Figure 4.

HxuCBA, SapA and HbpA participate in heme inter-bacterial donation. (a) Heme-starved NTHi 3655luxABCDE was used as a heme-recipient strain. Heme donation allowed recipient´s growth, measured as a means of luminescence. NTHi 3655luxABCDE co-culture with Hi RdKW20 WT rendered significant recipient growth, comparable to that of NTHi 3655luxABCDE grown in sBHI; in contrast, NTHi 3655luxABCDE co-culture with ΔhxuCBA, ΔsapA or ΔhbpA, did not promote recipient strain growth at the same extent (see growth time points 4, 5 and 6 h). HitABC does not contribute to surface-bound heme inter-bacterial donation. NTHi 3655luxABCDE co-culture with both Hi RdKW20 WT and ∆hitBC rendered significant recipient growth. White symbols, controls showing recipient strain growth in sBHI (square) or BHI-NAD (circle). Black symbols, recipient strain growth when co-cultured with WT (square) or mutant (circle) strains. *Indicates significant differences when comparing luminescence upon recipient co-culture with WT (square) or mutant (circle) strains. Data are shown as luminescence arbitrary units (AU) and represent mean ± SEM values. Statistical comparisons of the means were performed with two-way ANOVA (Bonferroni multiple comparisons test). (b) Semi-quantitative measure of heme binding on the surface of Hi RdKW20 WT and mutant strains. Normalized bacterial suspensions were 2-fold serially diluted (OD₆₀₀ = 0.1 and 0.05) and spotted on a PVDF filter. ECL was used for heme detection. Blots are representative images of at least three independent experiments.