Gene expression study using real-time PCR identifies an NTR gene as a major marker of resistance to benznidazole in Trypanosoma cruzi

<p>Abstract</p> <p>Background</p> <p>Chagas disease is a neglected illness, with limited treatments, caused by the parasite <it>Trypanosoma cruzi</it>. Two drugs are prescribed to treat the disease, nifurtimox and benznidazole, which have been previously reported to have limited efficacy and the appearance of resistance by <it>T. cruzi</it>. Acquisition of drug-resistant phenotypes is a complex physiological process based on single or multiple changes of the genes involved, probably in its mechanisms of action.</p> <p>Results</p> <p>The differential genes expression of a sensitive <it>Trypanosoma cruzi </it>strain and its induced <it>in vitro </it>benznidazole-resistant phenotypes was studied. The stepwise increasing concentration of BZ in the parental strain generated five different resistant populations assessed by the IC₅₀ranging from 10.49 to 93.7 μM. The resistant populations maintained their phenotype when the BZ was depleted from the culture for many passages. Additionally, the benznidazole-resistant phenotypes presented a cross-resistance to nifurtimox but not to G418 sulfate. On the other hand, four of the five phenotypes resistant to different concentrations of drugs had different expression levels for the 12 genes evaluated by real-time PCR. However, in the most resistant phenotype (TcR5x), the levels of mRNA from these 12 genes and seven more were similar to the parental strain but not for NTR and OYE genes, which were down-regulated and over-expressed, respectively. The number of copies for these two genes was evaluated for the parental strain and the TcR5x phenotype, revealing that the NTR gene had lost a copy in this last phenotype. No changes were found in the enzyme activity of CPR and SOD in the most resistant population. Finally, there was no variability of genetic profiles among all the parasite populations evaluated by performing low-stringency single-specific primer PCR (LSSP-PCR) and random amplified polymorphic DNA RAPD techniques, indicating that no clonal selection or drastic genetic changes had occurred for the exposure to BZ.</p> <p>Conclusion</p> <p>Here, we propose NTR as the major marker of the appearance of resistance to BZ.</p

High-Resolution Melting (HRM) of the Cytochrome B Gene: A Powerful Approach to Identify Blood-Meal Sources in Chagas Disease Vectors

Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate

Amplification results from field samples (Table S1) showing one or two peaks in the melting curve.

<p>Tm1: melting temperature of peak 1, Tm2: melting temperature of peak 2, SD: standard deviation, HRM %C: confidence percentage of HRM analysis, ND: not determined or not recognized, -: No secondary peak.</p

Blastn results of ten insects collected from the field.

<p>For each sample the identity with the standard species used for HRM identification is shown.</p

Source of the sample of the standard species used in the HRM analysis.

<p>-: sample not used.</p

Fourteen standard species included in the study and its respective Tm and HRM analysis.

<p>SD: standard deviation, CI: confidence interval, VC: variability correlation, %C: confidence percentage, %C SD: standard deviation of confidence percentage, Tm Genotype: Genotype according to Tm from all the replicas.</p

Samples from intestinal content and feces at different times after feeding.

<p>H: human, C: chicken, IC: intestinal content, F: feces, CG: chicken genotype, HG: human genotype, M: mixed feeding determined by two peaks in the melting curve, Tm1: melting temperature of peak 1, Tm2: melting temperature of peak 2, SD: standard deviation, HRM %C: confidence percentage HRM, %C SD standard deviation of confidence percentage, ND: not determined, -: No secondary peak.</p

Dissociation curve of 13 of the 14 genotypes included in the study.

<p>Dissociation curve of 13 of the 14 genotypes included in the study.</p

Tm distribution of 14 genotypes included in the study.

<p>Tm: melting temperature, Sp: species.</p