Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms

Abstract

Pseudomonas aeruginosa produces several phenazines including the recently described 5-methyl-phenazine-1-carboxylic acid (5MPCA), which exhibits a novel antibiotic activity towards pathogenic fungi such as Candida albicans. Here we characterize the unique antifungal mechanisms of 5MPCA using its analogue phenazine methosulphate (PMS). Like 5MPCA, PMS induced fungal red pigmentation and killing. Mass spectrometry analyses demonstrated that PMS can be covalently modified by amino acids, a process that yields red derivatives. Furthermore, soluble proteins from C. albicans grown with either PMS or P. aeruginosa were also red and demonstrated absorbance and fluorescence spectra similar to that of PMS covalently linked to either amino acids or proteins in vitro, suggesting that 5MPCA modification by protein amine groups occurs in vivo. The red-pigmented C. albicans soluble proteins were reduced by NADH and spontaneously oxidized by oxygen, a reaction that likely generates reactive oxygen species (ROS). Additional evidence indicated that ROS generation precedes 5MPCA-induced fungal death. Reducing conditions greatly enhanced PMS uptake by C. albicans and killing. Since 5MPCA was more toxic than other phenazines that are not modified, such as pyocyanin, we propose that the covalent binding of 5MPCA promotes its accumulation in target cells and contributes to its antifungal activity in mixed-species biofilms.

Fig. 1

Phenazines and phenazimiums. A. Biosynthetic pathway for P. aeruginosa PYO and its immediate precursors. B. Chemical structure of PMS, a 5MPCA analog (only the 5-methyl-phenazinium ion is shown).

Fig. 2

PMS and 5MPCA effects on C. albicans. A. Plate co-cultures of C. albicans lawns inoculated with P. aeruginosa PA14 (WT) or without P. aeruginosa (control) after 24 and 48 hours of incubation. B. C. albicans colonies grown at 30°C on YNBA medium with or without (control) 1 mM PMS. The PMS-supplemented medium remained yellow throughout the experiment. C. C. albicans viability was determined by methylene blue staining of samples collected at the indicated time points after growth on either control or PMS medium (n=2). D. C. albicans grown for 72 hours on 1mM PMS-containing medium was suspended in saline solution and then photographed after the addition of few crystals of Na₂S₂O₄, 10 μl of 30% H₂O₂ or without treatment. Data are representative of three independent experiments.

Fig. 3

Methylphenazinium modification by amino acids and cellular proteins. A. LC-MS/ES ionization analysis of the in vitro-produced red products from PMS reaction with amino acids. (A-insets) Proposed chemical structures of the corresponding (‡) adduct of PMS-arginine or PMS-lysine. B. Soluble proteins (SP) extracted from C. albicans cultured on medium with or without (control) 1 mM PMS and with P. aeruginosa PA14 (WT), phzS::TnM (phzS⁻) phzM::TnM (phzM⁻) for 96 and 48 hours, respectively. C. Absorption spectra of the in vitro-produced red compound formed by reacting PMS with arginine (PMS-arginine), compared with representative absorption spectra of the SP extracted from C. albicans co-cultured with P. aeruginosa WT (Ca-Pa WT), phzS::TnM (Ca-Pa phzS⁻) or phzM::TnM (Ca-Pa phzM⁻). To minimize artifacts caused by turbidity, these latter spectra were obtained from difference of the reduced (by adding 80 mM of Na₂S₂O₄) minus the oxidized spectra of each extract. D. Redox changes of absorption spectra of the red SP extracted from C. albicans cocultured with P. aeruginosa WT in its oxidized form (Ca-Pa WT) and after reduction with 2 mM NADH or 80 mM Na₂S₂O₄, and followed by reoxidation with 280 mM H₂O₂.

Fig. 4

Oxidative stress and cell death in C. albicans. A. Effect of PMS on ROS generation in C. albicans cells determined by DCFH-DA staining. Cells were grown on YNBA with or without (control) 1 mM PMS, and with 10 mM H₂O₂ at 30°C. Fungal cells were harvested after 18 hours growth, washed and incubated with DCFH-DA for 30 minutes. Green fluorescence was evaluated by flow cytometry. B. C. albicans CAT1/CAT1 (WT CU2) or cat1/cat1 were grown on YNBA medium alone (control) and with PMS for 72 hours. Fungal survival determined by MB staining (n=3). Data are representative of at least two independent experiments ***P<0.01.

Fig. 5

Intracellular fluorescence due to PMS uptake, modification and redox state. A. Microscopic view of epifluorescence (rhodamine) and DIC images of C. albicans grown for 72 hours in the presence of 1 mM PMS. B. Fluorescence emission spectra of PMS-arginine derivative. The red product obtained from the chemical reaction performed at pH 10.0 (see Supporting Information section for more details) was scanned before and after addition 80 mM Na₂S₂O₄ using a SpectraMax M2 spectrophotometer. Excitation was at 488 nm. C. Flow cytometry analysis of C. albicans grown for 24 hours on YNBA medium (gray histogram) or medium containing 1 mM PMS (open histogram). PMS-cultured cells exhibited either intermediate (I) or high (H) fluorescence in the phycoerythrin (PE) channel. D and E. Flow cytometry analysis of C. albicans pre-grown on YNBA without (control) or with 1mM PMS for 24 hours and then treated: D. Cells assessed after treatment with 100 mM of Na₂S₂O₄. E. C. albicans heat killed at 80°C for 30 minutes. Data are representative of two independent experiments. F. Viability analysis of C. albicans grown for 24 hours on YNBA medium alone (control) or with 1 mM PMS. Colony-forming units (CFUs) were determined for ~5×10⁴ sorted cells from C. albicans populations exhibiting either (I) or (H) fluorescence detected by FACS. ***P<0.01 (n=3).

Fig. 6

Reduction of PMS is required for its uptake and modification by C. albicans suspensions. C. albicans was grown on YNBA for 24 hours, and cells were then suspended in saline alone (control) or saline solution plus 100 mM ascorbic acid (AA), PMS (1mM) or PMS and AA (PMS+AA). A. Cells were analyzed by flow cytometry 4 hours post challenge to determine the levels of intracellular fluorescence. Mean fluorescence intensity (MFI) of each treatment is shown at the top of the FACS histogram. Data are representative of three independent experiments. B. C. albicans survival for each treatment as determined by CFU counts (n=3).

Fig. 7

Proposed model for the mechanisms mediating 5MPCA antifungal properties, based on observations with the structurally similar methylphenazinium, PMS. In mixed species C. albicans–P. aeruginosa colony biofilms, 5MPCA is secreted by P. aeruginosa. This methylphenazinium is either released in its reduced form, or reduced extracellularly by bacterial or fungal factors within the metabolically active biofilm. Reduced 5MPCA enters to C. albicans cells, where it can be oxidized (dotted arrow) to generate toxic by-products such as superoxide and H₂O₂ (ROS). In addition, 5MPCA reacts with cellular amines within macromolecules, such as proteins leading to the accumulation of red, fluorescent methylphenazinium derivatives within C. albicans. As metabolic activity decreases, oxygen (O₂) tension increases thereby causing more efficient redox cycling of 5MPCA itself (dotted arrow) and protein-bound 5MPCA derivatives. Oxidation of colorless, reduced 5MPCA and its derivatives (outline text) by O₂ causes the generation and accumulation of toxic levels of ROS, resulting in fungal cell death. Metabolic reducing equivalents, such as NADH, can re-reduce 5MPCA and 5MPCA-derivatives (filled text), thus closing the redox cycle. As the metabolism of biofilm cells slows or stops, the proportion of the 5MPCA derivatives in the oxidized state increases, conferring the characteristic red color and high level fluorescence that is apparent in fungal cells from P. aeruginosa-C. albicans co-cultures.