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

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. ### Competing Interest Statement The authors have declared no competing interest. * GlcNAc : N-acetylglucosamine GbpA : GlcNAc binding protein A LPMO : lytic polysaccharide monooxygenase CFS : cell free supernatant CV : conventionally reared GF : germ free WT : wild type CFU : colony forming unity TSB : tryptic soy broth TSA : tryptic soy agar LB : Luria broth EM : embryo medium MS-222 : tricaine methanesulfonate dpf : days post fertilization EdU : 5-ethynyl-2′-deoxyuridine SDS-PAGE : Sodium dodecyl-sulfate polyacrylamide gel electrophoresis ANOVA : analysis of variance.