Evolutionary analysis and protein family classification of chitin deacetylases in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen causing cryptococcal meningoencephalitis. Interestingly, the cell wall of C. neoformans contains chitosan, which is critical for its virulence and persistence in the mammalian host. C. neoformans (H99) has three chitin deacetylases (CDAs), which convert chitin to chitosan. Herein, the classification of the chitin-related protein (CRP) family focused on cryptococcal CDAs was analyzed by phylogenetics, evolutionary pressure (d/d), and 3D modeling. A phylogenetic tree of 110 CRPs revealed that they can be divided into two clades, CRP I and II with bootstrap values (> 99%). CRP I clade comprises five groups (Groups 1-5) with a total of 20 genes, while CRP II clade comprises sixteen groups (Groups 6-21) with a total of 90 genes. CRP I comprises only fungal CDAs, including all three C. neoformans CDAs, whereas CRP II comprises diverse CDAs from fungi, bacteria, and amoeba, along with other carbohydrate esterase 4 family proteins. All CDAs have the signal peptide, except those from group 11. Notably, CDAs with the putative O-gycosylation site possess either the glycosylphosphatidylinositol (GPI)-anchor motif for CRP I or the chitin-binding domain (CBD) for CRP II, respectively. This evolutionary conservation strongly indicates that the O-glycosylation modification and the presence of either the GPI-anchor motif or the chitin-binding domain is important for fungal CDAs to function efficiently at the cell surface. This study reveals that C. neoformans CDAs carrying GPI anchors have evolved divergently from fungal and bacterial CDAs, providing new insights into evolution and classification of CRP family.