Control of the O-antigen length in Brucella

A.M. ROMANI; M.G. DEL GIUDICE; A.C. CASABUONO; A.S. COUTO; C. CZIBENER; J.E. UGALDE

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedades de Biociencias

Bacteria of the genus Brucella are gram-negative coccobacilli that cause brucellosis, a world-wide distributed zoonosis. Lipopolysaccharide (LPS) is the main component of the extracellular side of the outer membrane of the bacteria and constitutes the first physical barrier that protects them from the environment and, therefore, is central for the virulence of many pathogens. The LPS is composed of three parts, the lipid A, the oligosaccharide nucleus and the terminal polysaccharide terminal O-antigen. Although several genes that participate in the synthesis of LPS have been identified, the mechanism that regulates the length of the O-antigen is currently not known. Previous results indicated that Brucella actively controls the length of the O-antigen and in the search for putative genes that could play a role in this control we identified wbkB, a gene with no known function, present in the O-antigen synthesis cluster that codes for a protein with a putative glycosiltransferase domain. A deletion mutant in wbkB exhibited longer O-antigen chains measured by western blot and MALDI-TOF. Topology studies using fusions to -galactosidase and to the alkaline phosphatase showed that the putative glycosiltransferase domain could be facing the citoplasmic side of the inner membrane strongly suggesting that this protein could be acting on the lipid-intermediate. A double mutant pgm/wbkB that accumulates the lipid-intermediate reinfoced this hypothesis. Additionally, infection assays with the wbkB mutant in cultured cells showed that the inability to control the O-antigen chain length affects virulence. In subsequent studies we will try to understand mechanistically how WbkB operates to regulate the of the O-antigen and how this fine tuned regulation affects the virulence process.