BipA : a tyrosine phosphorylated GTPase that mediates interactions betwen enteropathogenic Escherichia coli (EPEC) and epithelial cells

This thesis describes the functional characterisation of a novel GTPase termed BipA that directly or indirectly regulates a number of EPEC processes implicated in virulence and survival. The EPEC homologue of BipA appears to be phosphorylated while BipA homologues from both E. coli K-12 and S. typhimurium are not. Phosphoamino acid analysis and immunoblotting with phosphotyrosine-specific monoclonal antibodies has shown that EPEC BipA is phosphorylated on tyrosine, making it the first example of a tyrosine phosphorylated protein in E. coli. Although BipA is capable of autophosphorylation, a factor is present in EPEC particulate fractions that stimulates BipA phosphorylation. A hexahistidinyl-tagged derivative of BipA was constructed and used to confirm that BipA has GTPase and ATPase activity. Subsequent analysis showed that the nucleotide hydrolysing activity of BipA was stimulated by prior in vitro phosphorylation of the protein, suggesting a possible biological role for phosphorylation in this system.Specific inactivation of the bipA gene resulted in effects on diverse stress- and virulence-related processes, although cell growth in laboratory media was unaffected. EPEC strains lacking BipA do not show typical AE lesions whereas overproduction of BipA leads to hyperformation of actin-rich pseudopods and pedestals beneath bacteria. Epithelial cells infected with bacteria that either lack or overproduce BipA exhibit elevated levels of both inositol triphosphate and intracellular calcium, but also exhibit reduced levels of the tyrosine phosphorylated intimin receptor, Tir. Analysis of proteins secreted by a bipA null mutant showed that a component of the flagella filament, flagellin, was hyper-secreted. Subsequently, BipA was demonstrated to negatively regulate cell motility in EPEC and also to affect chemotaxis. BipA is also required for resistance to antibacterial components of the host defense system, which may constitute a major stress factor for enteric pathogens.Based on these results, it is proposed that BipA is a novel global regulator that controls diverse virulence-related processes in EPEC. Its striking sequence similarity to ribosome-binding GTPase suggests that it may use a novel mechanism to bring about its effects.