Fig. 4: Global regulation of pINV-encoded virulence proteins via tRNA modification and differential codon abundance.
a, Schematic of MnmE/G-dependent tRNA modifications. b, BO enteroids were infected with barcoded consortia comprising two wild-type (tagA, tagB), two ΔmxiD (tagC, tagD) and two mutant (tagE, tagF) strains at MOI 40 for 6 h. Left, quantification of relative tag abundance, normalized against the corresponding input (Extended Data Fig. 6d). Data for three independently generated consortia for each infection. Significance determined by two-sided paired t-test between normalized output and normalized input abundances. **P < 0.01. Right, colonization index for the ΔmnmE and ΔmnmG mutants (derived from data in left panel). Data shown as mean ± s.d. c,d, Volcano plots showing differentially abundant proteins in Shigella ΔmnmE versus wild-type (c) or ΔmnmG versus wild-type (d) strains. Each dot represents a protein. Differentially abundant proteins encoded on pINV shown in pink, on chromosome in black and all nonsignificant differentially abundant proteins in gray. Significance determined by two-sided limma analysis with Benjamini–Hochberg multiple comparison correction; log2FC ≥ 0.5; adjusted P value ≤ 0.01. e,f, Plots showing relative protein levels (log2FC as in c, ΔmnmE versus wild-type) versus AGA codon usage ratios for Arg (e) and GGA codon usage ratios for Gly (f) per protein. Differentially abundant proteins encoded on pINV shown in pink, on chromosome in black, and nonsignificant differentially abundant proteins in gray; log2FC ≥ 0.5; adjusted P value ≤ 0.01. Dark and light green dashed lines specify mean AGA/GGA codon usage ratios for all open reading frames on pINV or chromosome, respectively. g–i, ipaA (g), mnmE (h) and mnmG (i) mRNA levels (2–ΔΔCt) upon induction with 0.25 mM IPTG in the indicated strains carrying an inducible ipaA-3xFT plasmid, p-Empty or p-mnmE or p-mnmG, normalized to the corresponding mRNA levels in wild-type Shigella. j, Relative IpaA-3xFT protein levels (0.25 mM IPTG) in wild-type Shigella, ΔmnmE or ΔmnmG strains carrying plasmids as in g–i. Quantification by western blot of serially diluted samples with wt/ipaA-3xFT protein levels set as 1. Data shown as mean ± s.d. of three independent experiments (Extended Data Fig. 7a,b). k,l, BO enteroids were infected with barcoded consortia comprising the indicated strains at MOI 40 for 6 h. Shown is the quantification of relative tag abundance, normalized on the corresponding input (Extended Data Fig. 7c,d). Data for two independently generated consortia per infection. Significance determined by two-sided paired t-test between normalized output and normalized input abundances. Only consortia including the two mutant strains carrying the p-Empty or p-mnmE (k) p-mnmG (l) plasmids were used in the analysis. *P < 0.05, **P < 0.01, ***P < 0.001. m,n, mnmE (m) and mnmG (n) mRNA expression (2–ΔΔCt) in the indicated strains, normalized to mnmE (m) and mnmG (n) expression in wild-type Shigella. For g,h,i,m and n, n = 3 biological replicates. Data shown as mean ± s.d. Panel a partially created using BioRender.com.
