LONG TERM OBJECTIVE: To understand how Entamoeba histoytica, an enteric protozoan parasite, destroys host tissue. E. histolytica is a virulent, protozoan parasite of humans that exhibits contact-dependent killing of host cells and degradation of extracellular matrix. Both of these activities are dependent upon actin polymerization. The small GTP- binding protein Rho has been cloned from the ameba (EhRho). Rho is involved in the control of actin polymerization, adhesion, cytolysis, and invasion in mammalian cells. HYPOTHESIS: The small, GTP-binding protein Rho plays a role in the control of tissue destruction by the protozoan parasite E. histolytica. By determining the effect(s) of both inactive and constituitively active Rho in E. histolytica we seek to discover this role.
AIM 1 : To ascertain the effect of inactive EhRho on the adhesive, cytolytic, and degradative phenotypes of E. histolytica. Research Design: Using a newly developed system for inducible expression, the gene for the C3 exoenzyme from Clostridium botulinum will be expressed in the ameba to specifically and irreversibly inactivate EhRho. The resulting phenotype will then be assayed by observations of the structure and extent of actin morphologies and through tests of: adherence to fibronectin and collagen, adherence to target cells, cytolysis of target cells, phagocytosis of red blood cells, and phosphorylation of adhesion plate proteins.
AIM 2 : To determine the effect of constituitively active EhRho on the adhesive, cytolytic, and degradative phenotypes of E. histolytica. Research Design: Using the system for inducible expression, the gene for the constituitively active mutant EhRhoV29 will be expressed in the ameba. The resulting phenotype will be assayed as described in AIM 1.
