Caracterização comparativa da variabilidade genômica de isolados de Leishmania spp. obtidos de cães com leishmaniose visceral em diferentes áreas endêmicas no Brasil

Abstract

Leishmaniasis is a complex of diseases caused by protozoan parasites of the Leishmania genus, endemic in Brazil and in countries of the Old and New worlds. The clinical manifestations of the disease depend on the infectivity of the species involved and the host immune response. Leishmania (Leishmania) infantum is associated with the visceral form of the disease in humans and dogs, but some studies evidence the potential of visceralization of Leishmania (L.) amazonensis. Coinfection cases with the presence of more than one Leishmania species infecting the same host are less reported in the literature and may be underestimated. It is known that these trypanosomatids have great potential of adaptability in the face of the most adverse environmental conditions. The biological cycle of these parasites can undergo changes due to the environment in which they are exposed and, mainly, by anthroponotic changes in the ecosystem. The survival and perpetuation of Leishmania are related to modifications at the genetic level, which can modify its gene expression. Hence, there is great relevance in employing field samples from hosts, such as dogs and humans, from endemic areas for visceral leishmaniasis (VL), where parasites certainly undergo environmental pressures and can alter their genomic content and eventually their gene expression due to reservoirs, vectors and hosts availability. This work aims at performing a comparative analysis of Leishmania infantum isolates, predominantly from naturally infected dogs from different geographic regions of Brazil – Bahia, Governador Valadares, Januaria, Rio Grande do Norte and Santa Catarina. Through bioinformatics tools and molecular biomarkers, 85 isolates were analyzed for variation of chromosome number, variation of SNVs (Single Nucleotide Variants), phylogeny, population structure and gene enrichment. In addition, 24 field samples from dogs from the municipality of São Joaquim de Bicas, MG and from a Veterinary hospital in Belo Horizonte were characterized by employing subgenus-specific biomarkers, Viannia and Leishmania, in order to identify the circulating species in the region. The in silico analyses have demonstrated that there are intra and interpopulational variations between geographic areas and different time lapse for these non-clonal samples, which displays the dynamics of population variability that occurs defining distinct subpopulations over several years. The analyzes also point to the occurrence of more than one ancestral population among the isolates, suggesting the coexistence and interaction between parasites of different origins in the same environment increasing the genotypic and phenotypic diversity of these parasites. The CCNV (Chromossome Copy Number Variation) analyzes revealed aneuploidies more evident on chromosomes 8, 23 and 31, which contain enriched genes related to the metabolic activities, survival and virulence of the parasites. The second part of the work demonstrated the occurrence of atypical patterns of infection in five dogs from the metropolitan region of Belo Horizonte that had characterized samples, and four of them were found positive for both subgenus, Viannia and Leishmania, presenting a pattern of coinfection, and one of them was positive only for Viannia subgenus, suggesting a visceralization profile of species so far related to the tegumentary form of the disease, since the sample used was obtained through bone marrow aspiration. PCR products sequencing confirmed four of these results. Population and gene enrichment analyzes are very promising in the development of potential therapeutic targets and subpopulation markers, which will allow a greater understanding of circulating isolates in endemic regions for leishmaniasis. Moreover, the study of VL in field conditions associated to molecular tools and bioinformatics are of great relevance for better understanding population and adaptive dynamics of Leishmania, which may directly influence the VL control employed so far.