Recombinant expression, purification and biophysical characterisation of virulence factors from Gram-negative pathogens

Endotoxin are complex molecules that provide Gram-negative pathogens with antibiotic resistance. They contain a hydrophobic anchor called lipid A. Lipid A biosynthesis enzymes UDP-2,3 diacylglucosamine hydrolase (LpxH) and Lipid A disaccharide synthase (LpxB) have been identified as drug targets. Another enzyme target of importance is cholesterolalpha-glycosyltransferase (CGT) from H. pylori, which synthesises glucosyl-cholesterol protecting the bacterium from changing environmental conditions. Recombinant expression and biophysical studies on these enzymes indicates they are conformationally stable and monodisperse while bioinformatic studies predicted the membrane interaction region for substrate recognition. These studies lay the foundation for future work focussed on structure-based drug design.