Identification of divergent Leishmania (Viannia) braziliensis ecotypes derived from a geographically restricted area through whole genome analysis

Abstract

Leishmania braziliensis , the main etiological agent of cutaneous leishmaniasis (CL) in Latin America, is characterized by major differences in basic biology in comparison with better-known Leishmania species. It is also associated with a high phenotypic and possibly genetic diversity that need to be more adequately defined. Here we used whole genome sequences to evaluate the genetic diversity of ten L . braziliensis isolates from a CL endemic area from Northeastern Brazil, previously classified by Multi Locus Enzyme Electrophoresis (MLEE) into ten distinct zymodemes. These sequences were first mapped using the L . braziliensis M2904 reference genome followed by identification of Single Nucleotide Polymorphisms (SNPs). A substantial level of diversity was observed when compared with the reference genome, with SNP counts ranging from ~95,000 to ~131,000 for the different isolates. When the genome data was used to infer relationship between isolates, those belonging to zymodemes Z72/Z75, recovered from forested environments, were found to cluster separately from the others, generally associated with more urban environments. Among the remaining isolates, those from zymodemes Z74/Z106 were also found to form a separate group. Phylogenetic analyses were also performed using Multi-Locus Sequence Analysis from genes coding for four metabolic enzymes used for MLEE as well as the gene sequence coding for the Hsp70 heat shock protein. All 10 isolates were firmly identified as L . braziliensis , including the zymodeme Z26 isolate previously classified as Leishmania shawi , with the clustering into three groups confirmed. Aneuploidy was also investigated but found in general restricted to chromosome 31, with a single isolate, from zymodeme Z27, characterized by extra copies for other chromosomes. Noteworthy, both Z72 and Z75 isolates are characterized by a much reduced heterozygosity. Our data is consistent with the existence of distinct evolutionary groups in the restricted area sampled and a substantial genetic diversity within L . braziliensis .

Genomic Diversification, Structural Plasticity, and Hybridization in Leishmania (Viannia) braziliensis

“…This species is characterized by its ability to cause distinct forms of tegumentary leishmaniasis in humans (Jirmanus et al, 2012;Meireles et al, 2017) and animals (De Oliveira et al, 2013;Brilhante et al, 2019), and also for its variable infectivity, virulence, and response to anti-leishmanial therapy (Rego et al, 2018;Patino et al, 2019a). In addition, it is considered a zoonotic parasite circulating in a wide range of mammalian and vector host species (Kuhls et al, 2013;Roque and Jansen, 2014), characteristics that can contribute to the generation and maintenance of genetic diversity within the species (Cupolillo et al, 2003;Bruna et al, 2019).…”

Intraspecific Genomic Divergence and Minor Structural Variations in Leishmania (Viannia) panamensis

“…The last genomic analysis used to evaluate the genetic variability among the genomes included in this study was associated with the identification of nucleotide-level variations (SNPs). Initially, we observed that the number of SNPs per genome in all the clades analyzed was low (≈1800 to ≈3700 SNPs) ( Figure 4A), compared with the results obtained for other Leishmania (Viannia) species, such as L. braziliensis, where the number of variants observed in clinical isolates from Brazil ranged from ≈96,000 to ≈132,000 SNPs [26], in clinical isolates from Colombia was approximately 18,000 (data in preparation), and in L. peruviana isolates was approximately 27,000 [21]. These results together with the relationship between the number of homozygous/heterozygous SNP sites (39.785/21.603), observed in this study (Table S3), indicate not only a considerable genetic differentiation among species belonging to the same subgenus (L. braziliensis, L. peruviana, and L. panamensis) but also substantially lower variation in terms of SNPs in L. panamensis.…”

“…Although the extensive literature has revealed intra-and interspecific genetic variability of some Leishmania species (mainly L. donovani, L. major, L. tropica) [23,30,41,42], little is known about the genetic variability of Leishmania (Viannia) species. The reported studies so far have focused on L. braziliensis, where the majority report consistent findings in describing its high intraspecific genetic diversity [16,26,43] even in cell populations isolated from a single L. braziliensis strain ("sub-strain") [44], while in L. panamensis the results are not yet concordant, probably for the molecular approaches used. While studies using MLST reveal extreme genetic homogeneity within L. panamensis [10], others using MLEE [5], sequencing of specific genes (Cytb/HSP70) [6], AFLP [13], or microsatellites [14], have reported a high intraspecific genetic diversity in this species.…”

“…However, despite the number of studies reported to date, only few studies using the above-mentioned approaches have focused on evaluating such genomic variability in the clinical isolates of Leishmania species from the subgenus Viannia, such as the published study by de Figueiredo de Sá et al who highlighted the high genetic diversity of L. braziliensis within restricted or adjacent areas [26], the study published by Urrea et al who evaluated the genomic variations of four L. panamensis strains exhibiting different levels of virulence [27], and the recently published study by Restrepo et al who described the genetic variations occurred in two L. panamensis clinical samples [28].…”

Ecological divergence and hybridization of Neotropical Leishmania parasites

“…Our understanding of the genetics of diversification and hybridization in Leishmania is increasingly informed by the genomic revolution. Whole genome sequencing data of hundreds of isolates revealed the global 18 and local 19,20 genome diversity of the Old-World L. donovani species complex, although genome studies on New-World Leishmania species remain scarce and limited to a few isolates 21–23 . Genome-scale analyses also provided strong evidence for genome-wide patterns of recombination in presumed clonal Leishmania species 24–26 , including evidence of classical crossing over at meiosis 24 .…”