The T2SS of A. veronii is required for secretion of a pro-proliferative factor that stimulates intestinal epithelial cell proliferation. (a) Schematic of the larval zebrafish intestine, highlighting the proximal 210 μm region in which proliferative epithelial cells were quantified, as marked by incorporation of the nucleotide analog EdU. (b) Representative transverse section of the proximal zebrafish intestine stained to reveal cells that incorporated EdU.(c) Quantification of proximal intestinal epithelial cell proliferation in 8 dpf CV larvae and 8 dpf GF larvae untreated or exposed from 6 dpf to CFS from A. veronii with a functional or deleted T2SS. Boxplot whiskers represent range. Groups with different letter designations are statistically different with a p value of < 0.05 whereas groups with the same letter are not significantly different.

A. veronii gbpA encodes the secreted pro-proliferative factor that stimulates intestinal epithelial cell proliferation. (a0 The major protein constituents of ammonium sulfate fractions of A. veronii CFS separated by SDS-PAGE and visualized with Coomassie Brilliant Blue, with the corresponding proliferation and hemolysis activity of each fraction indicated below. (b) Schematic of the shared domain architecture of A. veronii and V. cholerae GbpA proteins, with the amino acid identity indicated for each of the four protein domains. (c) Quantification of proximal intestinal epithelial cell proliferation in 8 dpf CV larvae and 8 dpf GF larvae untreated or exposed from 6 dpf to CFS from WT or ∆gbpA A. veronii. Boxplot whiskers represent range. Groups with different letter designations are statistically different with a p value of < 0.05 whereas groups with the same letter are not significantly different.

Colonization of the zebrafish intestine by A. veronii and V. cholerae does not require gbpA. (a) Binding of WT and ∆gbpa A. veronii to chitin beads, quantified as the percent of total bacteria, after either 0.5 hr (white bars) or 1 hour (gray bars) of incubation. Bacterial strains were added to chitin beads alone (solid bars) or mixed with the other strain (striped bars). Boxplot whiskers represent range. Groups with different letter designations are statistically different with a p value of < 0.05 whereas groups with the same letter are not significantly different. (b) Competitive index of ∆gbpa versus WT A. veronii recovered from chitin beads. (c) A. veronii CFUs recovered at 6 dpf following inoculation of GF zebrafish with individual strains at 4 dpf. (d) Competitive index of ∆gbpa versus WT A. veronii recovered at 6 dpf following co-inoculation of GF zebrafish with the two strains at 4 dpf. (e) V. cholerae CFUs recovered at 6 dpf following inoculation of GF zebrafish with individual strains at 4 dpf. (f) Competitive index of ∆gbpa versus WT V. cholerae strains recovered at 6 dpf fish following co-inoculation of GF zebrafish with the two strains at 4 dpf. (g) Light sheet micrograph of zebrafish intestine colonized with WT (green) and ∆gbpa (purple) A. veronii in the proximal intestinal region indicated in the schematic. (h) Growth curves measuring OD₆₀₀ for each A. veronii strain grown in TSB. (i) Final OD₆₀₀ measurement for WT and ∆gbpa strains grown on colloidal chitin and GlcNAc.

Secreted GbpA proteins are sufficient to increase intestinal epithelial proliferation in GF zebrafish. (a) CSF from engineered E. coli expressing no recombinant protein or IPTG-inducible GbpA^Av and from the V. cholerae gbpA complementation strain expressing arabinose-inducible GbpA^Vc were incubated with chitin beads, rinsed, and the protein content of the flow through (FT) and chitin bead (C) fractions were separated by SDS-PAGE and visualized with Coomassie Brilliant Blue. (b) Quantification of proximal intestinal epithelial cell proliferation in 8 dpf CV larvae and 8 dpf GF larvae untreated or exposed from 6 dpf to CFS of E. coli expressing full length GbpA^Av, domain 1 of GbpA^Av, or purified full length GbpA^Av protein. (c) Quantification of proximal intestinal epithelial cell proliferation in 8 dpf CV larvae and 8 dpf GF larvae untreated or exposed from 6 dpf to CFS of WT V. cholerae, the gbpA deletion strain, and the gbpA complementation strain expressing arabinose-inducible GbpA^Vc. Boxplot whiskers represent range. Groups with different letter designations are statistically different with a p value of < 0.05 whereas groups with the same letter are not significantly different.