Differential susceptibility to microtubule agents has been demonstrated between mammalian cells and kinetoplastid organisms such as Leishmania spp. and Trypanosoma spp. The aims of this study were to identify and characterize the architecture of the putative colchicine binding site of Leishmania spp. and investigate the molecular basis of colchicine resistance. We cloned and sequenced the β-tubulin gene of Leishmania (Viannia) guyanensis and established the theoretical 3D model of the protein, using the crystallographic structure of the bovine protein as template. We identified mutations on the Leishmania   β-tubulin gene sequences on regions related to the putative colchicine-binding pocket, which generate amino acid substitutions and changes in the topology of this region, blocking the access of colchicine. The same mutations were found in the β-tubulin sequence of kinetoplastid organisms such as Trypanosoma cruzi, T. brucei, and T. evansi. Using molecular modelling approaches, we demonstrated that conformational changes include an elongation and torsion of an α-helix structure and displacement to the inside of the pocket of one β-sheet that hinders access of colchicine. We propose that kinetoplastid organisms show resistance to colchicine due to amino acids substitutions that generate structural changes in the putative colchicine-binding domain, which prevent colchicine access.

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We have previously identified a novel genomic sequence of 500 bp, the beta 500-DNA sequence, in the subgenus Leishmania (Viannia). This sequence was localized upstream of the beta-tubulin gene. Restriction fragment length polymorphism and hybridization analysis has shown that the beta 500-DNA sequence is specific to this subgenus. A polymerase chain reaction (PCR) assay confirmed this specificity. The beta 500-DNA sequence was apparently absent from the genomic deoxyribonucleic acid of L. colombiensis and L. equatoriensis. These results indicate that a PCR assay based on the beta 500-DNA sequence is likely to be of use to detect and identify Leishmania parasites of this subgenus in clinical samples with high sensitivity, specificity and reliability. The beta 500-DNA sequence can be considered a molecular marker for the subgenus Viannia.

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