Grant: Operating Grant

Shigella bacteria can cause severe intestinal infections and death, especially in children. These bacteria are highly infectious; they protect themselves with an outer coat of complex polysaccharides (O-antigens) that determines the infectious and antigenic properties of bacteria. Many of the genes responsible for the assembly of these polysaccharides are known but the enzymes involved have not been identified. Thus, the biosynthesis of these complex polysaccharides is still not understood, mainly due to the lack of appropriate methods. We have recently developed these methods and synthesized enzyme substrates that resemble the natural substrates. In this proposal we will use the Shigella strain B14 as a model to study the enzymes that sequentially assemble O-antigen polysaccharides. Having the methods and expertise to study the bacterial enzymes, as well as collaborators that are experts in bacterial genetics and protein structure determination, we propose to unravel the sequence of events in O-antigen assembly in Shigella. Using new synthetic compounds and methods based on previous success, we will identify and purify the enzymes required to assemble the O-antigen and thoroughly characterize their properties. The protein structures will be determined in order to understand the molecular mechanisms of their action and to design specific enzyme blockers. We will thus gain a better understanding of the functions of genes and enzymes in Shigella, as well as in other intestinal bacteria. This work is hoped to result in new anti-bacterial strategies. Knowledge of these enzymes will allow the synthesis of specific bacterial carbohydrates for the purpose of vaccines, and will reduce bacterial virulence.