Pseudomonas aeruginosa-Candida albicans interactions: localization and fungal toxicity of a phenazine derivative

Abstract

Phenazines are redox-active small molecules that play significant roles in the interactions between pseudomonads and diverse eukaryotes, including fungi. When Pseudomonas aeruginosa and Candida albicans were cocultured on solid medium, a red pigmentation developed that was dependent on P. aeruginosa phenazine biosynthetic genes. Through a genetic screen in combination with biochemical experiments, it was found that a P. aeruginosa-produced precursor to pyocyanin, proposed to be 5-methyl-phenazinium-1-carboxylate (5MPCA), was necessary for the formation of the red pigmentation. The 5MPCA-derived pigment was found to accumulate exclusively within fungal cells, where it retained the ability to be reversibly oxidized and reduced, and its detection correlated with decreased fungal viability. Pyocyanin was not required for pigment formation or fungal killing. Spectral analyses showed that the partially purified pigment from within the fungus differed from aeruginosins A and B, two red phenazine derivatives formed late in P. aeruginosa cultures. The red pigment isolated from C. albicans that had been cocultured with P. aeruginosa was heterogeneous and difficult to release from fungal cells, suggesting its modification within the fungus. These findings suggest that intracellular targeting of some phenazines may contribute to their toxicity and that this strategy could be useful in developing new antifungals.

FIG. 1.

P. aeruginosa phenazine biosynthetic genes and structures of pyocyanin and its immediate precursors. (A) P. aeruginosa has two redundant operons encoding the enzymes necessary for PCA production (phzABCDEFG). phzM and phzS are present as single copies. (B) Proposed biosynthetic pathway modified from reference . The 5MPCA intermediate has not been detected in P. aeruginosa cultures, while PCA and pyocyanin are readily detected in culture supernatants (4). Aeruginosin A has an amino substitution at position 7, and aeruginosin B has amino and sulfonate substitutions at positions 7 and 3, respectively.

FIG. 2.

Plate cocultures of C. albicans with P. aeruginosa strains. (A to C) C. albicans lawns were grown for 48 h at 30°C on YPD plates before point inoculation from LB agar-grown P. aeruginosa strains. Plates were photographed after coculture for 48 h. (A) C. albicans SC5314 with P. aeruginosa (P.a.) PA14 WT. (B) C. albicans tup1/tup1 mutant with P. aeruginosa PA14 WT. (C) C. albicans tup1/tup1 mutant with PA14 WT, flgK::Tn5, pqsA::TnM, phzS::TnM, and phzM::TnM strains. (D) Complementation of mutations in phzM::TnM and phzS::TnM strains in the coculture assay. P. aeruginosa strains were inoculated onto the C. albicans tup1/tup1 lawns as 10-μl drops and incubated at 30°C. Vector controls for WT, phzM::TnM, and phzS::TnM/(pUCP26) strains were also included. (E) Redox activity of fungal-associated pigment. C. albicans tup1/tup1 cells from two plate-grown P. aeruginosa PA14 WT cocultures were separated from the bacteria by centrifugation, resuspended in 5 ml 50 mM phosphate buffer, pH 7, and divided into three tubes. The suspensions were photographed several minutes after aeration (center), after the addition of 20 μl 3% hydrogen peroxide (left), and after the addition of dithionite (right).

FIG. 3.

Survival of C. albicans in coculture with strains of P. aeruginosa PA14. Established lawns of C. albicans SC5314 yeast were inoculated with P. aeruginosa suspensions. Core samples from plates were taken for counts of total cells by microscopy and of viable cells (CFU) after incubation for various times. (A) C. albicans methylene blue staining after incubation alone or with P. aeruginosa PA14 WT, Δphz (which lacks the genes necessary to synthesize PCA), phzM::TnM, and phzS::TnM strains. (B) Survival of C. albicans after 72 h alone (control) or in coculture with the P. aeruginosa PA14 WT, phzM::TnM, or phzS::TnM strain. Percent viability represents the fraction of the total cells that gave rise to visually detectable colonies within 24 h. These experiments were repeated as three completely separate experiments, with similar results each time.

FIG. 4.

Epifluorescence microscopy of C. albicans grown in coculture with P. aeruginosa or in medium containing the E. coli-synthesized PhzM product. For each pair of images, identical fields were photographed using differential interference contrast and Zeiss set 20 fluorescence optics for rhodamine. (A) C. albicans SC5314 from 72-h cocultures with P. aeruginosa PA14 WT, P. aeruginosa phzM::TnM, or P. aeruginosa phzS::TnM strain. (B) C. albicans tup1/tup1 mutant from 72-h cocultures with P. aeruginosa PA14 WT. No fluorescence was observed in similar cocultures with a P. aeruginosa ΔphzM strain (not shown). (C) C. albicans SC5314 after 24 h of incubation with either ∼200 μM 5MPCA prepared by incubating E. coli/pUCP-M (expressing the phzM gene) with PCA (+PhzM product) or extracts from a control preparation from E. coli/pUCP26 incubated with PCA (control).

FIG. 5.

Spectra of late intermediates in pyocyanin biosynthesis and the coculture pigment. All solutions were in 0.1 M NH₄HCO₃ and were normalized at the UV maximum. (A) Absorption spectra of PCA, the E. coli-synthesized PhzM product, and pyocyanin. (B) Absorption spectra of 5MPCA, aeruginosin A, and the partially purified coculture pigment. (C) Fluorescence emission spectra of partially purified red pigment from C. albicans cells grown in coculture with P. aeruginosa (solid lines). Excitation was done at 550 nm. The pigment was analyzed before (black line) and after (gray line) the addition of a few crystals of sodium dithionite (DT). A comparable fraction from C. albicans (C.a.) cells grown in the absence of P. aeruginosa was also analyzed (dashed line).