Early Diverging Insect-Pathogenic Fungi of the Order Entomophthorales Possess Diverse and Unique Subtilisin-Like Serine Proteases

Abstract

Insect-pathogenic fungi use subtilisin-like serine proteases (SLSPs) to degrade chitin-associated proteins in the insect procuticle. Most insect-pathogenic fungi in the order Hypocreales (Ascomycota) are generalist species with a broad host-range, and most species possess a high number of SLSPs. The other major clade of insect-pathogenic fungi is part of the subphylum Entomophthoromycotina (Zoopagomycota, formerly Zygomycota) which consists of high host-specificity insect-pathogenic fungi that naturally only infect a single or very few host species. The extent to which insect-pathogenic fungi in the order Entomophthorales rely on SLSPs is unknown. Here we take advantage of recently available transcriptomic and genomic datasets from four genera within Entomophthoromycotina: the saprobic or opportunistic pathogens Basidiobolus meristosporus, Conidiobolus coronatus, C. thromboides, C. incongruus, and the host-specific insect pathogens Entomophthora muscae and Pandora formicae, specific pathogens of house flies (Muscae domestica) and wood ants (Formica polyctena), respectively. In total 154 SLSP from six fungi in the subphylum Entomophthoromycotina were identified: E. muscae (n = 22), P. formicae (n = 6), B. meristosporus (n = 60), C. thromboides (n = 18), C. coronatus (n = 36), and C. incongruus (n = 12). A unique group of 11 SLSPs was discovered in the genomes of the obligate biotrophic fungi E. muscae, P. formicae and the saprobic human pathogen C. incongruus that loosely resembles bacillopeptidase F-like SLSPs. Phylogenetics and protein domain analysis show this class represents a unique group of SLSPs so far only observed among Bacteria, Oomycetes and early diverging fungi such as Cryptomycota, Microsporidia, and Entomophthoromycotina. This group of SLSPs is missing in the sister fungal lineages of Kickxellomycotina and the fungal phyla Mucoromyocta, Ascomycota and Basidiomycota fungi suggesting interesting gene loss patterns.

Keywords: Subtilase; early-diverging fungi; insect-pathogen; phylogenomics; proteases.

Figure 1

Maximum likelihood phylogeny calculated with RAxML and based on a 2,379 bp alignment of 152 subtilisin-like serine protease codon nucleotide sequences from Entomophthoromycotina that contain the peptidase S8/S53-subtilisin (PF00082) domain. Branches are colored for eachs species as Entomophthora muscae (Blue), Pandora formicae (Purple), Conidiobolus coronatus (Pink), C. thromboides (brown), C. incongruus (orange), and B. meristoporus (Green). For each SLSP, the accession number and protein domains additional to PF00082 are shown. The three identified clusters: Protease K cluster (A), Pyrolysin/osf protease cluster (B), and the new bacillopeptidase-like Entomophthorales cluster (C), are marked in the gray circle surrounding the tree and with gray background for cluster B and C.

Figure 2

Active site and domain co-occurrence variability of the three Tribe-MCL clusters identified among 152 Entomophthoromycotina subtilisin-like serine proteases. The columns DTG, GHGTH, and SGTS represents the closest amino acid sequence for each of the amino acids from the DHS catalytic triad. A. Amino acid alignment of the active site residues for the three identified groups (A-C) of SLSPs within Entomophthoromycotina. Accession codes are color coded as: Orange – C. incongruus, Blue – E. muscae, and Purple – P. formicae. B. Sequence motifs of the active site residues for each group.

Figure 3

Mid-point rooted maximum likelihood phylogeny calculated with RAxML and based on a (479 amino acid) alignment of 413 protein subtilisin-like serine protease sequences, which belonged to group C in the Tribe-MCL analysis (see text for details). Bootstrap values >70 from 1000 iterations are shown for non-terminal deeper nodes.

Figure 4

Schematic phylogeny and classification of the early-diverging fungi and related taxonomic groups principally based on Spatafora et al. (2016). Branch lengths are not proportional to genetic distances.

Figure 5

Venn diagram showing taxonomic distribution of subtilisin-like serine protease clusters of major insect and nematode-pathogenic fungal genera. Black circles correspond to clusters with number of S8A proteases for each cluster, and the placement within the Venn diagram correspond to the taxonomic groups contributing sequences to a specific cluster. The asterisk (*) marks the 11 members of the new bacillo-peptidase like cluster C described within the order Entomophthorales (subphylum: Entomophthoromycotina). Entomophthoromycotina encompasses SLSP’s found in the genera: Basidiobolus, Conidiobolus, Entomophthora, and Pandora. Hypocrealean entities consist of SLSP’s in the MEROPS database from the genera: Cordyceps, Metarhizium, Ophiocordyceps, and Nematode trapping fungi are MEROPS SLSP’s found in the genera: Arthrobotrys and Monacrosporium.

Figure 6

Maximum likelihood phylogeny calculated with RAxML and based on a (276 amino acid) alignment of 500 protein subtilisin-like serine protease sequences. SLPSs within Entomophthoromyctina from group B and C, and 10 representative SLSPs from group A are included together with the 10 most similar SLSPs in the MEROPS database for each group A, B, and C, respectively. To show the relationship between the three Entomophthoromycotina SLSP groups, representative SLSPs from insect-pathogenic and nematode-trapping fungi were included (see text for details). Bootstrap values >70 from 100 iterations are shown for non-terminal nodes. The tree is rooted with the S8B subfamily type Kexin from S. cerevisiae (UniProt: P13134)