Fig. 7: Spatial distribution of toxin gene expression during murine infection.
a–c Single-cell quantification of toxin gene-specific fluorescence (PtcdA::mNG) normalized to the constitutive Pcwp2::LP-mScI3-G228R signal was performed across C. difficile cells localized to the lumen, mucosa, and epithelium of infected mice (* denotes dual reporter strains carrying Pcwp2::LP-mScI3-G228R and PtcdA::mNG). Each gray point represents an individual bacterium. Superplots with colored points denote the median fluorescence value per mouse. Horizontal black lines indicate the mean and standard deviation of medians. Dashed lines mark the negative-control threshold used to define toxin-ON cells on day 2 (blue) and day 14 (red). d–f The % Toxin-ON (green dots in a–c) was determined by the proportion of cells with mNeonGreen signal greater than the highest PtcdA::mNG signal in the ΔtcdR* dual reporter strain. Values that are separated above the clustering of points within the ΔtcdR* datasets are considered toxin-ON. For Day 2 infections, n = 8 mice per strain were analyzed. For mice infected with the WT dual reporter strain, a total of 1219 cells were counted, including 500 luminal, 373 mucosal, and 346 epithelial bacteria. For mice infected with the ΔtcdR dual reporter strain, a total of 831 cells were counted, comprising 325 luminal, 282 mucosal, and 224 epithelial bacteria. For mice infected with the ΔrstA dual reporter strain, a total of 1162 cells were counted, including 555 luminal, 344 mucosal, and 263 epithelial bacteria. For Day 14 infections, n = 4 mice for WT and ΔrstA and n = 3 mice for ΔtcdR were analyzed. For mice infected with the WT dual reporter strain, a total of 409 cells were counted, comprising 189 luminal, 139 mucosal, and 81 epithelial bacteria. For mice infected with the ΔtcdR dual reporter strain, a total of 275 cells were counted, including 150 luminal, 75 mucosal, and 50 epithelial bacteria. For mice infected with the ΔrstA dual reporter strain, a total of 497 cells were counted, comprising 200 luminal, 150 mucosal, and 147 epithelial bacteria. Statistical significance was determined using a one-way ANOVA and Tukey’s test on median values. Only statistically significant comparisons are shown. To see per mouse counts at each location, see Supplementary Fig. 6.
