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.
Highlights
Hexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell
In silico analyses of T. cruzi hexose transporter gene (TcrHT) gene A similarity search of the TcrHT sequence showed that 15 sequences within T. cruzi CL Brener Esmeraldo-like (TcChr-S - 7 sequences) and non-Esmeraldo-like (TcChrP – 8 sequences) matched the search criteria (Additional file 1: Table S1)
As part of an on-going project dedicated to the elucidation of the molecular basis of drug resistance in T. cruzi, we have studied a selection of genes that are differentially expressed in BZ resistant and susceptible populations of the parasite [30,31,50]
Summary
Hexose transporters (HT) are membrane proteins involved in the uptake of energy-supplying glucose and other hexoses into the cell. There are a number of issues associated with the use of these drugs, including the low percentage cure rate in the chronic phase (8%) compared with that in the acute phase (76%) [2], the age-dependent efficacy [3,4], and the undesirable side effects [5]. Another factor for concern is the appearance of parasite populations that are naturally resistant to NFX or BZ, and some with cross-resistance to both drugs [6,7,8,9]. The problems associated with the available drugs, and the lack of alternative medications, highlight the urgent need to develop new strategies for chemotherapy against Chagas disease [10]
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