Fasciola hepatica expresses multiple alpha- and beta-tubulin isotypes

We have identified five alpha-tubulin and six beta-tubulin isotypes that are expressed in adult Fasciola hepatica. Amino acid sequence identities ranged between 72 and 95% for fluke alpha-tubulin and between 65 and 97% for beta-tubulin isotypes. Nucleotide sequence identity ranged between 68-77% and 62-80%, respectively, for their coding sequences. Phylogenetic analysis indicated that two of the alpha-tubulins and two of the beta-tubulins were distinctly divergent from the other trematode and nematode tubulin sequences described in this study, whereas the other isotypes segregated within the trematode clades. With regard to the proposed benzimidazole binding site on beta-tubulin, three of the fluke isotypes had tyrosine at position 200 of beta-tubulin, two had phenylalanine and one had leucine. All had phenylalanine at position 167 and glutamic acid at position 198. When isotype RT-PCR fragment sequences were compared between six individual flukes from the susceptible Cullompton isolate and from seven individual flukes from the two resistant isolates, Sligo and Oberon, these residues were conserved.