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. 2018 Feb 14:9:205.
doi: 10.3389/fmicb.2018.00205. eCollection 2018.

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

Kelli Monteiro da Costa  1 , Raphael C Valente  2 , Eduardo J Salustiano  1 , Luciana B Gentile  1 , Leonardo Freire-de-Lima  1 , Lucia Mendonça-Previato  1 , José O Previato  1
Affiliations

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

Kelli Monteiro da Costa et al. Front Microbiol. .

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.

Keywords: ABC transporters; Chagas disease; P-glycoprotein; Trypanosoma cruzi; multidrug resistance phenotype; multidrug resistance protein; thiol transporter.

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Figures

Figure 1
Figure 1
Inhibition of ABCC-like activity by MK-571 in epimastigote forms of the T. cruzi Y strain. ABCC-like activity was evaluated by the carboxyfluorescein (CF) efflux assay. 106 epimastigotes were incubated in medium containing 5 μM CFDA in the presence or absence of 100, 200, or 300 μM MK-571 for 30 min. Parasites were then centrifuged and incubated in medium in the presence or absence of MK-571 for another 30 min. After incubation, parasites were centrifuged, suspended in PBS and kept on ice for acquisition by flow cytometry. Graphs in (A,B) represent the median fluorescence intensity (MFI) for CF, (C,D) the percentages of CF+ parasites at 27 and 37°C, respectively. The Friedman statistical test was used with n = 10 (27°C) and n = 8 (37°C) independent experiments. Lines represent the median for each group and the significance values were represented by *p < 0.05; **p < 0.01, and ***p < 0.001.
Figure 2
Figure 2
Inhibition index of CF efflux (Δ) by ABCC modulators in epimastigote forms of the T. cruzi Y strain. Inhibition index of CF efflux (Δ) was calculated by the ratio between the CF MFI of the parasite in the presence of the modulator and the CF MFI in the absence of the modulator (CTL). Graphs in (A,B) represent the Δ values in the presence of the modulators at 27 and 37°C, respectively. Comparison between Δ indexes at 27 and 37°C are shown in (C) for 7.5 mM probenecid, in (D) for 600 μM indomethacin and in (E) for 200 μM MK-571. The Kruskal–Wallis and (C–E) Mann–Whitney (A,B) statistical tests were performed with n = 10 (27°C) and n = 8 (37°C) independent experiments. Bars represent the mean ± standard error and the significance values were represented by *p < 0.05, **p < 0.01, and ***p < 0.001.
Figure 3
Figure 3
Inhibition of ABCC-like activity by MK-571 in trypomastigote forms of the T. cruzi Y strain. ABCC-like activity was evaluated by the carboxyfluorescein (CF) efflux assay. 106 trypomastigotes were incubated in medium containing 2.5 μM CFDA in the presence or absence of 7.5 mM probenecid, 600 μM indomethacin or 200 μM MK-571 for 30 min. Parasites were then centrifuged and incubated in medium in the presence or absence of the modulators for another 30 min. After incubation, parasites were centrifuged, suspended in PBS and kept on ice for acquisition by flow cytometry. Graph in (A) represents the median fluorescence intensity (MFI) for CF, in (B) the percentages of CF+ parasites for the assay performed at 37°C. Inhibition index of CF efflux (Δ) was calculated by the ratio between the CF MFI of the parasite in the presence of the modulator and the CF MFI in the absence of the modulator (CTL). Comparison between Δ indexes are shown in the graphs in (C) for modulators and in (D) trypomastigote and epimastigote forms. The Friedman (A–C) and Man–Whitney (D) statistical test were used with n = 11 independent experiments. Lines represent the median for each group and the significance values were represented by *p < 0.05, **p < 0.01, and ***p < 0.001.
Figure 4
Figure 4
Inhibition of ABCC-like activity after ATP depletion in epimastigote forms of the T. cruzi Y strain. ABCC-like activity was evaluated by the carboxyfluorescein (CF) efflux assay. 106 epimastigotes were preincubated for 60 min at 27 or 37°C with 40 mM sodium azide (NaN3) or 1 mM iodoacetic acid (C2H3IO2) in PBS. Parasites were then centrifuged and suspended in PBS containing 5 μM CFDA. After 30 min, parasites were centrifuged and incubated in PBS for another 30 min. After incubation, parasites were centrifuged, suspended in PBS and kept on ice for acquisition by flow cytometry. Graphs shown in (A,B) represent the CF MFI, (C,D) the percentages of CF+ parasites at 27 and 37°C, respectively. The Friedman statistical test was used with n = 8 independent experiments. Lines represent the median for each group and the significance values were represented by *p < 0.05; **p < 0.01, and ***p < 0.001.
Figure 5
Figure 5
Reduced ABCC-like activity in absence of an energy source in epimastigote forms of the T. cruzi Y strain. ABCC-like activity was evaluated by the carboxyfluorescein (CF) efflux assay. 106 epimastigotes were incubated in medium in the presence or absence of glucose containing 5 μM CFDA in the accumulation step. After 30 min, parasites were centrifuged and incubated in RPMI or PBS for another 30 min. After incubation, parasites were centrifuged, suspended in PBS and kept on ice for acquisition by flow cytometry. The Friedman statistical test was used with n = 8 independent experiments. Bars represent the mean ± standard error and the significance values were represented by **p < 0.01 and ***p < 0.001.
Figure 6
Figure 6
Depletion of non-protein thiols in epimastigote forms of the T. cruzi Y strain. 106 epimastigotes were preincubated with 100 μM N-ethylmaleimide (NEM) in PBS for 60 min or with 3 mM buthionine sulfoximine (BSO) in medium for 48 h at 27°C. Then, parasites were centrifuged and suspended in PBS containing 1 μM CMFDA at 27 or 37°C. After 15 min of incubation, parasites were centrifuged, suspended in PBS and kept on ice for acquisition by flow cytometry. Graphs shown in (A,B) represent the median fluorescence intensity (MFI) for TMF after preincubation with NEM, (C,D) the TMF MFI after preincubation with BSO at 27 and 37°C, respectively. The Wilcoxon statistical test was used with 11 ≤ n ≤ 13 independent experiments. Lines represent the median for each group and the significance values were represented by ***p < 0.001.
Figure 7
Figure 7
Inhibition of ABCC-like activity after depletion of non-protein thiols in epimastigote forms of the T. cruzi Y strain. ABCC-like activity was evaluated by the carboxyfluorescein (CF) efflux assay. 106 epimastigotes preincubated in PBS for 60 min with 100 μM N-ethylmaleimide (NEM) at 27°C. Parasites were then centrifuged and suspended in PBS containing 5 μM CFDA at 27 or 37°C. After 30 min, parasites were centrifuged and incubated with PBS for another 30 min. After incubation, parasites were centrifuged, suspended in PBS and kept on ice for acquisition by flow cytometry. Graphs in (A,B) represent the median fluorescence intensity (MFI) for CF, (C,D) the percentages of CF+ parasites at 27 and 37°C, respectively. The Wilcoxon statistical test was used with n = 12 (27°C) and n = 13 (37°C) independent experiments. Lines represent the median for each group and the significance values were represented by ***p < 0.001.
Figure 8
Figure 8
Inhibition of ABCC-mediated efflux of a thiol-conjugated compounds by indomethacin in epimastigote forms of the T. cruzi Y strain. ABCC-like activity was evaluated by the thiol-conjugated methylfluorescein (TMF) efflux assay. 106 epimastigotes were incubated in PBS containing 1 μM CMFDA in the presence or absence of 600 μM indomethacin for 30 min. Parasites were then centrifuged and incubated in medium in the presence or absence of indomethacin for another 30 min. After incubation, parasites were centrifuged, suspended in PBS and kept on ice for acquisition by flow cytometry. Graphs in (A,B) represent the median fluorescence intensity (MFI) for TMF, (C,D) the percentages of TMF+ parasites at 27 and 37°C, respectively. The Wilcoxon statistical test was used with n = 18 (27°C) and 16 (37°C) independent experiments. Lines represent the median for each group and the significance values were represented by ***p < 0.001.
Figure 9
Figure 9
Absence of ABCB1-like activity after treatment of epimastigote forms of the T. cruzi Y strain with transport modulators. ABCB1-like activity was evaluated by the Rhodamine 123 (Rho 123) efflux assay. 106 epimastigotes were incubated in medium containing 100 nM Rho 123 in the presence or absence of 2 or 50 μM cyclosporin A (CsA), 10 or 50 μM verapamil (VP) or 2 or 10 μM trifluoperazine (TFP) for 30 min. Parasites were then centrifuged and incubated in medium in the presence or absence of the modulators for another 30 min. After incubation, parasites were centrifuged, suspended in PBS and kept on ice for acquisition by flow cytometry. The graphs in (A,B) represent the median fluorescence intensity (MFI) for Rho 123, (C,D) the percentages of Rho 123+ parasites at 27 and 37°C, respectively. The Friedman statistical test was used with n = 8 independent experiments. Lines represent the median for each group and the significance values were represented by *p < 0.05, **p < 0.01, and ***p < 0.001.
Figure 10
Figure 10
Absence of ABCB1-like activity after ATP depletion in epimastigote forms and after treatment with transport modulators in trypomastigote forms of the T. cruzi Y strain. ABCB1-like activity was evaluated by the Rhodamine 123 (Rho 123) efflux assay. (A,B) 106 epimastigotes were preincubated for 60 min at 27°C with 40 mM sodium azide or 1 mM iodoacetic acid (C2H3IO2) in PBS. Parasites were then centrifuged and suspended in PBS containing 100 nM Rho 123. After 30 min, parasites were centrifuged and incubated in PBS for another 30 min. (C,D) 106 trypomastigotes were incubated in medium containing 100 nM Rho 123 in the presence or absence of 2 μM cyclosporin A (CsA) or 10 μM of either verapamil (VP) or trifluoperazine (TFP) for 30 min. Parasites were then centrifuged and incubated in medium in the presence or absence of the modulators for another 30 min. After incubation, parasites were centrifuged, suspended in PBS and maintained on ice for acquisition by flow cytometry. Graphs in (A,C) represent the median fluorescence intensity (MFI) for Rho 123 and in (B,D) the percentages of Rho 123+ parasites at 37°C. The Friedman statistical test was used with n = 8 independent experiments. Lines represent the median for each group and the significance values were represented by **p < 0.01 and ***p < 0.001.
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