A Novel Extracellular Metallopeptidase Domain Shared by Animal Host-Associated Mutualistic and Pathogenic Microbes

Abstract

The mucosal microbiota is recognised as an important factor for our health, with many disease states linked to imbalances in the normal community structure. Hence, there is considerable interest in identifying the molecular basis of human-microbe interactions. In this work we investigated the capacity of microbes to thrive on mucosal surfaces, either as mutualists, commensals or pathogens, using comparative genomics to identify co-occurring molecular traits. We identified a novel domain we named M60-like/PF13402 (new Pfam entry PF13402), which was detected mainly among proteins from animal host mucosa-associated prokaryotic and eukaryotic microbes ranging from mutualists to pathogens. Lateral gene transfers between distantly related microbes explained their shared M60-like/PF13402 domain. The novel domain is characterised by a zinc-metallopeptidase-like motif and is distantly related to known viral enhancin zinc-metallopeptidases. Signal peptides and/or cell surface anchoring features were detected in most microbial M60-like/PF13402 domain-containing proteins, indicating that these proteins target an extracellular substrate. A significant subset of these putative peptidases was further characterised by the presence of associated domains belonging to carbohydrate-binding module family 5/12, 32 and 51 and other glycan-binding domains, suggesting that these novel proteases are targeted to complex glycoproteins such as mucins. An in vitro mucinase assay demonstrated degradation of mammalian mucins by a recombinant form of an M60-like/PF13402-containing protein from the gut mutualist Bacteroides thetaiotaomicron. This study reveals that M60-like domains are peptidases targeting host glycoproteins. These peptidases likely play an important role in successful colonisation of both vertebrate mucosal surfaces and the invertebrate digestive tract by both mutualistic and pathogenic microbes. Moreover, 141 entries across various peptidase families described in the MEROPS database were also identified with carbohydrate-binding modules defining a new functional context for these glycan-binding domains and providing opportunities to engineer proteases targeting specific glycoproteins for both biomedical and industrial applications.

Recognition of protein-linked glycans as a determinant of peptidase activity

“…S2). These results were consistent with the reported activity of BT4244 and IMPa on glycoprotein substrates (11,12), while revealing comparable properties for ZmpB_m. In all cases, EDTA treatment of the enzymes abolished activity, supporting their assignment as metallopeptidases, as did mutation of the putative catalytic glutamate in the gluzincin motif ( Fig.…”

“…The amino acid sequence signature upon which this family is built includes the tryptophan and asparagine residues that are present in the core GalNAc binding subsite of BT4244, ZmpB, and IMPa. This finding suggests the potential for most, if not all, of the family members to recognize O-glycans and generally supports the proposal of Nakjang et al that this is a glycoprotein-specific family of peptidases (11). Indeed, this family, which is better described as a superfamily, includes three MEROPS families (M60, M88, and M98) and likely contains several additional unclassified families.…”

“…comprise glycoprotein-specific peptidases (11). Although the Pfam classification of these three peptidases indicates an evolutionary relationship between them, BT4244, IMPa, and SslE are classified into the separate MEROPS families M60, M88, and M98, respectively.…”

Enterotoxigenic Escherichia coli Secretes a Highly Conserved Mucin-Degrading Metalloprotease To Effectively Engage Intestinal Epithelial Cells

“…AcfD has antivibriocidal activity (33) and is required for efficient intestinal colonization of Vibrio cholerae (34). Given the presence of an enhancin-like protease domain, it has been suggested that AcfD is a mucinase (40). To our knowledge, this has not been tested experimentally.…”

“…Interestingly, enhancin, the prototype molecule of the family of proteases most closely related to YghJ, was isolated from an insect virus and targets insect intestinal mucins (35,39). It bears a canonical HEXXH metalloprotease motif within a domain (M60-like pfam13402) that is strongly associated with pathogens and commensal organisms that colonize mucosal surfaces (40).…”