FIG 7.

Effects of P. aeruginosa siderophores on A. fumigatus iron metabolism. (A) A. fumigatus strain AfS77 conidia were grown at 37°C in 15 ml of liquid 2YT medium for 15 h. Subsequently, 10 ml of the culture supernatant was replaced with 10 ml of wild-type or pvdA pvdE mutant PA14 PCF and incubated for 3 h. A Northern blot analysis was performed for genes inducible by iron starvation (hapX, sidA, and mirB) and genes repressed by iron starvation (sreA, cccA, cycA, and acoA). Measurement of rRNA served as a loading control. (B) A. fumigatus strain 10AF conidia were incubated in the presence of RPMI 1640 (striped bar) or RPMI 1640 supplemented with 10 μM pyoverdine (gray striped bar) for 24 h. Supernatants were sterile filtered. As a control, 10 μM pyoverdine (PYOV) solution in RPMI 1640 was prepared (solid gray bar). A CAS assay measuring siderophore content in A. fumigatus supernatants was performed, and the results were normalized to those of an XTT assay prepared in parallel with the CAS assay measuring fungal metabolism. Siderophore production by A. fumigatus was regarded as 100% and compared to all other bars. Other comparisons are indicated by the ends of the brackets above the bars. ***, P ≤ 0.001. Pyoverdine itself as a siderophore was measurable by CAS assay. (C) RPMI 1640 or PA14 PCF with or without increasing amounts of FeCl₃ was subjected to a BCAM assay measuring effects on the metabolism of A. fumigatus strain 10AF forming biofilm. A. fumigatus strain 10AF metabolism was evaluated by XTT assay. Metabolism in the presence of RPMI 1640 alone was regarded as 100% A. fumigatus strain 10AF metabolic activity. The data shown are the mean ± SD from four replicates. **, P ≤ 0.01; ***, P ≤ 0.001 (RPMI 1640 versus RPMI 1640 containing FeCl₃ [left side] or PCF versus PCF containing FeCl₃ [right side]). Other comparisons are indicated by the ends of the brackets above the bars. (D) Agar and A. fumigatus strain 10AF conidia were distributed into 96-well cell culture plates that were loaded with wild-type or mutant PAO1 PCF and incubated at 37°C for 24 h. A. fumigatus strain 10AF metabolism was evaluated by XTT assay. Metabolism in the presence of RPMI medium alone was compared to metabolism in the presence of PCF. Other comparisons are indicated by the ends of the brackets above the bars. ***, P ≤ 0.001.