Fig. 3

Distinct regulatory mechanisms and metabolic cues control the expression of PltB and PltC. a Schematic representation of the FAST-INseq genetic screen used to identify S. Typhi genes that influence pltC expression in infected host cells. A large library of random transposon mutants was generated in the S. Typhi pltC:gfp strain and used to infect Henle-407 cells. Sixteen hours post-infection the bacteria were isolated and sorted by FACS into pools exhibiting high and low GFP fluorescence. INseq was then used to identify mutants that stimulate (green dot in plot) or reduce (red dot) pltC expression during infection. b Overview of the results of the FAST-INseq screen. Plot shows the normalized numbers of sequencing reads of transposon insertions within each S. Typhi gene in the high fluorescence vs. low fluorescence pools. c, d Expression levels of pltB:lacZ and pltC:lacZ reporters in infected human cells for wild-type S. Typhi (WT) and the indicated deletion mutant strains. Henle-407 cells were infected with the indicated strains for 24 h, after which the β-galactosidase activity from bacterial lysates was measured and normalized to the numbers of CFU recovered. Values indicate mean values  ± S.D. for six independent determinations taken over two separate experiments. Asterisks denote statistically significant differences relative to the corresponding wild-type sample determined using unpaired two-tailed t-tests. ****p < 0.0001, *p < 0.05, n.s.s. not statistically significant. e Flow cytometry analysis of pltB:gfp and pltC:gfp expression of the indicated S. Typhi strains 24 h post-infection. Histograms show the GFP fluorescence intensities of individual bacteria for the indicated strains. Gates were established to show the percentage of bacteria exhibiting high, low and intermediate (int) fluorescence. The percentage of bacteria with fluorescence intensities within these gates is shown (bottom). Gating strategy provided in Supplementary Fig. 5b. f Overview of the identified factors that differentially affect the expression of pltB and pltC and thus are likely to be important for controlling relative abundance of the two typhoid toxins produced by S. Typhi upon encountering different environments during infection