Cellular location and function of the P-glycoproteins (EhPgps) in Entamoeba histolytica multidrug-resistant trophozoites.
We have studied the cellular location and the efflux pump function of the Entamoeba histolytica P-glycoproteins (EhPgps) in drug-sensitive and -resistant trophozoites. Polyclonal antibodies against the EhPgp384 polypeptide (375-759 amino acids) revealed a 147-kDa protein by Western blot. The band intensity correlated with the emetine-resistance of the trophozoites. Through the confocal microscope, using the anti-EhPgp384 and fluorescein secondary antibodies, the EhPgps were found in a complex vesicular network, in the plasma membrane and outside of the cells. Transmission electron microscopy assays confirmed that drug-resistant trophozoites presented four to five times more EhPgps than sensitive cells. Fluorescence co-localization experiments using rhodamine-123 (R123) and the anti-EhPgp384 antibodies suggested the interaction between EhPgps and the drug. R123 efflux kinetics evidenced that the emetine-resistant trophozoites displayed a drug efflux kinetic four times higher than the drug-sensitive trophozoites, which was reduced by verapamil in both cases. EhPgps may participate in avoiding drug accumulation in the trophozoites by two putative mechanisms: (1) the direct extrusion of the drug from the plasma membrane, and (2) an indirect transport mechanism in which the drug is trapped by EhPgps and concentrated within vesicles that drive the drug to the plasma membrane.
Bañuelos
Cecilia
C
Department of Experimental Pathology, CINVESTAV-IPN. A. P. 14-740, Mexico 07300, D.F.
Orozco
Esther
E
Gómez
Consuelo
C
González
Arturo
A
Medel
Olivia
O
Mendoza
Leobardo
L
Pérez
D Guillermo
DG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000276:immunology; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002121:Calcium Channel Blockers / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D004748:Entamoeba histolytica / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D007120:Immunochemistry; D007700:Kinetics; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014700:Verapamil / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000953:Antigens, Protozoan; D000981:Antiprotozoal Agents; D002121:Calcium Channel Blockers; D012333:RNA, Messenger; D014700:Verapamil