Cyclic ß(1-2) glucans are involved in virulence and intracellular multiplication of Brucella abortus

A. E. CIOCCHINI, G. BRIONES IANNINO, F. IANNINO, R. A. UGALDE Y N. IÑÓN DE IANNINO

Villa Carlos Paz, Cordoba, Argentina

Congreso; XXXVII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB 2001); 2001

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

Cyclic b-1,2-glucan is synthesized by the cyclic glucan synthase (Cgs) by a novel mechanism in which the enzyme, an integral inner membrane protein of 316 KDa, acts itself as protein intermediate. Cgs uses UDP-glucose as sugar donor and has the three enzymatic activities required for the synthesis of the polysaccharide: initiation, elongation and cyclization.. B. abortus  cgs mutants, obtained by transposon insertion, displayed reduced virulence in mice and were defective in intracellular replication in HeLa cells. Complementation of the cgs mutants with a plasmid containing a wild-type copy of the cgs (pBAI9) restored to wild-type phenotype. The addition of purified cyclic b-1,2-glucan in the B. abortus Bvl129 cgs mutant restored the ability to replicate in HeLa cells. No complementation was obtained with lineal glucans of Xhantomonas or Ralstonea solanacearum. These results suggest that the attenuated phenotype observed in the mutant is due to the lack of cyclic glucan itself and not to a pleotropic effect caused by the absence of the Cgs in the inner membrane. It has been reported that various families of glycosyltransferases share a common motif consisting of DXD, described as the UDP-glucose binding site. Nucleotide sequence revealed that two of these motifs are conserved in the amino-terminal region of the protein. By site-directed mutagenesis in which aspartic acid residues 636 and 638 were exchanged to alanine a mutant of the second DXD motif was constructed. Less than 10% of cyclic glucan was produced by the mutant, indicating that this site is involved in the binding of UDP-glucose.