Molecular characterization of the hexose transporter gene in benznidazole resistant and susceptible populations of Trypanosoma cruzi

Abstract

BackgroundHexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell. Previous studies employing the Differential Display technique have shown that the transcription level of the HT gene from T. cruzi (TcrHT) is higher in an in vitro-induced benznidazole (BZ)-resistant population of the parasite (17 LER) than in its susceptible counterpart (17 WTS).MethodsIn the present study, TcrHT has been characterized in populations and strains of T. cruzi that are resistant or susceptible to BZ. We investigated the copy number and chromosomal location of the gene, the levels of TcrHT mRNA and of TcrHT activity, and the phylogenetic relationship between TcrHT and HTs from other organisms.ResultsIn silico analyses revealed that 15 sequences of the TcrHT gene are present in the T. cruzi genome, considering both CL Brener haplotypes. Southern blot analyses confirmed that the gene is present as a multicopy tandem array and indicated a nucleotide sequence polymorphism associated to T. cruzi group I or II. Karyotype analyses revealed that TcrHT is located in two chromosomal bands varying in size from 1.85 to 2.6 Mb depending on the strain of T. cruzi. The sequence of amino acids in the HT from T. cruzi is closely related to the HT sequences of Leishmania species according to phylogenetic analysis. Northern blot and quantitative real-time reverse transcriptase polymerase chain reaction analyses revealed that TcrHT transcripts are 2.6-fold higher in the resistant 17 LER population than in the susceptible 17 WTS. Interestingly, the hexose transporter activity was 40% lower in the 17 LER population than in all other T. cruzi samples analyzed. This phenotype was detected only in the in vitro-induced BZ resistant population, but not in the in vivo-selected or naturally BZ resistant T. cruzi samples. Sequencing analysis revealed that the amino acid sequences of the TcrHT from 17WTS and 17LER populations are identical. This result suggests that the difference in glucose transport between 17WTS and 17LER populations is not due to point mutations, but probably due to lower protein expression level.ConclusionThe BZ resistant population 17 LER presents a decrease in glucose uptake in response to drug pressure.

Genome Plasticity in Cultured Leishmania donovani: Comparison of Early and Late Passages

“…Increased transcript abundance of 3-hydroxyisobutyryl-coenzyme a hydrolase-like protein in the early passages relative to the late is in consistency with the preparedness of the infection ready parasites to use beta-oxidation of fatty acids, as observed by others ( Alcolea et al, 2009 ) (Supplementary Data Sheet S4 ). As opposed to the earlier observations ( Moreno et al, 2014 ), level of tyrosine amino transferase presented a reverse trend, as it was slightly downregulated in the early passage, although protein expression may reveal different results as observed previously for Trypanosoma cruzi ( dos Santos et al, 2012 ). Purine acquisition and inter-conversion is an important determinant of environmental sensing and morphogenesis in Leishmania .…”

Molecular characterization of Cyclophilin (TcCyP19) in Trypanosoma cruzi populations susceptible and resistant to benznidazole

“…Overexpression of enzymatic activity related to antioxidant response, like that of tryparedoxin peroxidase, ascorbate peroxidase, and iron-superoxide dismutase, has been detected in T. cruzi populations resistant to BZ (Nogueira et al, 2006(Nogueira et al, , 2009(Nogueira et al, , 2012. In addition, the hexose transporter activity was 40% lower in a BZ-resistant T. cruzi population than in the susceptible control (dos Santos et al, 2012). Differences in susceptibility to BZ and NFX between T. cruzi strains (Filardi and Brener, 1987;Toledo et al, 2004) and/or the genetic diversity of the host (Filardi and Brener, 1987) might explain, in part, the variations in the efficacies of antiparasitic drugs.…”

Transcriptomic analysis of benznidazole-resistant and susceptible Trypanosoma cruzi populations

“…In a previous study, the transcript level of the HT gene was observed to be more highly expressed in the BZ-resistant T. cruzi population. However, the authors observed a 40% reduction in transporter activity in this population, suggesting the existence of a regulatory mechanism at the protein activity level [ 52 ].…”