Polymerase Chain Reaction: A Powerful New Approach for the Diagnosis of Human Brucellosis

Cyclic β-l,2-glucans are unique periplasmatic polysaccharides produced by Brucella, Rhizobium , and Agrobacterium , bacteria that belong to the a Proteobacteria group and display intimate interactions with plant or animal eukaryotic cells. Cyclic β-1,2-glucans are homopolymers with a degree of polymerisation ranging from 17 to 24 glucose residues substituted with a variety of non-glycosidic residues. Under low osmolarity conditions cyclic glucans are highly accumulated in the periplasmatic space reaching values ranging from 5 to 20 % of the total cellular dry weight, thus suggesting that they may play a role in adaptation and/or protection of the bacterial cells under certain environmental conditions. Moreover, Rhizobium and Agrobacterium cyclic β-1,2-glucans are required for effective plant interaction (symbiosis and virulence, respectively). In addition, Brucella cyclic β-1,2-glucan mutants displayed reduced intracellular multiplication and virulence, thus suggesting that cyclic β3-l,2-glucan may be a common feature required for effective interaction of the bacteria with animal and plant cells. Cyclic β-1,2- glucans are synthesised by a glucosyl transferase, cyclic glucan synthase (Cgs), that uses UDP-glucose as sugar donor. The synthesis proceeds through a novel mechanism in which Cgs, an integral membrane protein of 300 kDa, acts itself as protein intermediate and contains all the enzymatic activities required for the synthesis: i.e. initiation, elongation and cyclization. Cyclic glucans are synthesised on the cytoplasmatic side of the inner membrane and secreted into the periplasmic space by inner membrane-bound ABC-transporters ( S. meliloti ndvA, A. tumefaciens chvA or B. abortus cgt). A. tumefaciens, S. meliloti and B. abortus cyclic glucan ABC-transporters are highly similar and functionally interchangeable. Moreover ndvA, chvA and cgt null mutants, unable to transport cyclic glucan into the periplasm, display defective host interaction, thus suggesting that secretion into the periplasmic space is required to exert its action. Little is known on the mechanism of action of cyclic glucan. It has been proposed that the glucan itself may be required for effective cell attachment. Other authors have proposed that the observed phenotype might be associated to the protection of the cells under stressing conditions, as for example low osmolarity. However, several evidences suggest that the mechanism may be more complicated than that originally thought. 194 For example several pleiotropic effects on the structure and function of cellular envelope were observed in cgs mutants: i) defective assembly of the flagella in motile species like Rhizobium and Agrobacterium , ii) increased sensitivity to antibiotics and detergents, and iii) reduced stability of several components of the type IV secretion system. These results suggest that cyclic β-1,2-glucan may be involved in the correct assembly of membrane macromolecular structures required for effective host interaction.