The goal of the proposed work is to investigate mechanism of drug resistance in Entamoeba histolytica, the protozoan parasite that causes human amoebiasis. Drug treatment is presently the major means of controlling E. histolytica infection, which is widespread in the developing world and among homosexual men in the United States, including AIDS patients. The availability of drug resistant mutants of El histolytica growing in axenic culture make it an excellent model system for the study of drug resistance in parasites. These studies also may have relevance to the clinically important area of drug resistance by tumors. The model system used to study drug resistance in amoeba is based on cloned isolates of E. histolytica, one of which (C2) was selected for resistance to emetine but was found to be cross-resistant to colchicine. This resistance to a drug not selected for is reminiscent of multidrug resistance in cultured mammalian cells. There, cells are resistant to many chemotherapeutic drugs based upon an ATP-dependent efflux of the drugs, which is inhibited by calcium ion channel blockers such as verapamil. The increased drug efflux is secondary to amplification of the mRNA and sometime DNA of the multidrug resistance (mdr) gene, which codes for a 170 kd plasma membrane protein called the P-glycoprotein. Our preliminary results suggest that increased drug efflux may also be involved in drug resistance of E. histolytica parasites. First, the increased resistance of the mutant clone C2 to emetine and colchicine is reversed by verapamil. Second, using the polymerase chain reaction and oligonucleotide primers to conserved amino sequences near the ATP binding site of the mammalian mdr gene, a 330 bp fragment of E. histolytica DNA was identified that shows a 54% amino acid identity with the mammalian mdr gene. Thus, it appears that this readily manipulated parasite may possess an mdr-like gene similar to that of tumor cells. Tests of the idea that resistance of mutant amoebae to emetine is based on increased drug efflux will include a) measurements of uptake and efflux of radiolabeled drugs by amoebae, b) use of the 330 mdr-like amoeba DNA segment to identify and clone the entire mdr gene of E. histolytica and to determine whether the gene is amplified in emetine resistant organisms, and c) identification by radiolabeling of the P- glycoprotein if it is present on the mutant amoeba.
Showing the most recent 10 out of 16 publications
