Figure 4. The decreased virulence of WT V. cholerae is recapitulated in a ΔhaprR mutant through tryptophan supplementation or inhibition of tryptophan uptake.
(A) Polar metabolomics of LB broth as well as the intracellular and extracellular milieus of WT V. cholerae and the ΔhapR mutant cultured for 8 h in LB broth. The mean of log2 of the normalized intensity (NI) of biological triplicates is shown. A one-way ANOVA with Fisher’s least significant difference (LSD) post hoc analysis was used to assess significance. Only significantly different metabolites with between WT and ΔhapR FDR < 0.05 are shown.
(B) Fractional survival of OreR flies infected with WT V. cholerae or a ΔhapR mutant and supplemented with the indicated concentrations of tryptophan. Log-rank analysis was used to calculate statistical significance.
(C) qRT-PCR analysis of 20E and IMD pathway-regulated genes in the intestines of uninfected OreR flies fed LB broth supplemented with the indicated concentrations of tryptophan. The mean of biological triplicates is shown. Error bars represent the standard deviation. A Student’s t test was used to calculate significance.
(D) Fractional survival of OreR flies infected with WT V. cholerae or a ΔhapR mutant alone or with deletions of the indicated genes hypothesized to be involved in tryptophan transport.
(E) High-performance liquid chromatography (HPLC) analysis of tryptophan concentrations in LB broth or the supernatants of the indicated V. cholerae strains harvested after 8 h of culture in LB broth. The mean of biological triplicates is shown. Error bars represent the standard deviation. A one-way ANOVA with Tukey’s multiple comparisons test was used to calculate statistical significance.
Floating symbols indicate significance with respect to the ΔhapR strain. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; ns, not significant. See also Figure S6.