Chitinases catalyze the β-1,4-glycoside bond hydrolysis reaction of N-acetylglucosamine residues present in chitins and chitodextrins (Cohen-Kupiec and Chet, 1998). Amino acid sequence similarity analysis indicated that these enzymes cluster in the GH18 and GH19 glycosyl hydrolase families. Chitinase and N-acetylglucosaminidase activities in Leishmania were initially found in promastigote supernatant cultures of L. (Leishmania) major. Apparently these enzymes were not secreted through the sand fly gut (Schlein, Jacobson, and Shlomai, 1991), thereby indicating chitinolytic action secreted by a specific parasite (Rogers, Chance, and Bates, 2002). The activity of both enzymes was observed in L. donovani, L. infantum, L. braziliensis, Leptomonas seymouri, Crithidia fasciculata and Trypanosoma lewisi. The molecular approach led to the identification and biochemical characterization of the gene encoding a GH18 chitinase from L. donovani (Ld Cht1). This sequence was found to be well distributed within the Leishmania genus (L. major, L. infantum, L. donovani and L. braziliensis) (Shakarian et al., 2010).
Homologous episomal overexpression of chitinase in both amastigotes and promastigotes of L. mexicana revealed an increase in vector transmission rate and increased pathogenicity in the vertebrate host, thereby indicating that chitinase plays an important role in parasite development, survival and transmission in mammalian hosts (Joshi et al., 2005; Rogers et al., 2008). However, the presence and role of this protein in human blood and tissues of leishmaniasis patients remain unknown. Given the importance of chitinase, its conservation across the Leishmania genus, species-specific amino acid and nucleotide sequence expression in all parasite developmental stages, in this work we geared our focus towards the study of chitinase-encoding gene as a molecular phylogenetic marker.
The genomic locus of GH18 chitinase encoding gene is conserved among basal trypanosomatids, including B. saltans and is absent in the Trypanosoma genus and also it was not found in genomic sequences of parasites from genus Phytomonas. In addition, amino acid sequence comparison among GH18 chitinases from trypanosomatids using public genome database revealed 35% identity of GH18 chitinases from marine protozoa and bacteria to the corresponding B. saltans ortholog. These results strongly suggest that the GH18 chitinase from the Kinetoplastida derived from a common marine ancestor, harboring the primitive enzyme. The phylogenetic reconstruction of basal trypanosomatids, based on the GH18 chitinase, corroborated the most accepted theory for Leishmania origin, the Supercontinent hypothesis (Harkins et al., 2016), which was based on a multigene analysis, with more than 200,000 nucleotides as informative sites. Thus, the GH18 chitinase, present in basal trypanosomatids, can be used as a molecular marker to identify unknown microorganisms, related to Leishmania genus, contributing to investigate the diversity and the evolutive history of this group.
The phylogenetic position of subgenus Sauroleishmania according to the Supercontinent hypothesis indicates the switch of its Leishmania ancestors from mammalian to reptilian hosts (Akhoundi et al., 2016a). In considering a probable marine environment emergence of the trypanosomatid GH18 chitinase, it is possible to explore that the Sauroleishmania subgenus could diverge from an ancestor before the rise of mammals, during the transition of animals from marine to the terrestrial environment. In this case, parasites with similarity to basal groups of trypanosomatids could be found in fish and amphibians. Considering the conservation of the chitinase-encoding gene in Leishmania, the diagnostic method developed in this work can be used to investigate this hypothesis directly on biological samples, circumventing the isolation difficulties of unknown Leishmania related parasites.
Nucleic acid detection techniques in samples from people and/or animals infected with Leishmania, such as PCR, are used for detection and identification of the parasite since the 1980's. PCR include amplification of fragments of the gene encoding the small ribosomal RNA subunit (SSU rDNA,(van Eys et al., 1992), the transcribed internal ribosomal DNA spacer (ITS) (Schonian et al., 2003), sequences corresponding to kinetoplast (kDNA) (Cortes et al., 2004), mini-exon (Paiva et al., 2004), the gene encoding the heat shock protein HSP70, among others (da Silva et al., 2010). In spite of the high sensitivity of PCR and, depending on the molecular target, high specificity, it is more used in epidemiological studies than as a routine diagnostic method, and the gold standard method to diagnose Leishmania is the observation of the parasite by microscopic analysis (Thakur, Joshi, and Kaur, 2020). In addition, to achieve high sensitivity in the methodologies evaluated so far, PCR complementation with other techniques including nested PCR and hybridization is required. For identification of Leishmania species, the methodologies include restriction fragment size analysis of PCR products obtained, and as most of gene targets have multiple copies, interpretation of the results increases the difficulty of using these techniques in the clinical routine (Rogers et al., 2011; Ubeda et al., 2008). In addition, false positives are possible due to contamination with other post-PCR amplified samples or DNA fragments and cross-reaction with other pathogens, including Trypanosoma (Degrave et al., 1994; Viol et al., 2012).
The differential diagnosis showed in this study, based on the detection of the GH18 chitinase gene, presents advantages over other molecular methods, since it employs a single copy gene, absent in Trypanosoma genus, enabling specific detection of Leishmania parasites. Also, the sensitivity of the method, regarding the large size of the amplified fragment supports post-PCR analysis after a single PCR reaction performed directly from biological samples. Restriction analysis of the 953 bp Leishmania chitinase PCR fragment with PstI permitted the identification of medically important species in Latin America where three different Leishmania subgenera circulates in animal reservoirs, human and sand flies (Fig. 2). Given the specificity of the Leishmania chitinase-encoding gene, the molecular diagnostic method can also be used to identify isolated parasites from biological samples, with high specificity, by restriction analysis and/or sequencing (Suzuki et al., 2016). Also, using the restriction enzyme Dde I on the 953 bp chitinase PCR fragment, it is possible to differentiate L. major from all others Old World Leishmania subgenus species, which is of clinical importance in Oriental TL endemic countries (Hijjawi et al., 2016) (Online resource 1 and 2).
Leishmania chitinase is present in basal groups of trypanosomatids genera, probably derived from an ancestor living in a marine environment, and unique in the human pathogen group. To the best of our knowledge, there are no Leishmania chitinase or homologous proteins described with a molecular structure associated to biochemical characterization. Considering the biological importance and the specificity of this protein to the Leishmania genus, molecular studies to define its biochemical function are warranted. Additionally, the diagnostic method described in this work enables detection of basal groups of trypanosomatids, directly from biological sources, helping in the identification of unknown species which may contribute to the Kinetoplastida evolutive history.