Secreted Aeromonas GlcNAc binding protein GbpA stimulates epithelial cell proliferation in the zebrafish intestine

Abstract

In response to microbiota colonization, the intestinal epithelia of many animals exhibit increased rates of cell proliferation. We used gnotobiotic larval zebrafish to identify a secreted factor from the mutualist Aeromonas veronii that is sufficient to promote intestinal epithelial cell proliferation. This secreted A. veronii protein is a homologue of the Vibrio cholerae GlcNAc binding protein GbpA, which was identified as a chitin-binding colonization factor in mice. GbpA was subsequently shown to be a lytic polysaccharide monooxygenase (LPMO) that can degrade recalcitrant chitin. Our phenotypic characterization of gbpA deficient A. veronii found no alterations in the mutant cells’ biogeography in the zebrafish intestine and only a modest competitive disadvantage in chitin-binding and colonization fitness when competed against the wild type strain. These results argue against the model of GbpA being a secreted adhesin that binds simultaneously to bacterial cells and GlcNAc, and instead suggests that GbpA is part of a bacterial GlcNAc utilization program. We show that the host proliferative response to GbpA occurs in the absence of bacteria upon exposure of germ-free zebrafish to preparations of native GbpA secreted from either A. veronii or V. cholerae or recombinant A. veronii GbpA.Furthermore, domain 1 of A. veronii GbpA, containing the predicted LPMO activity, is sufficient to stimulate intestinal epithelial proliferation. We conjecture that intestinal epithelia upregulate their rates of renewal in response to secreted bacterial GbpA proteins as an adaptive strategy for coexisting with bacteria that can degrade glycan constituents of the protective intestinal lining.

A mucin-regulated adhesin determines the intestinal biogeography and inflammatory character of a bacterial symbiont

“…3E ). GbpA is a copper-dependent lytic polysaccharide monooxygenases (LPMO) first identified as a GlcNAc-binding protein from Vibrio cholerae that recognizes GlcNAc moieties on mucin and has since been shown to degrade GlcNAc-containing polymers such as chitin (Bhowmick et al, 2008; Loose et al, 2014) and stimulate intestinal epithelial proliferation in the gnotobiotic zebrafish intestine (Banse et al, 2022).…”

“…Similarly, in Pseudomonas aeruginosa mucin glycans act through the sensor kinase RetS to inhibit GacS and Type-6 secretion system (T6SS) activity and toxin secretion (Wang et al, 2020). RNA-seq analysis of WT Aer01 and the MB4 isolate exposed to GlcNAc in culture indicate MbkS activity modulates the transcription of mbpA , motility-associated genes (ie, fliD and fliE ), and several mucin-degrading enzyme genes ( stcE and gbpA ), the latter of which induces proliferative responses in the zebrafish intestinal epithelium (Banse et al, 2022). In the gut, the motile MB4 isolate resists formation of large aggregates characteristic of its WT counterpart and elicits more intestinal inflammation.…”

“…3E). GbpA is a copper-dependent lytic polysaccharide monooxygenases (LPMO) first identified as a GlcNAc-binding protein from Vibrio cholerae that recognizes GlcNAc moieties on mucin and has since been shown to degrade GlcNAc-containing polymers such as chitin (Bhowmick et al, 2008;Loose et al, 2014) and stimulate intestinal epithelial proliferation in the gnotobiotic zebrafish intestine (Banse et al, 2022). Like gbpA from Vibrio, Aer01 gbpA contains a Cyclic di-GMP-I riboswitch (RF01051) in its putative 5'-UTR, which, in Vibrio increases translation of gbpA when cdG levels are low (Sudarsan et al, 2008).…”