Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis
Fig 5
C. albicans secreted factors inhibit P. aeruginosa pyochelin and pyoverdine gene expression and pyoverdine production in vitro.
A) Pyochelin and pyoverdine gene expression by RT qPCR of P. aeruginosa PAO1 grown in vitro to mid-log phase in iron-limited GGP media ± live C. albicans (grown in YPD media) or YPD media alone control. P. aeruginosa and C. albicans co-cultures were mixed in a 1:1 ratio and co-incubated at 37°C for 10 minutes before RNA isolation. B) Total iron content of media (YPD, LB, GGP, PBS), supernatant of C. albicans cultures grown in YPD, and C. albicans supernatant protein preparations (pre and post iron depletion). Total iron content (Fe2+and Fe3+) was determined by ferrozine assay [48]. All data shown are means ± SEM. Assays were performed in triplicate. Statistical analysis was performed by unpaired Student’s t-test. C, D, E) Pyochelin and pyoverdine gene expression by RT qPCR of P. aeruginosa PAO1 grown in vitro to mid-log phase in iron-limited GGP media with or without the following: C) Heat-killed (HK) C. albicans cells (suspended in PBS) or PBS alone control. D) C. albicans supernatant (stationary cultures grown in YPD at 30°C), C. albicans supernatant protein (supernatants of C. albicans stationary cultures grown in YPD; precipitated with ammonium sulfate, desalted, and dialyzed against PBS precipitation; final concentration of 100 μg/mL), and YPD subjected to the same protein purification process as C. albicans supernatant proteins. E) C. albicans supernatant proteins ± physical (boiled for 60 minutes) or chemical (treated with protease from Streptomyces griseus for 60 minutes) denaturation. HK C. albicans cells, PBS, and C. albicans supernatant were mixed in a 1:1 ratio with live P. aeruginosa culture. C. albicans supernatant protein was added to a final concentration of 100 ug/mL. All co-cultures were incubated at 37°C for 10 minutes before RNA isolation. All data shown for pyoverdine and pyochelin RT qPCR assay (Fig 5A, 5C, 5D and 5E) are means ± SEM. Assays were performed in triplicate. Statistical analysis was performed by unpaired Student’s t-test. F) Dose dependent effect of C. albicans supernatant protein on pyoverdine production (as determined by measuring fluorescence at 400±10/460±40 nm excitation/emission and normalizing to cell density measured at 600 nm) by P. aeruginosa PAO1 grown in GGP media at 37°C over 24 hours. G, H) Representative picture of (G) and pyoverdine levels of (H) a wild-type PAO1 stationary culture (after 24 hours of growth in GGP media) compared to cultures of pyochelin deletional mutant PAO1ΔpchBA, pyoverdine deletional mutant PAO1ΔpvdS, pyochelin/pyoverdine double mutant PAO1ΔpchBA ΔpvdS, PAO1 ± C. albicans SC5314 supernatant protein (final concentration 100 ug/mL), PAO1 ± C. albicans SC5314 supernatant protein boiled, and PAO1 ± C. albicans SC5314 supernatant protein treated with Streptomyces griseus protease. Bars shown are the means ± SEM. Assays were performed in triplicate. Statistical analysis performed by unpaired Student’s t-test, * p< 0.05; ** p<0.01, *** p<0.0001; ns, not significant.