Identificação de marcadores moleculares de diagnóstico e virulência em Leishmania por bioinformática

Abstract

After 10 years of publication of the first complete genome of Leishmania genus, advances in sequencing technology have provided a large accumulation of genomic, transcriptomic and proteomic data of these taxa. The convergence of these -omics data combined with bioinformatics tools allows the identification of new molecular markers and determination of specific characteristics of the parasite. Thus, the focus of this work was to integrate these data with bioinformatics resources to contribute to a better understanding of important biological aspects of this parasite: identification of new biomarkers for taxa differentiation and associated with virulence. For this purpose, we developed a web tool able to receive complete genomes to identify sets of taxon-specific primers for genotyping by multiplex PCR. This tool, named TipMT, was able to identify 19,314 pairs of primer using the genomic data from distinct Leishmania species as input. The experimental validation verified the efficiency of a set of primers designed by the tool, which were able to differentiate three species: L. major, L. braziliensis and L. infantum. TipMT offers a combination of features that are not present in other available web applications developed for this purpose. TipMT supports multiple sequences as input, identifies target regions automatically, tests the specificity of the primers and generates a text file with general information of the primers and virtual electrophoresis gel as output. In a second part of this work, we evaluated the difference in expression profile of L. amazonensis promastigotes with high and low infectivity by RNA-seq. In this transcriptome study, we compared the expression profile of parasites freshly isolated from experimentally infected mice and parasites that were cultured after 30 in vitro passages. The evaluation of infectivity of the samples confirmed the decrease of infection rate after several passages in axenic culture. Sequencing by Illumina HiSeq 2000 platform generated 27.35 million of reads and these transcripts were mapped to the reference genome of L. amazonensis with an average percentage of 86.57%. We have identified 626 genes with significant differential expression, 66.13% with decreased expression, after serial passages in in vitro culture. After 30 passages in vitro, we have identified a significant decrease in the expression of genes already described as involved in infectivity, as GP63 metallo-peptidase, tryparedoxin peroxidase and heatshock protein HSP70. The identification of metabolic pathways showed that the loss of infectivity is related to changes in metabolism, data corroborated by previous studies using proteomic techniques. Considering these results, the Leishmania virulence could be associated with the efficiency of three processes: parasite-host interaction mediated by parasite surface proteins; response to oxidative stress; and metabolism of amino acids and fatty acids. This study disclosed several other genes that are likely to be associated with Leishmania virulence and are good candidates for further functional studies.