Sialic Acid Transport in Pathogenic Bacteria: A Novel Role for TRAP Transport Systems

A feature of many bacterial pathogens that live on mucosal surfaces is that they synthesise a virulence factor called sialidase. This enzyme removes sialic acid from host cell surfaces and the bacteria can use the free sialic acid for two important cellular functions that aid survival in this biological niche. Both processes require uptake of this monocarboxylate sugar into the bacterial cell, a mechanism that is poorly studied in these mucosal pathogens and this project aims to characterise sialic acid uptake in the bacteria Haemophilus influenzae and Vibrio cholerae. One emerging family of microbial transporters is the tripartite ATP-independent periplasmic (TRAP) transporters. These secondary transporters are unusual in containing an extracytoplasmic solute receptor (ESR) protein that binds ligand before it is delivered to the membrane components. The genes for an uncharacterised TRAP transporter are associated with the genes for sialic acid utilization in both H. influenzae and V. cholera and we aim to characterise this transporter both in vitro and in vivo to determine its role in uptake of sialic acid. <P>
A secondary objective will be to test mutants in the TRAP transporter for effects on the virulence of these pathogens. The ESR from the H. influenzae TRAP transporter has been expressed and purified from E. coli and preliminary evidence suggests that it does indeed bind sialic. The biochemical properties of this sialic acid binding protein will be determined using protein fluorescence and other biophysical techniques which we have used to characterize other TRAP transporters. Strains of both organisms will be constructed with deletions in the TRAP transporter which will be used to assess the contribution of this system to sialic acid transport and virulence of the microbes using animal models. Significantly, a strain of the avian mucosal pathogen Pasteurella multocida with greatly reduced virulence has been isolated recently, which contains a deletion of the homologous uncharacterised TRAP transporter, providing supporting evidence for an important role of this transporter in the biology of the related human pathogens.