Comparative proteomic analysis of antimony-resistant and -susceptible Leishmania braziliensis and Leishmania infantum chagasi lines.

The emergence of drug-resistant Leishmania species is a significant problem in several countries. A comparative proteomic analysis of antimony-susceptible and antimony-resistant Leishmania braziliensis (LbSbR) and Leishmania infantum chagasi (LcSbR) lines was carried out using two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (LC/MS/MS) for protein identification. Out of 132 protein spots exclusive or up-regulated submitted to MS, we identified 80 proteins that corresponded to 57 distinct proteins. Comparative analysis of data showed that most of the protein spots with differential abundance in both species are involved in antioxidant defense, general stress response, glucose and amino acid metabolism, and cytoskeleton organization. Five proteins were commonly more abundant in both SbIII-resistant Leishmania lines: tryparedoxin peroxidase, alpha-tubulin, HSP70, HSP83, and HSP60. Analysis of the protein abundance by Western blotting assays confirmed our proteomic data. These assays revealed that cyclophilin-A is less expressed in both LbSbR and LcSbR lines. On the other hand, the expression of pteridine reductase is higher in the LbSbR line, whereas tryparedoxin peroxidase is overexpressed in both LbSbR and LcSbR lines. Together, these results show that the mechanism of antimony-resistance in Leishmania spp. is complex and multifactorial.

Selection and phenotype characterization of potassium antimony tartrate-resistant populations of four New World Leishmania species.

In the present study, we selected in vitro populations of Leishmania Viannia guyanensis, L.V. braziliensis, L. Leishmania amazonensis and L.L. infantum chagasi that were resistant to potassium antimony tartrate (SbIII). The resistance index of these populations varied from 4- to 20-fold higher than that of their wild-type counterparts. To evaluate the stability of the resistance phenotype, these four resistant populations were passaged 37 to 47 times in a culture medium without SbIII. No change was observed in the resistance indexes of L.V. guyanensis (19-fold) and L.L. infantum chagasi (4-fold). In contrast, a decrease in the resistance index was observed for L.V. braziliensis (from 20- to 10-fold) and L.L. amazonensis (from 6- to 3-fold). None of the antimony-resistant populations exhibited cross-resistance to amphotericin B and miltefosine. However, the resistant populations of L.V. braziliensis, L.L. amazonensis and L.L. infantum chagasi were also resistant to paromomycin. A drastic reduction was observed in the infectivity in mice for the resistant L.V. guyanensis, L.L. amazonensis and L.V. braziliensis populations. The SbIII-resistant phenotype of L.V. braziliensis was stable after one passage in mice. Although the protocol of induction was the same, the SbIII-resistant populations showed different degrees of tolerance, stability, infectivity in mice and cross-resistance to antileishmanial drugs, depending on the Leishmania species.

Characterization of a gene encoding alcohol dehydrogenase in benznidazole-susceptible and -resistant populations of Trypanosoma cruzi.

Alcohol dehydrogenases (ADH) are a class of oxidoreductases that catalyse the reversible oxidation of ethanol to acetaldehyde. In the human parasite Trypanosoma cruzi the TcADH gene was identified through microarray analysis as having reduced transcription in an in vitro induced benznidazole (BZ)-resistant population. In the present study, we have extended these results by characterizing the TcADH gene from 11 strains of T. cruzi that were either susceptible or naturally resistant to benznidazole and nifurtimox or had in vivo selected or in vitro induced resistance to BZ. Sequence comparisons showed that TcADH was more similar to prokaryotic ADHs than to orthologs identified Leishmania spp. Immunolocalisation using confocal microscopy revealed that TcADH is present in the kinetoplast region and along the parasite body, consistent with the mitochondrial localization predicted by sequence analysis. Northern blots showed a 1.9kb transcript with similar signal intensity in all T. cruzi samples analysed, except for the in vitro selected resistant population, where transcript levels were 2-fold lower. These findings were confirmed by quantitative real-time PCR. In Western blot analysis, anti-TcADH polyclonal antisera recognised a 42kDa protein in all T. cruzi strains tested. The level of expression of this polypeptide was approximately 2-fold lower in the in vitro induced benznidazole-resistant strain, than in the susceptible parental strain. The chromosomal location of the TcADH gene was variable, but was not associated with the zymodeme or with the drug resistance phenotype. The data presented here show that the TcADH enzyme has a decreased level of expression in the in vitro induced BZ-resistant T. cruzi population, a situation that has not been observed in the in vivo selected BZ-resistant and naturally resistant strains.

Trypanosoma cruzi: multiplex PCR to detect and classify strains according to groups I and II.

A multiplex PCR was developed for simultaneous detection of Trypanosoma cruzi DNA and classification of the parasite strain into groups I and II. As little as 10fg of T. cruzi DNA could be detected by multiplex PCR. The technique was shown to be specific for T. cruzi DNA, since no PCR amplification products were obtained with DNA from other tripanosomatid species. Multiplex PCR was validated by assaying genomic DNA from 34 strains of T. cruzi that had been previously characterized; 24 blood samples from experimentally-infected mice and non-infected controls; 20 buffy coat samples from patients in the acute phase of Chagas disease and non-infected individuals, and 15 samples of feces from naturally-infected Triatoma infestans. T. cruzi samples from patients and from Y strain-infected mice were classified by multiplex PCR as T. cruzi II and samples from T. infestans and Colombiana strain-infected mice as T. cruzi I.

Differential gene expression in Trypanosoma cruzi populations susceptible and resistant to benznidazole.

Differential gene expression in three pairs of Trypanosoma cruzi populations or clones susceptible or resistant to benznidazole (BZ) was investigated by differential display (DD) and representation of differential expression (RDE). GenBank searches of 14 genes selected by DD showed that four sequences corresponded to different hypothetical proteins and the others were very similar to T. cruzi genes encoding mucin (TcMUC), dihydrolipoamide dehydrogenase (TcLipDH), the hexose transporter (TcHT), or a ribosomal protein. Sequence analysis was performed on 34 clones obtained by RDE; approximately half of these clones encoded 14 different hypothetical proteins and the other half encoded proteins involved with stress response, antioxidant defence, metabolism, transporter proteins, surface proteins, ribosomal proteins and others. The mRNA levels of eight T. cruzi genes obtained by RDE and DD were analysed by northern blotting to confirm the differential expression of these sequences. For six of the eight genes, TcLipDH, TcHT, TcFeSOD-A (iron superoxide dismutase-A), TcHSP70, TcHSP100 (heat shock protein) and Tc52 (thiol-transferase), mRNA levels in the drug-resistant T. cruzi population were at least twice those in the susceptible population. Further analysis of TcHSP70 showed that although the levels of TcHSP70 mRNA were four-fold higher in T. cruzi BZ-resistant population, no corresponding increase was observed in the levels of TcHSP70 protein expression. The results suggest that TcHSP70 is not directly associated with the T. cruzi drug resistance phenotype.

Arylfurans as potential trypanosoma cruzi trypanothione reductase inhibitors.

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 microM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 microg/ml (59 and 90 microM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 microg/ml (0.6-1.5 mM).

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 æM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60 percent at 20 æg/ml (59 and 90 æM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 æg/ml (0.6-1.5 mM).