Genomic Analyses and Transcriptional Profiles of the Glycoside Hydrolase Family 18 Genes of the Entomopathogenic Fungus Metarhizium anisopliae

Abstract

Fungal chitin metabolism involves diverse processes such as metabolically active cell wall maintenance, basic nutrition, and different aspects of virulence. Chitinases are enzymes belonging to the glycoside hydrolase family 18 (GH18) and 19 (GH19) and are responsible for the hydrolysis of β-1,4-linkages in chitin. This linear homopolymer of N-acetyl-β-D-glucosamine is an essential constituent of fungal cell walls and arthropod exoskeletons. Several chitinases have been directly implicated in structural, morphogenetic, autolytic and nutritional activities of fungal cells. In the entomopathogen Metarhizium anisopliae, chitinases are also involved in virulence. Filamentous fungi genomes exhibit a higher number of chitinase-coding genes than bacteria or yeasts. The survey performed in the M. anisopliae genome has successfully identified 24 genes belonging to glycoside hydrolase family 18, including three previously experimentally determined chitinase-coding genes named chit1, chi2 and chi3. These putative chitinases were classified based on domain organization and phylogenetic analysis into the previously described A, B and C chitinase subgroups, and into a new subgroup D. Moreover, three GH18 proteins could be classified as putative endo-N-acetyl-β-D-glucosaminidases, enzymes that are associated with deglycosylation and were therefore assigned to a new subgroup E. The transcriptional profile of the GH18 genes was evaluated by qPCR with RNA extracted from eight culture conditions, representing different stages of development or different nutritional states. The transcripts from the GH18 genes were detected in at least one of the different M. anisopliae developmental stages, thus validating the proposed genes. Moreover, not all members from the same chitinase subgroup presented equal patterns of transcript expression under the eight distinct conditions studied. The determination of M. anisopliae chitinases and ENGases and a more detailed study concerning the enzymes’ roles in morphological or nutritional functions will allow comprehensive insights into the chitinolytic potential of this highly infective entomopathogenic fungus.

Molecular diversity of LysM carbohydrate-binding motifs in fungi

“…Fungal chitinases can be divided into three subgroups, A, B and C (Seidl 2008 ) and further extensions to this classification have been proposed to include GH family 18 proteins with different functions that have more recently been described, e.g. endo- N -acetylglucosaminidases (Junges et al 2014 ). Subgroup C chitinases are—based on the open reading frames of their genes—large proteins of 150–250 kDa and contain multiple CBMs of families CBM 50 (LysM) and CBM 18 (chitin-binding) (Gruber et al 2011a , b ; Tzelepis et al 2012 , 2014 ).…”

Secondary metabolite gene clusters in the entomopathogen fungus Metarhizium anisopliae: genome identification and patterns of expression in a cuticle infection model

“…During infection, several hydrolytic enzymes, such as chitinases, proteases and lipases, act as important virulence determinants [2]. These enzymes not only facilitate nutritional processes but also morphogenesis and autolytic processes in fungal development [10]. In addition to hydrolytic enzymes, secondary metabolites (SMs) are also produced by Metarhizium to overcome and kill the host [11].…”

A Plumieridine-Rich Fraction From Allamanda polyantha Inhibits Chitinolytic Activity and Exhibits Antifungal Properties Against Cryptococcus neoformans

“…Chitinases are responsible for the hydrolysis of the chitin β-1,4 linkage resulting in monomers and oligomers of GlcNAc ( Howard et al, 2003 ). These enzymes have been reported in a variety of organisms beside fungi, including plants, bacteria and humans ( Grover, 2012 ; Junges et al, 2014 ; Rathore and Gupta, 2015 ). In C. neoformans , chitinases have been reported to be essential for sexual development but not vegetative growth and asexual reproduction ( Baker et al, 2009 ).…”