Functional Characterization of ABCC Proteins from Trypanosoma cruzi and Their Involvement with Thiol Transport

Abstract

Chagas disease is a neglected disease caused by the protozoan Trypanosoma cruzi and affects 8 million people worldwide. The main chemotherapy is based on benznidazole. The efficacy in the treatment depends on factors such as the parasite strain, which may present different sensitivity to treatment. In this context, the expression of ABC transporters has been related to chemotherapy failure. ABC transporters share a well-conserved ABC domain, responsible for ATP binding and hydrolysis, whose the energy released is coupled to transport of molecules through membranes. The most known ABC transporters are ABCB1 and ABCC1, involved in the multidrug resistance phenotype in cancer, given their participation in cellular detoxification. In T. cruzi, 27 ABC genes were identified in the genome. Nonetheless, only four ABC genes were characterized: ABCA3, involved in vesicular trafficking; ABCG1, overexpressed in strains naturally resistant to benznidazole, and P-glycoprotein 1 and 2, whose participation in drug resistance is controversial. Considering P-glycoprotein genes are related to ABCC subfamily in T. cruzi according to the demonstration using BLASTP alignment, we evaluated both ABCB1-like and ABCC-like activities in epimastigote and trypomastigote forms of the Y strain. The transport activities were evaluated by the efflux of the fluorescent dyes Rhodamine 123 and Carboxyfluorescein in a flow cytometer. Results indicated that there was no ABCB1-like activity in both T. cruzi forms. Conversely, results demonstrated ABCC-like activity in both epimastigote and trypomastigote forms of T. cruzi. This activity was inhibited by ABCC transport modulators (probenecid, indomethacin, and MK-571), by ATP-depleting agents (sodium azide and iodoacetic acid) and by the thiol-depleting agent N-ethylmaleimide. Additionally, the presence of ABCC-like activity was supported by direct inhibition of the thiol-conjugated compound efflux with indomethacin, characteristic of ABCC subfamily members. Taken together, the results provide the first description of native ABCC-like activity in T. cruzi epimastigote and trypomastigote forms, indicating that the study of the biological role for that thiol transporter is crucial to reveal new molecular mechanisms for therapeutic approaches in the Chagas disease.

Thiol efflux mediated by an ABCC-like transporter participates forTrypanosoma cruziadaptation to environmental and chemotherapeutic stresses

“…Our results demonstrated the lack of ABCB1-like activity in epimastigote and trypomastigote forms from Y strain (31). In order to confirm that this transporter is not involved in the results obtained in the present work, the ABCB1-like mediated efflux assay was performed with the fluorescent substrate rhodamine 123 (Rho 123).…”

“…Previous results have shown that ABCC was capable of transporting thiol-conjugated compounds in epimastigote forms of T. cruzi Y strain (31). Similarly, GSH, GSSG and hemin increased the accumulation of carboxyfluorescein (CF), a fluorescent substrate transported by ABCC subfamily members ( Fig.…”

“…Transporters from ABCC subfamily are mainly responsible for the efflux of molecules conjugated to or in cotransport with thiol, as observed in protozoans as Leishmania and Plasmodium (27) and in mammals (24). In our previous work, we identified an ABCC-like transport in trypomastigote and epimastigote forms of T. cruzi Y strain (31). Although we demonstrated that the efflux of fluorescent molecules conjugated to thiol were inhibited by indomethacin, this inhibitor is not specific, being reported in the literature as ABCB1 inhibitor as well (44).…”

“…Knowing that ABC proteins are overexpressed in the MDR phenotype, we decided to evaluate whether the ABCC-like activity could relate to cell protection in naturally or in benznidazole-induced resistant strains. To estimate natural resistance, three T. cruzi strains were evaluated for ABCC-like transport and compared to Y strain analyzed in our previous work (31). CL Brener and Berenice were employed as models of naturally sensitive strains while the Y and Colombiana as strains naturally resistant to benznidazole.…”

“…At both temperatures, Berenice and CL Brener presented higher Δ values than Y and Colombiana strains, suggesting a greater aptitude for CF efflux in those strains and, consequently, the fact that ABCC inversely correlates to natural resistance to benznidazole. The Δ values of Y strain were obtained from da Costa et al (31) and included for comparison.…”