Ameobic colitis, caused by Entamoeba histolytica, is a major world health burden, resulting in an estimated 100,000 deaths per year. Invasion of the intestinal mucosa by the infectious amoeba trophozoites depends on a dynamic cytoskeleton that is regulated by Rho family GTPases. The Siderovski lab has recently identified the first functional heterotrimeric G proteins (EhG123) and a regulator of G protein signaling (RGS) domain-containing Rho guanine nucleotide exchange factor (GEF) from the E. histolytica genome. My own preliminary data indicates that EhRGS-RhoGEF binds EhG1 in a nucleotide-dependent manner and accelerates the latter protein's GTPase activity. A heterotrimeric G protein signaling pathway, similar to the G112/13 pathway in mammals, likely regulates Rho GTPase activity and thus intestinal invasion by E. histolytica. Furthermore, G protein signaling pathways frequently provide excellent drug targets.
In Specific Aim 1, the molecular components of this G protein signaling pathway will be determined by identifying a Rho GTPase substrate for EhRGS-RhoGEF. Biochemical and structural mechanisms for their activation wil be elucidated, using nucleotide exchange assays and X-ray crystallography, respectively.
Specific Aim 2 will determine roles for EhG1 and EhRGS-RhoGEF in amoebic intestinal invasion and destruction. The proposed experiments will test whether over- or under-expression of either protein alters the ability of trophozoites to cap surface receptors, invade extracelular matrix, kil intestinal epithelial cels, or invade the intestinal walls of infected mice. These experiments are designed to elucidate a pathogenically important celular signaling cascade in E. histolytica that may be exploited for drug discovery. This work will also provide a potential mechanism for amoebic response to extracellular cues in the intestine, furthering our understanding of the host-pathogen interaction during amoebic colitis. The proposed training and research plans will provide the skills and knowledge base necessary for a successful career as a gastroenterologist physician-scientist.
PROJECT NARRATIVE Amoebic colitis and other complications arising from infection with the pathogen E. histolytica results in considerable morbidity and mortality worldwide, particularly in developing countries. We propose to study a new G-protein signaling pathway within E. histolytica and its relevance to pathogenicity and intestinal invasion. Like many analogous signaling pathways in humans, detailed structural and functional knowledge of a G-protein signaling pathway controlling E. histolytica pathogenicity is likely to greatly facilitate drug discovery.
