Characterization of GH18 chitinase in Leishmania braziliensis: expression, structural insights, and implications for vaccine and therapeutic development.

Chitinases, a group of glycosyl hydrolases (GHs), catalyze the degradation of chitin by releasing N-acetylglucosamine subunits. GHs can be found in all three domains of life. Among the three GH families (GH18, GH19, and GH20), GH18 chitinases are the most conserved and extensively studied. These enzymes have been implicated in nutrition, immune modulation, invasion, and virulence across diverse pathogens. In some parasitic protists, GH18 chitinases are essential for transmission. However, in kinetoplastids, including Leishmania spp., these enzymes remain poorly characterized. This study aimed to identify and characterize chitinase across kinetoplastids, with a particular focus on GH18 chitinase in Leishmania braziliensis, the causative agent of muco-cutaneous leishmaniasis. A bioinformatic pipeline was implemented to retrieve and annotate chitinase genes from multiple databases. Catalytic domains and subcellular localization were identified using dedicated computational tools. Phylogenetic relationships were reconstructed in MEGA12 using maximum likelihood and neighbor-joining methods. RNA-seq data were analyzed to evaluate stage-specific expression. Structural models of L. braziliensis GH18 chitinase (Lbr_ChGH18) were created with AlphaFold and subsequently refined. We identified GH18 chitinases genes across Leishmania species and other kinetoplastids, together with GH19 and GH20 chitinase genes. Lbr_ChGH18 was detected across all life-cycle stages, with peak levels in amastigotes. Docking analyses identified closantel, argifin, and argadin as potential inhibitors of Lbr_ChGH18. Epitope prediction revealed conserved B- and T-cell epitopes in GH18 chitinases from Old and New World Leishmania, capable of binding multiple HLA class I and II molecules, highlighting their potential as diagnostic and vaccine candidates. The discovery of GH20 chitinase-like genes in Trypanosoma species offers new insights into the evolution and functional diversification of GHs in kinetoplastids. The high conservation and expression of GH18 chitinases underscore their potential as promising targets for drug and vaccine development against leishmaniasis.