Changes at the genomic level of clinical isolates of Leishmania (Viannia) braziliensis given by in vitro maintenance.

Parasites of the genus Leishmania, etiological agents of Leishmaniasis, have been extensively studied due to the wide spectrum of clinical manifestations that they generate in humans, the impact of the disease in affected populations and the low availability of treatments. Most of the studies on this parasite, in both promastigote and amastigote stages, have been developed using cultures of the parasite kept in vitro by long time. Leishmania spp. presents a high genetic plasticity, due to variations at chromosomal level (rearrangements, aneuploidy, etc.) or in specific regions such as genes (copy number variation). This genomic mosaicism in Leishmania spp. can act as an essential adaptive mechanism that allows rapid selection of a particular genome structure under specific conditions. This plasticity means that the in vitro maintenance of parasite isolates can induce genotypic and eventually phenotypic alterations such as loss of virulence and infectivity. In this sense, this study presents an analysis of the genomic and phenotypic variation in vitro growth of promastigotes and [in vitro susceptibility to Amphotericin B (AmB) in promastigotes and amastigotes], resulting from the in vitro maintenance of promastigote stages of a clinical isolate of L. (V). braziliensis. Here we perform a comparison of the same parasite isolate between few culture passages after parasite isolation from patient (<10, early) vs multiple culture passages (>20, late). Phenotypic characterization data show that the in vitro doubling time during the logarithmic phase was 9.2 h (95% CI, 8.283 to 10.31) for the early isolate and 8.3 h (95% CI, 7.769 to 8.887) for the late version. Susceptibility to AmB determined from the IC50, was 0.101 μg/mL (95% CI, 0.09063 to 0.1129) for the early version and 0.057 μg/mL (95% CI, 0.05200 to 0.06357) for the late version with a significant difference between them (F-Test, p<0.0001). Regarding genomic analysis, a significant variation in the number of copies of different gene families was observed. Within the genes with known IDs, a loss in 484 gene families and a gain of 291 gene families were observed for late version with respect to early one. When comparing with the reference genome, a certain degree of similarity between losses and gains resulting from in vitro selection was observed. To understand the biological processes associated with the expansion/contraction in gene copy number, a gene ontology (GO) enrichment analysis was carried out, showing that gene losses resulting from in vitro growth were mainly associated with long-chain fatty acid metabolism, tryptophan catabolism and glycolysis. Gene gains are mainly associated with microtubule movements, vesicular localization and response to oxidative stress. The results obtained suggest that the in vitro maintenance of L. (V.) braziliensis isolates leads to important variations with respect to the natural genotype and phenotype. [ABSTRACT FROM AUTHOR]