Figure 1

SpaC pilin subunit is responsible for zebrafish intestinal mucosa damage. (A) One‐month old zebrafish were immersed with either LGG or PB22 at 10⁷ CFUs/mL for 7 or 14 days. (B) Representative intestinal histology images by H&E staining. Scale bar 100 μm. (C) Total histological score measuring the severity of the intestinal injury at 7 and 14 days (Day 7: F_2,6 = 16.545; Day 14: F_2,6 = 13.971; n = 3). Quantitative data was obtained by the indicators including: disorganized microvilli, edema or inflammatory infiltrate in lamina propria, vacuolar degeneration of IECs, cell shedding and necrosis. (D) Serum LPS levels (Day 7: F_2,6 = 5.185; Day 14: F_2,6 = 37.515; n = 3, pool of three zebrafish per sample). (E) Representative electron micrographs of intestinal sections at Day 14. Scale bar 2 μm (up). Scale bar 1 μm (below). Arrows indicate dying or dead intestinal epithelial cells. Triangular arrows indicate microvilli damage. (F) One‐month old zebrafish were immersed with recombinant SpaA, SpaB, or SpaC at concentrations of 10 μg/mL for Day 7 or 14, respectively. (G) Serum LPS levels (Day 7: F_3,24 = 13.562; Day 14: F_3,20 = 2.543; n = 6 or 7, pool of 3 zebrafish per sample). (H) A representative western blot analysis showing Caspase‐3 activation, GSDMEa cleavage, Caspy2 activation and GSDMEb cleavage. Numbers of biologically independent samples are labeled on the violin plots. Horizontal line represents median in the violin plots. Statistics: one‐way ANOVA followed by Duncan's test. Treatments in plots labeled with different letters on top represent statistically significant results (p < 0.05). Caspase, cysteinyl aspartate specific proteinase; CFUs, colony forming units; GSDM, gasdermin; H&E, Hematoxylin and eosin; IECs, intestinal epithelial cells; LGG, Lactobacillus rhamnosus GG; LPS, lipopolysaccharide.