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

Abstract

Background: Hexose 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).

Methods: In 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.

Results: In 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.

Conclusion: The BZ resistant population 17 LER presents a decrease in glucose uptake in response to drug pressure.

Figure 1

Maximum likelihood phylogenetic tree based on sequences of hexose transporters ofTrypanosoma cruziand other organisms. In the consensus bootstrap tree (1000 replicates) shown, the numbers above each branch represent the bootstrap confidence percentage and the GenBank accession numbers are provided for each species included in the tree.

Figure 2

Levels of transcription of theTcrHTgene in BZ resistant and susceptible T. cruzipopulations and strains showing: (A) Northern blot profiles of total RNA extracts obtained using a ³²P-labeled TcrHT-specific probe; (B) the quantitative control in which the same membrane was exposed to a T. cruzi 24 S ribosomal RNA probe; and (C) levels of TcrHT mRNA as determined quantitatively (relative to the single-copy housekeeping gene TcHGPRT) by real-time RT-PCR. Mean values of the copy number ratio TcrHT/TcHGPRT ± standard deviations from three independent experiments are indicated. The mean values for 17 WTS and 17 LER are significantly different (* p <0.001), whilst the BZR versus BZS and the CL versus Colombiana mean values show no difference.

Figure 3

Southern blot analyses of theTcrHTgene from BZ resistant and susceptible T. cruzipopulations and strains. Preparations of genomic DNA (14 μg) were digested with endonucleases SalI (A) or EcoRI (B), separated by electrophoresis on a 1% agarose gel and hybridized with a ³²P-labeled TcrHT-specific probe. The molecular weight markers were derived from λ phage DNA digested with HindIII and ϕX 174 DNA digested with Hae III.

Figure 4

Chromosomal locations of theTcrHTgene in BZ resistant and susceptibleT. cruzipopulations and strains showing: (A) chromosomal bands that had been separated by pulsed-field gel electrophoresis and stained with ethidium bromide; and (B) Southern blots obtained by hybridizing the chromosomal bands with a ³²P-labeled TcrHT-specific probe. Whole chromosomes from Saccharomyces cerevisiae were employed as molecular weight markers.

Figure 5

Comparison of hexose transporter activities among BZ resistant and susceptibleT. cruzipopulations and strains showing the quantities of 6-deoxy-D-[6-³ H] glucose transported per mg of total protein as a function of time. The values shown represent the means of three independent experiments and the error bars indicate the standard error of the mean (± SEM). The mean values for 17 WTS and 17 LER are significantly different (p <0.001), whilst the BZR versus BZS and the Colombiana versus CL mean values show no such differences (p = 0.072 and p = 0.5, respectively).

Figure 6

Multiple sequence alignment of TcrHR amino acid sequences from four BZ-resistant or –susceptibleT. cruzistrains. The figure shows characteristics of the HT that are conserved in kinetoplastid: boxes indicate 12 transmembrane domains, asterisks indicate conserved arginine residues and diamonds indicate cysteine residues. Amino acid residues in red colour and arrows indicate substitution.