Diversification of Fungal Chitinases and Their Functional Differentiation in Histoplasma capsulatum

Chitinases enzymatically hydrolyze chitin, a highly abundant and utilized polymer of N-acetyl-glucosamine. 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 (GH18) domain. This large-scale analysis does not support the previous division of fungal chitinases into three major clades (A, B, C) as chitinases previously assigned to the “C” clade are not resolved as distinct from the “A” clade. 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 (including the LysM domain) do not define specific clades, but instead are found more broadly across clades of chitinases. To gain insight into biological function diversity, we characterized all eight chitinases (Cts) from the thermally dimorphic fungus, Histoplasma capsulatum: six A clade, one B clade, and one formerly classified C clade 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. These results suggest chitinase activity is variable even within subclades and that predictions of functionality require more sophisticated models.

Diversification of fungal chitinases and their functional differentiation in Histoplasma capsulatum

ABSTRACTChitinases 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.

Identification and characterization of the glycoside hydrolase family 18 genes from the entomopathogenic fungus Isaria cicadae genome

Fungal chitinases play essential roles in chitin degradation, cell wall remodeling, chitin recycling, nutrition acquisition, autolysis, and virulence. In this study, 18 genes of the glycoside hydrolase 18 (GH18) family were identified in the Isaria cicadae genome. Seventeen of the genes belonged to chitinases and one was an endo-β-N-acetylglucosaminidase (ENGase). According to phylogenetic analysis, the 17 chitinases were designated as subgroups A (7 chitinases), B (7), and C (3). The exon–intron organizations of these genes were analyzed. The conserved regions DxxDxDxE and S/AxGG and the domains CBM1, CBM18, and CBM50 were detected in I. cicadae chitinases and ENGase. The results of analysis of expression patterns showed that genes ICchiA1, ICchiA6, ICchiB1, and ICchiB4 had high transcript levels in the different growth conditions or developmental stages. Subgroup A chitinase genes had higher transcript levels than the genes of all other chitinases. Subgroup B chitinase genes (except ICchiB7) presented higher transcript levels in chitin medium compared with other conditions. ICchiC2 and ICchiC3 were mainly transcribed in autolysis medium and in blastospores, respectively. Moreover, ICchiB1 presented higher transcript levels than genes of other chitinases. This work provides an overview of the GH18 chitinases and ENGase in I. cicadae and provides a context for the chitinolytic potential, functions, and biological controls of these enzymes of entomopathogenic fungi.

Chitinase genes from Metarhizium anisopliae for the control of whitefly in cotton

Entomopathogenic fungi produces endochitianses, involved in the degradation of insect chitin to facilitate the infection process. Endochitinases ( Chit1 ) gene of family 18 glycosyl hydrolyses were amplified, cloned and characterized from genomic DNA of two isolates of Metarhizium anisopliae. Catalytic motif of family 18 glycosyl hydrolyses was found in Chit1 of M. anisopliae , while no signal peptide was found in any isolate, whereas substrate-binding motif was found in Chit1 of both isolates. Phylogenetic analysis revealed the evolutionary relationship among the fungal chitinases of Metarhizium . The Chit1 amplified were closely related to the family 18 glycosyl hydrolyses. Transient expressions of Chit1 in cotton plants using Geminivirus-mediated gene silencing vector of Cotton Leaf Crumple Virus (CLCrV) revealed the chitinase activity of Chit1 genes amplified from both of the isolates of M. anisopliae when compared with the control. Transformed cotton plants were virulent against fourth instar nymphal and adult stages of Bemisia tabaci which resulted in the mortality of both fourth instar nymphal and adult B. tabaci. Thus, the fungal chitinases expressed in cotton plants played a vital role in plant defence against B. tabaci . However, further studies are required to explore the comparative effectiveness of chitinases from different fungal strains against economically important insect pests.

Chitinolytic fungi from the Birjand plain of Southern Khorasan Province in Eastern Iran

Fungal chitinases play important roles in the decomposition of wastes, mycoparasitism, and biocontrol of nematodes and plant pathogens through chitin biodegradation. This study was conducted during 2013–2017 to investigate the presence of chitinase genes in <em>Trichoderma</em> and <em>Clonostachys</em> species from the Birjand plain, and to evaluate their ability to degrade chitin. Fungal spores and soil suspensions were cultured on minimal medium containing 1% colloidal chitin from crab bodies to isolate chitinolytic fungi. Chitinolytic ability of the isolates was evaluated on this medium by staining with 1% Lugol’s iodine solution and screening for the production of a bright halo around the colonies. Fifty-two isolates capable of degrading chitin were recovered. DNA extracted from the isolates was amplified using Chit2 or DECH degenerative primers that are related to the chitinase gene, and their sequences were aligned using the NCBI GenBank database. The Chit2 and DECH primers amplified 600-bp and 250-bp fragments, respectively, and according to sequence alignment, the isolates had sequences similar to that of the <em>chi18</em> chitinase genes. Morphological and molecular characterization allowed identifying the isolates as belonging to the species <em>Trichoderma harzianum</em> (<em>n</em> = 41), <em>T. longibrachiatum</em> (<em>n</em> = 1), <em>T. virens</em> (<em>n</em> = 3), <em>T. brevicompactum</em> (<em>n</em> = 1), <em>Clonostachys rosea</em> (<em>n</em> = 5), and <em>C. rogersoniana</em> (<em>n</em> = 1), some of which may potentially be used as biocontrol agents of pathogenic nematodes and fungi. This is the first report of isolation of fungi capable of chitin biodegradation from the South Khorasan Province in Eastern Iran.