https://orcid.org/0000-0001-6004-9955
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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 these 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 propose that intestinal epithelial tissues 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.
Acknowledgments
We thank Joerg Graf for the generous gift of A. veronii strains, Ron Taylor for the generous gift of V. cholerae strains, JT Neal for the micrograph of EdU-labelled intestine, Erika Mittge for assistance with gnotobiotic zebrafish experiments, W. Zac Stephens for assistance with A. veronii genetics, the UO Histology Facility for tissue sectioning and Rose Sockol and the UO Zebrafish Facility for maintenance of zebrafish lines.
Disclosure statement
AVB and KG are patent holders for the use of GbpA, patent number 9,044,434, issued 06/02/2015.
Author contributions
AVB: conceptualization, methodology, investigation, original draft preparation; SV: visualization, original draft preparation; TJS: investigation (chitin-binding assay), manuscript review and editing; SLL: investigation (light sheet microscopy), visualization; KG: conceptualization, funding acquisition, supervision, original draft preparation, review and editing.
Data availability statement
The authors confirm that the data supporting the findings of this study are available within the article and the supplementary materials.
Abbreviations
| 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 |
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19490976.2023.2183686