Sequence analysis of the 3'-untranslated region of HSP70 (type I) genes in the genus Leishmania: its usefulness as a molecular marker for species identification

Abstract

Background: The Leishmaniases are a group of clinically diverse diseases caused by parasites of the genus Leishmania. To distinguish between species is crucial for correct diagnosis and prognosis as well as for treatment decisions. Recently, sequencing of the HSP70 coding region has been applied in phylogenetic studies and for identifying of Leishmania species with excellent results.

Methods: In the present study, we analyzed the 3'-untranslated region (UTR) of Leishmania HSP70-type I gene from 24 strains representing eleven Leishmania species in the belief that this non-coding region would have a better discriminatory capacity for species typing than coding regions.

Results: It was observed that there was a remarkable degree of sequence conservation in this region, even between species of the subgenus Leishmania and Viannia. In addition, the presence of many microsatellites was a common feature of the 3'-UTR of HSP70-I genes in the Leishmania genus. Finally, we constructed dendrograms based on global sequence alignments of the analyzed Leishmania species and strains, the results indicated that this particular region of HSP70 genes might be useful for species (or species complex) typing, improving for particular species the discrimination capacity of phylogenetic trees based on HSP70 coding sequences. Given the large size variation of the analyzed region between the Leishmania and Viannia subgenera, direct visualization of the PCR amplification product would allow discrimination between subgenera, and a HaeIII-PCR-RFLP analysis might be used for differentiating some species within each subgenera.

Conclusions: Sequence and phylogenetic analyses indicated that this region, which is readily amplified using a single pair of primers from both Old and New World Leishmania species, might be useful as a molecular marker for species discrimination.

Figure 1

Schematic map of theLeishmania HSP70locus (fragment), showing the region amplified in this study. The map was drawn to scale [39]. PCR primer annealing sites are indicated by arrows. CDR, protein coding region; 5’- and 3’-UTR, untranslated regions; IR, intergenic region.

Figure 2

Microsatellite distribution in the 3’-UTR ofHSP70-Igenes in severalLeishmaniaspecies. The repeated motifs correspond to those found in the sense strand. Note that the drawing scale is not proportional for the different Leishmania species; however, for a given sequence, the relative positions of the microsatellites are scaled.

Figure 3

Phylogeny ofLeishmaniaaccording to the Neighbor-joining (panel A) and Minimum Evolution (panel B) methods based on the multiple sequence alignment of 3’UTR ofHSP70-Igene. Numbers above the branch represent percent recovery in 1,000 bootstrap pseudoreplicates analysis (only values above 90 are shown). All positions containing gaps and missing data were eliminated from the dataset (complete deletion option). There were a total of 493 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 [32]. Distance represents the number of base substitutions per site. Species are abbreviated as follows: Lae, L. aethiopica; Lam, L. amazonensis; Lb, L. braziliensis; Ld, L. donovani; Lg, L. guyanensis; Li, L. infantum; Ll, L. lainsoni; Lmj, L. major; Lp, L. peruviana; Lpa, L. panamensis; Lt, L. tropica. See Table 1 for additional information of the strains.