Proline Modulates the Trypanosoma cruzi Resistance to Reactive Oxygen Species and Drugs through a Novel D, L-Proline Transporter

Abstract

Trypanosoma cruzi, the etiological agent of Chagas' disease, has a metabolism largely based on the consumption of glucose and proline. This amino acid is essential for host cells infection and intracellular differentiation. In this work we identified a proline transporter (TcAAAP069) by yeasts complementation assays and overexpression in Trypanosoma cruzi epimastigotes. TcAAAP069 is mono-specific for proline but presents an unusual feature; the lack of stereospecificity, because it is competitively inhibited by the D- enantiomer. Parasites overexpressing TcAAAP069 have an increased intracellular proline concentration, 2.6-fold higher than controls, as a consequence of a higher proline transport rate. Furthermore, augmented proline concentration correlates with an improved resistance to trypanocidal drugs and also to reactive oxygen species including hydrogen peroxide and nitric oxide, emulating natural physiological situations. The IC50s for nifurtimox, benznidazole, H2O2 and NO. were 125%, 68%, 44% and 112% higher than controls, respectively. Finally, proline metabolism generates a higher concentration (48%) of ATP in TcAAAP069 parasites. Since proline participates on essential energy pathways, stress and drug resistance responses, these results provide a novel target for the development of new drugs for the treatments for Chagas' disease.

Experimental Resistance to Drug Combinations in Leishmania donovani: Metabolic and Phenotypic Adaptations

“…Proline has been shown to enhance cell survival during environmental stress (e.g., drug pressure). In Trypanosoma cruzi, it was shown to be vital during metacyclogenesis, a process that has been linked to nutrient stress (49), and to modulate resistance toward ROS and drugs (e.g., nifurtimox and benznidazole) (50). This finding was confirmed by a recent study showing that the overexpression of T. cruzi proline dehydrogenase led to resistance to hydrogen peroxide (51).…”

“…The generation of intracellular reactive oxygen species (ROS) with or without drug pressure was measured using the cell-permeable nonfluorescent probe dichlorofluorescein diacetate (H 2 DCFDA), as described in Manzano et al (37) 50 ) and resistance index (EC 50 ratio for resistant and WTM parasites) to menadione and H 2 O 2 were determined for the WTM, A, M, P, S, AS, and SP lines using an MTT colorimetric assay after incubating the promastigotes for 72 h at 28°C in the presence of increasing concentrations of menadione or H 2 O 2 , as described previously (38,39).…”

“…Significant differences versus the WTM were determined using Student's t test (*, P Ͻ 0.01; **, P Ͻ 0.005; ***, P Ͻ 0.001). The black line indicates the threshold at which the levels of EC 50 have been modified versus WTM.…”

Role of Δ1-Pyrroline-5-Carboxylate Dehydrogenase Supports Mitochondrial Metabolism and Host-Cell Invasion of Trypanosoma cruzi

“…Accession code numbers are detailed under "Experimental Procedures." The alignment was performed using the ClustalW method (default parameters) (41 11 , and Ala 13 ) residues and the putative Thr 17 (arrow) cleavable site. C, bioinformatic analysis for the transmembrane region found for TcP5CDH.…”

Caracterização molecular e bioquímica das enzimas envolvidas na biossíntese de prolina em <i>Trypanosoma cruzi</i>.

“…Esse aminoácido e/ou os intermediários envolvidos no seu metabolismo induzem a metaciclogênese (CONTRERAS et al, 1985). Ademais, mostrou-se que a L-Pro participa na diferenciação das formas epimastigotas intracelulares a tripomastigotas que ocorre no interior da célula TcAAAP069, e sugere que os dois sistemas A e B são realizados por esse transportador de acordo com as condições extracelulares (SAYE et al, 2014).…”