Molecular evolution and transcriptional profile of GH3 and GH20 β-N-acetylglucosaminidases in the entomopathogenic fungus Metarhizium anisopliae

Abstract

Cell walls are involved in manifold aspects of fungi maintenance. For several fungi, chitin synthesis, degradation and recycling are essential processes required for cell wall biogenesis; notably, the activity of β-N-acetylglucosaminidases (NAGases) must be present for chitin utilization. For entomopathogenic fungi, such as Metarhizium anisopliae, chitin degradation is also used to breach the host cuticle during infection. In view of the putative role of NAGases as virulence factors, this study explored the transcriptional profile and evolution of putative GH20 NAGases (MaNAG1 and MaNAG2) and GH3 NAGases (MaNAG3 and MaNAG4) identified in M. anisopliae. While MaNAG2 orthologs are conserved in several ascomycetes, MaNAG1 clusters only with Aspergilllus sp. and entomopathogenic fungal species. By contrast, MaNAG3 and MaNAG4 were phylogenetically related with bacterial GH3 NAGases. The transcriptional profiles of M. anisopliae NAGase genes were evaluated in seven culture conditions showing no common regulatory patterns, suggesting that these enzymes may have specific roles during the Metarhizium life cycle. Moreover, the expression of MaNAG3 and MaNAG4 regulated by chitinous substrates is the first evidence of the involvement of putative GH3 NAGases in physiological cell processes in entomopathogens, indicating their potential influence on cell differentiation during the M. anisopliae life cycle.

Figure 1. Modular domain structure from M. anisopliae NAGase genes. NAGase genes exhibit specific conserved domains with different compositions. Coding exonic sequences are depicted as boxes (color codes are indicated) and introns as thin lines. Domains were identified using Conserved Database Domain (at NCBI), dbCAN and InterProScan. Signal peptide sequences were predicted using SignalP 4.1. Blank protein regions indicate the absence of characterized domains.

Figure 2. Phylogenetic relationships among GH3 NAGases from filamentous fungi, bacteria and zygomycetes. Putative and characterized fungal GH3 β-glucosidases were included as an outgroup. The phylogenies were obtained using MrBayes 3.2.5 (left side) and PhyML 3.1 (right side). ★: NAGase from the zygomyceteRhizomucor miehei. ●: Nodes with support values below 0.8 were collapsed into polytomies.

Figure 3. Phylogenetic relationships among GH20 NAGases from filamentous fungi. The phylogenies were obtained using MrBayes 3.2.5 (left side) and PhyML 3.1 (right side). ▲: Trichoderma NAG1. ★: Trichoderma NAG2.

Figure 4. Relative expression of GH20 and GH3 NAGase genes in M. anisopliae, considering MCc as the reference condition. Transcriptional profiles of GH20 NAGase genes (MaNAG1 and MaNAG2) and GH3 NAGase genes (MaNAG3 and MaNAG4) in seven different conditions (mycelium growth on different carbon source media, autolysis, and different cell types), using tef1α as a reference gene and applying the 2^-△△Ct method. A) nag1; B) nag2; C) nag3; D) nag4. Standard error bars are indicated. Different letters indicate statistically significant differences (p < 0.05) among studied conditions.