Extended Data Fig. 4: Contribution of the AcrABTolC efflux pump system to Shigella colonization of enteroids, and resilience to environmental conditions relevant to the infection assay.
(a) BO enteroids were infected with barcoded consortia comprising two Shigella wt (tagA, tagB), two ΔmxiD (tagC, tagD) and two mutant (tagE, tagF) strains at MOI 40 for 6 h in Organoid growth medium (OGM). Mut refers to mutant specified on x-axis. Left panel, percentage of each strain in the input population. Middle panel, relative tag abundance in the intracellular population, normalized against the corresponding input (see Left panel). Right panel, Colonization Index for the indicated mutants (derived from data in the middle panel and calculated as 1-(wt/mut)). Data in right panel shown as Mean +/- SD. Data for two independently generated consortia per infection. Grey shading indicates detection limit. Note that relative abundances of ΔacrA, ΔacrB, ΔacrAB, and ΔtolC mutants fall under the detection limit and are ~1000-fold lower than the non-invasive ΔmxiD strain. (b–f) Barcoded competition assay with a consortium comprising two wt (tagA, tagB), two ΔmxiD (tagC, tagD) and two ΔacrAB or ΔtolC mutant (tagF, tagG) strains grown in different media (LB broth, Organoid growth medium (OGM) or DMEM-F12/0.25%BSA) (b), or LB broth with increasing concentration of Na-deoxycholate (c), Bile salts (d), gentamicin (e), or LL-37 (f). Shown is the relative tag abundance under each condition. Relative tag abundances normalized to the consortia grown in LB. (g) BO enteroids infected with barcoded consortia as in a, but in DMEM-F12/0.25%BSA medium. Data for three independently generated consortia per infection. Significance determined by two-sided paired t-test between normalized output and input abundances (see Methods). **p < 0.01. Data in right panel shown as Mean +/- SD. Interpretation: ΔacrA, ΔacrB, ΔacrAB and ΔtolC mutants were not only sensitive to bile salts/deoxycholate, but also to the OGM medium used for infections (see b-d). Therefore, the low Log2FC value observed for these mutants in the TraDIS screen does not exclusively reflect decreased enteroid colonization, but also generalized sensitivity to multiple infection condition stressors (a–f). Nevertheless, an epithelial colonization defect was validated in the absence of OGM and Na-deoxycholate. Here, the Colonization Indexes for ΔacrA, ΔacrB, ΔacrAB mutants were ~10-fold lower and for ΔtolC mutant ~20-30-fold lower than the wt (g; compare to a).
