Analysis of the IGS rRNA Region and Applicability for Leishmania (V.) braziliensis Characterization

Abstract

The causative species is an important factor influencing the evolution of American cutaneous leishmaniasis (ACL). Due to its wide distribution in endemic areas, Leishmania (V.) braziliensis is considered one of the most important species in circulation in Brazil. Molecular targets derived from ribosomal RNA (rRNA) were used in studies to identify Leishmania spp.; however, the Intergenic Spacer (IGS) region has not yet been explored in parasite species differentiation. Besides, there is a shortage of sequences deposited in public repositories for this region. Thus, it was proposed to analyze and provide sequences of the IGS rRNA region from different Leishmania spp. and to evaluate their potential as biomarkers to characterize L. braziliensis. A set of primers was designed for complete amplification of the IGS rRNA region of Leishmania spp. PCR products were submitted to Sanger sequencing. The sequences obtained were aligned and analyzed for size and similarity, as well as deposited in GenBank. Characteristics of the repetitive elements (IGSRE) present in the IGS rRNA were also verified. In addition, a set of primers for L. braziliensis identification for qPCR was developed and optimized. Sensitivity (S), specificity (σ), and efficiency (ε) tests were applied. It was found that the mean size for the IGS rRNA region is 3 kb, and the similarity analysis of the sequences obtained demonstrated high conservation among the species. It was observed that the size for the IGSRE repetitive region varies between 61 and 71 bp, and there is a high identity between some species. Fifteen sequences generated for the IGS rRNA partial region of nine different species were deposited in GenBank so far. The specific primer system for L. braziliensis showed S = 10 fg, ε = 98.08%, and logσ = 10³ for Leishmania naiffi; logσ = 10⁴ for Leishmania guyanensis; and logσ = 10⁵ for Leishmania shawi. This protocol system can be used for diagnosis, identification, and quantification of a patient's parasite load, aiding in the direction of a more appropriate therapeutic management to the cases of infection by this etiological agent. Besides that, the unpublished sequences deposited in databases can be used for multiple analyses in different contexts.

Figure 1

Forming subunits of the Leishmania spp. ribosomal RNA. Repeating unit of an RNA locus, showing the 18S (orange), 5.8S (green), and 28S (blue) regions. In the intergenic space (IGS) between the repetition units, there is an element of repetition called IGSRE. It is estimated that the IGS region size is between 2 and 3 kb.

Figure 2

Agarose gel (1.0%) showing PCR reaction result for IGS rRNA region amplification of different Leishmania spp. IGS—Intergenic Spacer; rRNA—ribosomal RNA; bp—base pairs; PM—marker 100 bp (Promega); NTC—nontemplate control. Boxes show bands extracted for purification.

Figure 3

Reaction efficiency with the Lb/IGS system shown in the standard curve. Standard curve resulting from the amplification reaction of Leishmania braziliensis (Lb) DNA in six points or concentrations, demonstrating a detection limit of up to 10 fg for this specie. On the ordinate axis is the threshold cycle or cut-off point (C_t), and on the abscissa axis is the amount of template DNA in fentograms (fg). IGS—Intergenic Spacer; rRNA—ribosomal RNA.