Fig. 3: T3SS effector contributions to Shigella IEC colonization across different cell geometries and intestinal segments.
a, Heat map showing log2FC for T3SS effector gene mutants, as informed by the TraDIS screen in Fig. 2f; **FDR < 0.1; *FDR < 0.2. b–e, Representative confocal fluorescent microscopy images of a BO enteroid (b), AO enteroid (c), BO colonoid (d) and AO colonoid (e) infected with wild-type Shigella harboring cytosolic reporter puhpT-GFP at MOI 40. Experiments were repeated at least twice with imaging of at least ten enteroids or colonoids per experiment. f–i, Shigella CFU counts (left) and percentage of inoculum (right) upon coinfection with wild-type and ΔmxiD (noninvasive) strains for 6 h at MOI 40 of BO enteroids (f, same plots as in c), AO enteroids (g), BO colonoids (h) and AO colonoids (i); n = 7 biological replicates pooled from two to three independent experiments. Data shown as mean ± s.d. Significance determined by two-sided Mann–Whitney U-test; ***P < 0.001. j–m, BO and AO enteroids (j) and BO and AO colonoids (l) were infected with a barcoded consortium comprising two wild-type (tagA, tagB), two ΔmxiD (tagC, tagD) and two mutant (tagE, tagF) strains at MOI 40 for 6 h. Shown is the quantification of relative tag abundance, normalized against the corresponding input (Extended Data Fig. 5a,b). Data represent two independently generated consortia for each infection. Significance determined by two-sided paired t-test for each specific mutant between normalized output and normalized input abundances (Methods). *P < 0.05, **P < 0.01, ***P < 0.001. k–m, Bargraph showing the colonization index for each mutant in BO (dark gray) and AO (light gray) enteroid infections (k, derived from data in j and calculated as 1 – (WT/mutant); n as in j) and BO (dark gray) and AO (light gray) colonoid infections (m, derived from data in l; n as in l). Data shown as mean ± s.d. Wild-type shown as 0.
