Figure 3.

(a) Growth of S.Tm upon addition of a PIC (N = 3, n = 2). (b) Effect of the individual protease inhibitors of the PIC (excluding Bestatin hydrochloride, which was not available due to shortness of supplies, AEBSF: 346.67 µM, E64: 4.67 µM, Aprotinin: 0.27 µM, Leupeptin: 6.67 µM, Pepstatin A: 5 µM), on the growth of S.Tm with 5 µM of ferritin-bound iron (N = 3, n = 1). Growth of (c) WT S.Tm (N = 15, n = 5) and (d) the ΔentC strain upon addition of AEBSF to the medium (N = 12, n = 4). Growth of (e) the WT strain (N = 15, n = 4) and (f) the ΔentC mutant (N = 12, n = 4) upon addition of ferrous or ferritin bound iron with or without AEBSF (triangles and circles, respectively). (a,b) were conducted in ambient air and (c–f) were conducted in 5% O₂, 5% CO₂ atmosphere. Growth data were logarithmized and then normalized to (a,c,d) the iron free control, (b) supplementation with 5 µM of ferritin-bound iron, or (e,f) their corresponding controls: no supplement (circles) or AEBSF (triangles), to account for the iron-independent growth inhibition of AEBSF. Graphs are depicted as mean ± SD. For statistical analysis, either Student’s t-test (c,d) or one-way ANOVA (e,f) corrected for multiple comparisons, and the Holm-Sidak post hoc test was also used (*: p < 0.05; ****: p < 0.0001, numbers indicate a non-significant p-value > 0.05). N: number of biological replicates, n: number of experiments conducted, PIC: protease inhibitor cocktail, WT: wildtype S.Tm, ΔentC: S.Tm enterobactin/salmochelin knockout mutant strain, AEBSF: 4-(2-Aminoethyl)benzenesulfonyl fluoride.