FIG. 4.

(A) Sequence alignment of the Topo-I protein (LdTOP1A) at positions 185 and 325. Parasite DNA was extracted with DNAzol reagent, and the LdTOP1A gene (1,905 bp) was sequenced with specific primers as described in Materials and Methods. L. donovani C1 and LdRCPT.160 LdTOP1A sequences were compared with L. donovani infantum (Ldi) LdTOP1A sequence (GenBank accession number AF145121). G, glycine; D, aspartic acid; R, arginine; E, glutamic acid. (B) Effect of CPT on LdTOP1A mutant parasites. Parasites were grown in the presence of increasing concentrations of CPT (0 to 80 μM), and optical density was monitored for 6 days. L. donovani C1 was transfected with the mutated gene coding for the double-substituted LdTOP1A protein (L. donovani Topo-I Gly185Arg/Asp325Glu) or with the mutated genes coding for the single-substituted LdTOP1A proteins independently (L. donovani Topo-I Gly185Arg and L. donovani Topo-I Asp325Glu). L. donovani C1 overexpressing the wild-type LdTOP1A gene (no mutation) was used as a control. Proliferation of L. donovani C1 in the absence of any treatment was considered as a maximal growth control. The parasite growth is expressed as a percentage of the optical density compared with that for the control. Results (percent over control ± standard error) are representative of three experiments performed in triplicate.