Increase in mRNA of multiple Eh pgp genes encoding P-glycoprotein homologues in emetine-resistant Entamoeba histolytica parasites

Abstract

With the goal of understanding possible mechanisms of drug resistance by the protozoan parasite Entamoeba histolytica (Eh), two novel Eh P-glycoprotein (Pgp) genes (Eh pgp5 and Eh pgp6) were sequenced, and the expression of four Eh pgp genes determined in wild-type (wt) clone A and emetine-resistant (EmR) clone C2 amebae. The Eh pgp5 gene encodes a 1301-amino acid (aa) protein that is similar to those of Eh pgp1 (64% aa identity), Eh pgp2 (61%), Eh pgp6 (39%) and Homo sapiens MDR (multidrug-resistance-encoding)(Hs MDR1; 38%) genes. The 1282-aa Eh pgp6 open reading frame (ORF), which is 19-28 aa shorter than those encoded by other Eh pgp, is also similar to those of Eh pgp1 (46% aa identity), Eh pgp2 (38%), and Hs MDR1 (39%). Both Eh pgp5 and Eh pgp6 ORF predict two ATP-binding cassettes and twelve hydrophobic alpha-helices, which form the putative transmembrane channel. EmR clone C2 amebae, growing at all concentrations of drug, show increased amounts of Eh pgp1 and Eh pgp6 mRNA when compared to wt clone A amebae. In contrast, only clone C2 amebae selected for growth at the highest concentrations of emetine (100-200 micrograms/ml) show increased Eh pgp5 mRNA, while mRNA of both clone C2 and clone A Eh amebae fail to bind an Eh pgp2-specific probe. It appears then that multiple Pgp may contribute to amebic Em resistance in vitro.