Evolutive History of Leishmania Genus and Differential Diagnosis of Clinical Important Species Based on a Unique Kinetoplastida Chitinase

Abstract

Introduction: Leishmaniasis, a neglected infectious disease affecting humans, domestic, and wild animals, is caused by 20 from 53 Leishmania genus species and transmitted by sandflies. Leishmania genus, belonging to the Trypanosomatidae family and Kinetoplastida order, are grouped into five subgroups according to the biogeographic and evolutive history of parasites and hosts, leading to incongruences and paraphyly. The GH18 Leishmania chitinase, which is encoded by a species-specific single-copy gene, conserved in the basal groups of trypanosomatids, and absent in the genus Trypanosoma, was evaluated as a phylogenetic marker and a diagnostic target. Methods: Primers were designed to detect Leishmania in its host biological samples and obtain the chitinase sequence of species that are unavailable in public databanks. The GH18 chitinase gene and its genomic context were evaluated phylogenetically. A protocol was developed to discriminate Leishmania subgenera by adopting polymerase chain reaction (PCR) and restriction fragment length polymorphism and using in silico tools. The adopted PCR method for detecting a partial 953 bp GH18 chitinase-encoding gene represented high sensibility and specificity on DNA of isolated parasites and was used as negative controls, Trypanosoma cruzi, and DNA from Leishmania hosts. Results: Preservation of the chitinase locus in the aquatic free-living protozoan Bodo saltans disclosed a primitive common origin. Based on the comparative analysis, the amino acid sequence of GH18 trypanosomatidae chitinase demonstrated its high similarity to that of chitinase from marine prokaryotes and protozoans. Phylogenetic reconstruction based on chitinase corroborated the Supercontinent Origins Theory for Leishmania. Conclusion: The chitinase-encoding gene was effectively detected in biological samples and thus could be considered for differential molecular diagnosis among Leishmania clinical important species worldwide.