Amebiasis is a tissue invasive disease caused by the protozoan parasite, Entamoeba histolytica. It debilitates as much as 1% of the world's population and causes as many as 50,000 deaths each year, mostly in impoverished countries. The long term objectives of this research are to help explain the mechanism of destructive interaction between the parasite and host target cells. The focus is on the amoeba actin cytoskeleton, which has an essential dynamic function in the destructive process. Contact with target cells and lipids prepared from target membranes triggers rapid polymerization of E. histolytica actin and phagocytosis.
The specific aims of the proposed research will address the role of lipids in the structure and function of the parasite actin cytoskeleton. In these studies radioisotopic labeling and analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), chemical degradation and HPLC will be used to test preliminary indications that E. histolytica actin is fatty acylated. Lipids stimulate formation of actin-rich complexes in cell-free extracts, and these appear to contain a number of cytoskeleton associated proteins. The role and specificity of lipids in the stimulation of these complexes will be evaluated. Metabolic labeling with specific radioisotopic precursors and analysis by PAGE and autoradiography will be used to seek proteins of the actin complexes that may be associated with the parasite membrane or otherwise interact with lipids. Toward the same end, antibodies will be prepared against PAGE purified actin complex proteins and used in immunofluorescent assays to seek cytoskeleton or membrane associated antigens. Finally, sequence information for selected proteins identified in these studies will be obtained. A primary strategy for this aim will be microsequencing of peptides obtained from actin complex proteins purified by PAGE and use of the amino acid sequence information to design oligonucleotide primers for the polymerase chain reaction (PCR). Gene specific PCR products will be sequenced and also used to screen DNA libraries to obtain the corresponding genes for sequencing. At each stage the sequence information obtained will be compared to that in computerized databases in order to help identify and functionally characterize the proteins analyzed. The information gained in this research should contribute to the understanding of the molecular organization of the E. histolytica actin cytoskeleton and the role this dynamic cellular apparatus plays in the pathogenicity of amebiasis.
