Diversification of fungal chitinases and their functional differentiation inHistoplasma capsulatum

Abstract

Chitinases enzymatically hydrolyze chitin, a highly abundant biomolecule with many potential industrial and medical uses in addition to their natural biological roles. Fungi are a rich source of chitinases, however the phylogenetic and functional diversity of fungal chitinases are not well understood. We surveyed fungal chitinases from 373 publicly available genomes, characterized domain architecture, and conducted phylogenetic analyses of the glycoside hydrolase family 18 (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C). The chitinases previously assigned to the “C” clade are not resolved as distinct from the “A” clade in this larger phylogenetic analysis. Fungal chitinase diversity was partly shaped by horizontal gene transfer, and at least one clade of bacterial origin occurs among chitinases previously assigned to the “B” clade. Furthermore, chitin binding domains (CBD) including the LysM domain do not define specific clades but instead are found more broadly across clades of chitinase enzymes. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade (3 A-V, 1 A-IV, and two A-II), one B clade (B-I), and one formerly classified C clade (C-I) chitinases. Expression analyses showed variable induction of chitinase genes in the presence of chitin but preferential expression of CTS3 in the mycelial stage. Activity assays demonstrated that Cts1 (B-I), Cts2 (A-V), Cts3 (A-V), Cts4 (A-V) have endochitinase activities with varying degrees of chitobiosidase function. Cts6 (C-I) has activity consistent with N-acetyl-glucosaminidase exochitinase function and Cts8 (A-II) has chitobiase activity. This suggests chitinase activity is variable even within sub-clades and that predictions of functionality require more sophisticated models.

Endophyte genomes support greater metabolic gene cluster diversity compared with non-endophytes inTrichoderma

“…Chitinase sequences were first identified by hmmsearch for proteins containing Glyco_hydro_18 (PF00704) domain in hmmer-3.1b2 [175]. Chitinase genes from all Trichoderma species were then incorporated into a multifasta file with diverse representatives of each chitinase class in Goughenour et al ( 2020) [65]. The sequences were then aligned in mafft v7.487 [169] and analyzed in FastTree v2.1.10 with default settings [176].…”

“…Chitinases are a complex group of carbohydrate-active enzymes important for mycoparasitism, so we profiled chitinase repertoires across all genomes separate from the mycoparasitism genes analysis. The chitinase genes in our dataset are grouped into nine chitinase classes as delimited in [65], including classes AII (76), AIV (79), AV (107), BI (215), BII (105), BV (77), CI (107), CII (194), and ChitD (38), which collectively bear 153 lysin domains (LysM) and 374 chitin-binding domains (CBD) (Fig. 4).…”

“…We used an updated kingdom-level GH-18 chitinase ontology [65] to classify chitinases across the dataset. Chitinase sequences were first identified by hmmsearch for proteins containing Glyco_hydro_18 (PF00704) domain in hmmer-3.1b2 [175].…”

Endophyte genomes support greater metabolic gene cluster diversity compared with non-endophytes in Trichoderma

“…Chitinases are a complex group of carbohydrate-active enzymes important for mycoparasitism, so we profiled chitinase repertoires across all genomes separate from the mycoparasitism genes analysis. The chitinase genes in our dataset are grouped into nine chitinase classes as delimited in [68], including classes AII (76), AIV (79), AV (107), BI (215), BII (105), BV (77), CI (107), CII (194), and ChitD (38), which collectively bear 153 lysin domains (LysM) and 374 chitin-binding domains (CBD) (Fig 4). Each Trichoderma genome contains two AII chitinases and one ChitD chitinase gene each.…”

“…We used an updated kingdom-level GH-18 chitinase ontology [68] to classify chitinases across the dataset. Chitinase sequences were first identified by hmmsearch for proteins containing Glyco_hydro_18 (PF00704) domain in hmmer-3.1b2 [179].…”