Benzimidazole (BZ) resistance in Haemonchus controtus : specific interactions of BZs with tubulin

Abstract

The mechanism of benzimidazole (BZ) anthelmintic resistance in Haemonchus contortus was investigated. The total binding (TB), low-affinity binding (LAB) and high-affinity (specific) binding (HAB) of ($sp3$H) BZs (mebendazole (MBZ), oxibendazole (OBZ), albendazole (ABZ) and oxfendazole (OFZ)) in supernatants derived from BZ-susceptible (S) and BZ-resistant (R) strains were examined and compared. The TB of all ($sp3$H) BZs was reduced for the R strain. The TB of OBZ, MBZ and ABZ was separated into LAB and HAB. However, OFZ bound with low-affinity. The binding affinity, K$sb{ rm a},$ and maximum binding, B$sb{ rm max},$ for the HAB of OBZ and MBZ were calculated using computer programs. Compared with the S strain, the B$sb{ rm max}$ of the R strain was reduced but the K$sb{ rm a}$ was not affected. LAB to parasite preparations devoid of tubulin was observed but HAB occurred to preparations containing tubulin only. The HAB per mg protein decreased from egg through larva to adult stage. It was shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and enzyme-linked immunosorbent assay (ELISA) analysis that the tubulin content per mg protein decreased from egg, through larva to adult worm. The ability of various BZs--OBZ, MBZ, ABZ, OFZ, fenbendazole (FBZ), albendazole sulphoxide (ABZSO), albendazole sulphone (ABZSO$sb2),$ and thiabendazole (TBZ)--to bind tubulin was compared by displacement analysis and their IC$sb{50}$ ( (BZ) required to inhibit 50% of the ($sp3$H) BZ binding) and K$sb{ rm a}$ values were determined. The IC$sb{50}$ and K$sb{ rm a}$ values approximately correlated with the known anthelmintic potency (recommended therapeutic doses) of the BZs except for OFZ and ABZSO. Tubulin bound BZs at 4$sp circ$C with lower K$sb{ rm a}$ than at 37$sp circ$C. Western blot of tubulin separated by 2-dimensional electrophoresis showed that the $beta$-tubulin isoform pattern of the S and R strains were dissimilar whi