninety two positive clones, and five candidates (from clones containing different inserts derived from the same gene, “overlapping inserts”, originated after three rounds of panning) were selected. The presence of the fusion proteins was corroborated by screening for expression of the E-tag, and specificity of the binding was determined for each individual clone. The possible role as adhesins of the candidates was further analized.