AIEC LF82 colonizes the larval zebrafish intestine better than MG1655. (A) AIEC-loaded paramecia sampled from 0 to 6 hours post incubation, and CFU/paramecia was calculated. AIEC half-life (τ) in paramecia is 2.1 hours. Data are means ± SEM, n = 3. (B) Bacterial colonies from tissue homogenates grown on CHROMagar O157. The zebrafish microbiota (white colonies) can be distinguished from AIEC LF82 (dark blue colonies) and E. coli MG1655 (mauve colonies). (C) Quantification of LF82 and MG1655 CFUs/fish. Fish with CFU below the detection limit (10 CFU/fish, dashed line) were annotated as 1 CFU. Data are from individual fish (n = 14) and means ± SEM. (D) Colonized larvae (%) are the percentage of fish with a burden above the detection limit; n = 14. Non-linear regression, first-order decay, ROUT outlier test with Q = 0.2%, paired t-test and Wilcoxon test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001, ns, not significant. (E) Images of larvae colonized with E. coli (red), (Ei) whole larva at 10× magnification with intestinal segments (foregut (F), midgut (M), hindgut (H)) marked. (Eii–vii) Sagittal views of the midgut of larvae colonized with MG1655 (Eii–iv) and LF82 (Ev–vii) at 2, 24, and 30 hpi. The dotted white line outlines the intestinal epithelium and separates it from the lumen, indicated by *, and the blood vessel below the basement membrane (V). a to p marks anterior to posterior orientation; Scale bars = 100 um, E. coli (red), phalloidin (cyan, cell outline), nuclei (4′,6-diamidino-2-phenylindole, white), images are representative of n = 3.

Larval zebrafish treated with 0.5% DSS have decreased survival and intestinal growth rates. (A) Schematic outlining timeline of DSS administration (red, 3–6 dpf) and survival experiments (blue, 1–7 days post exposure). (B) Survival of larvae administered 0.5% DSS (black circles) relative to untreated (UT) controls (empty circles). Data were analyzed using a Kaplan-Meier plot and Mantel-Cox test; ****, P ≤ 0.0001, n = 20. (C) Quantification of swim bladder defects in UT or DSS-treated larvae. Group differences were analyzed using Mantel-Cox test; ****, P ≤ 0.0001, n = 20. (D) Gut-to-body length ratio was analyzed by linear regression; ns = not statistically significant. Data are means ± SEM from n = 20. (E) Representative images of untreated (Ei) and DSS-treated (Eii) larvae at 6 dpf (3 days post DSS exposure), with the swim bladder (teal) and the intestine (red) outlined. Scale bar = 0.3 mm.

DSS causes intestinal epithelial damage and inflammation consistent with enterocolitis. (A) Representative hematoxylin- and eosin-stained longitudinal sections (n = 4) of the anterior, mid, and posterior intestine from untreated (Ai–iii) and DSS-treated (Aiv–vi) larvae at 6 dpf; scale bars = 50 µm. (B) Representative confocal images of live UT (i, iii) and DSS-treated (ii, iv) Tg(mpo::egfp) larvae at 6 (i-ii) and 7 (iii-iv) dpf; neutrophils (green); larvae were imaged for 18 hours (3–20 hpi). Scale bars = 200 µm. (C) Quantification of neutrophils in the intestine at 6 and 7 dpf (3 and 4 days post DSS treatment); unpaired two-tailed t-test, n ≥ 11. (D) qRT-PCR analyses of cxcl8, il1b, mmp9, and tnfa in DSS-treated larvae relative to untreated controls at 6 dpf and (E) 7 dpf; n = 3. Unpaired two-tailed t-test. Mean ± SEM, *, P ≤ 0.05; **, P ≤ 0.01; ns, not significant.

Pre-existing intestinal inflammation enhances the colonization and invasion of AIEC LF82. (A) Timeline of DSS administration, infection of larvae, and sampling for CFU counts. (B) Quantification of LF82 CFUs per larvae with and without DSS treatment, n ≥17; fish with CFU below the detection limit (10 CFU/fish, dashed line) were annotated as 1 CFU. (C) Colonized larvae (%) are percentage of fish with a burden of AIEC above the detection limit; non-linear regression first-order decay, ROUT outlier test with Q = 0.2%. (D) LF82 (red) in the mid-intestine of UT (Di–iii) and DSS-treated (Div–vi) larvae relative to the basement membrane (blue) from 2 to 48 hpi or 6–8 dpf. The dotted white line outlines the intestinal epithelium and separates it from the lumen, indicated by *, and the blood vessel below the basement membrane (V). Scale bars = 10 µm. (E) Quantification of red fluorescence intensity (AFU) (representing AIEC) in the vasculature (V) at 2, 24, and 48 hpi, n = 6; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not statistically significant.

AIEC LF82 exacerbates intestinal inflammation in DSS-treated larvae. (A–D) H&E-stained longitudinal sections of the mid-intestine of larvae without (A and B) and with (C and D) prior DSS treatment, fed paramecia (para.) alone (A, C) or paramecia containing LF82 (B, D) at 2 (i), 24 (ii), and 48 (iii) hpi, n = 3. Black arrows point to goblet cells. Scale bars = 50 µm. (E) Representative confocal images of Tg(mpo::egfp) larvae fed paramecia only (i, iii) or LF82 (ii, iv) at 6 dpf. Larvae were imaged for 18 hours (3–20 hpi), neutrophils (green) and bacteria (red). Scale bars = 200 µm. (F) Quantification of neutrophils per intestine in UT- and DSS-treated fish fed with paramecia only (black) or para. containing LF82 (red); n ≥10. (G) qRT-PCR analyses of cxcl8, il1b, mmp9, and tnfa in DSS-treated (red) larvae infected with LF82- and DSS-treated larvae fed paramecia (black) relative to UT paramecia controls (onefold) at 6 dpf, n = 7. Unpaired two-tailed t-test. Mean ± SEM. (H) Survival of DSS-treated larvae that were uninfected (black), fed paramecia control (blue), or para. containing AIEC (red). N = 17. Kaplan-Meier and Mantel-Cox test, followed by a Bonferroni correction test. **, P ≤ 0.01; ***, P ≤ 0.001; ns, not statistically significant.

Effects of fimH and ibeA deletion on larval survival, bacterial burden, and bacterial clearance. Survival of larvae infected with (A) LF82 wild-type (WT), LF82ΔfimH, LF82ΔfimH:fimH or (B) LF82, LF82ΔibeA, LF82ΔibeA:ibeA at 2, 24, and 48 hpi. Kaplan-Meier and Mantel-Cox test, followed by a Bonferroni correction test, n = 20. Quantification of bacterial burden and clearance of (C, E) LF82, LF82ΔfimH, LF82ΔfimH:fimH, or (D, F) LF82ΔibeA, and LF82ΔibeA:ibeA in DSS-treated larvae from 2 to 48 hpi. Fish with CFU below the detection limit (10 CFU/fish, dashed line) were annotated as 1 CFU. Significance of difference in burden was analyzed using a Kruskal-Wallis test, n ≥16. Bacterial clearance (percent of fish with a burden of AIEC above the detection limit) was analyzed using a log-rank test. Non-linear regression, first-order decay graph used to model bacterial clearance. *, P ≤ 0.05; **, P ≤ 0.01; ns, not significant; Experiments for the WT and both mutant strains were performed in parallel, and thus, data for the WT strain are duplicated between panels A and B, C and D, and E and F, respectively.

Deletion of ibeA, but not fimH, results in aggregation and retention of AIEC LF82 on the epithelial surface. Representative sections (n = 3) of the mid-intestine of larvae infected with (A) LF82 WT, (B) LF82ΔfimH, (C) LF82ΔfimH:fimH, (D) LF82ΔibeA:ibeA, or (E) LF82ΔibeA:ibeA at (i) 2, (ii) 24, and (iii) 48 hpi. AIEC LF82 (red), laminin (cell surface, cyan), nuclei (DAPI, white). The dotted white line outlines the intestinal epithelium and separates it from the lumen, indicated by *, and the blood vessel below the basement membrane (V). a to p marks anterior to posterior orientation; Scale bars represent 10 µm.

Deletion of fimH and ibeA in AIEC LF82 results in decreased tissue damage and neutrophil recruitment to the intestine compared to LF82. H&E longitudinal sections of the mid-intestine of larvae infected with (A) LF82 WT, (B) LF82ΔfimH, (C) LF82ΔfimH:fimH, (D) LF82ΔibeA, and (E) LF82ΔibeA:ibeA at 2 (i), 24 (ii), and 48 (iii) hpi. Representative images for n = 3. Scale bars = 50 µm. (F) Quantification of neutrophils per intestine for DSS-treated fish infected with abovementioned LF82 strains or paramecia-only control. Kruskal-Wallis test. n ≥11. *, P ≤ 0.05; **, P ≤ 0.001; ****P ≤ 0.0001; ns, not significant.