Figure 4.

Analysis of TcsL secretion in a holin mutant of P. sordellii. (a) Levels of TcsL in the TY culture supernatant of the isogenic strains. At T = 22 h, culture supernatants were collected and assayed using a TcdB-specific ELISA detection kit (tgcBiomics). The concentrations of TcsL detected relative to a TcdB standard protein curve are shown, having been standardised to total protein concentration in the supernatant as determined by a BCA assay. n ≥ 4, error bars indicate SEM. Statistical analysis was conducted using a Mann–Whitney U Test * p < 0.05, ** p < 0.01. (b) Growth curve of the TY cultures over 22 h. After dilution in TY broth, an OD₆₀₀ reading was taken at T = 1, followed by every 2 h until T = 11. A final reading was taken at T = 22. n ≥ 3, error bars indicate SEM. (c) Relative levels of tcsL transcription among the isogenic panel of mutants. At T = 10 h, cells were collected from TY cultures; RNA was extracted and cDNA synthesized. Concentrations of tcsL and rpoA (housekeeping control) transcript were determined from the cDNA using ddPCR. Displayed is the relative expression of tcsL as a percentage of rpoA (housekeeping gene) expression for each mutant. n = 4, error bars indicate SEM. Statistical analysis was conducted using a Mann–Whitney U Test.