Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

Abstract

Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, Leishmania amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of α-and β-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly-disassembly cycle in 1% overall recovery based on total cellular protein. L. tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae α-and β-tubulin genes were 99.6% and 99.4% identical to the corresponding amino acid sequences from the L. major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.

Inhibitors of Tubulin Assembly Identified through Screening a Compound Library

“…The use of L. tarentolae , which grows to higher cell density in a less expensive growth medium compared with other Leishmania species, as a source of tubulin means lower collection volumes are required for tubulin isolation (22). We have previously shown that the use of diethylaminoethyl–Sepharose chromatography together with one cycle of assembly–disassembly allows for the ready isolation of assembly competent L. tarentolae tubulin of excellent purity as assessed by SDS‐PAGE (22). However, isolating sufficient tubulin to screen 10 000 compounds at the volumes and concentrations reported in the previous 96‐well plate assay was undesirable.…”

Interaction of ascaridole, carvacrol, and caryophyllene oxide from essential oil of Chenopodium ambrosioides L. with mitochondria in Leishmania and other eukaryotes

“…L. tarentolae is a parasite of geckos of the species Tarentola annularis , belonging to the genus Sauroleishmania (Lainson & Shaw, ), and is not pathogenic for humans (Raymond et al, ). L. tarentolae has been widely used in pharmacological studies for (a) the screening of natural and synthetic products (Taylor et al, ), (b) the purification and characterization of proteins that are used for the screening of drugs with potential antileishmanial activity (Fritsche et al, ; Yakovich, Ragone, Alfonzo, Sackett, & Werbovetz, ), and (c) the amplification of genes involved in the resistance to certain antileishmanial drugs such as amphotericin B (Singh, Papadopoulou, & Ouellette, ) and sodium stibogluconate (Haimeur & Ouellette, ).…”

Novel drug design for Chagas disease via targeting Trypanosoma cruzi tubulin: Homology modeling and binding pocket prediction on Trypanosoma cruzi tubulin polymerization inhibition by naphthoquinone derivatives

“…T. cruzi tubulin polymerization assays were performed according to the procedures of Yakovich et al [22]. Polymerization reactions were conducted in buffer containing 0.1M PIPES (pH 6.9), 1mM EGTA, 5mM MgCl 2 , 1.2 mg/ml purified T. cruzi tubulin, and different concentrations of the 3-chloro,2- imido-substituted 1,4- naphthoquinones (IMNDQ) (0.6-40 μM in 50% DMSO).…”