Phenazines Pseudomonas aeruginosa Formation by Metabolism and Biofilm Candida albicans Control of 2013

Candida albicans has developmental programs that govern transitions between yeast and filamentous morphologies and between unattached and biofilm lifestyles. Here, we report that filamentation, intercellular adherence, and biofilm development were inhibited during interactions between Candida albicans and Pseudomonas aeruginosa through the action of P. aeruginosa-produced phenazines. While phenazines are toxic to C. albicans at millimolar concentrations, we found that lower concentrations of any of three different phenazines (pyocyanin, phenazine methosulfate, and phenazine-1-carboxylate) allowed growth but affected the development of C. albicans wrinkled colony biofilms and inhibited the fungal yeast-to-filament transition. Phenazines impaired C. albicans growth on nonfermentable carbon sources and led to increased production of fermentation products (ethanol, glycerol, and acetate) in glucose-containing medium, leading us to propose that phenazines specifically inhibited respiration. Methylene blue, another inhibitor of respiration, also prevented the formation of structured colony biofilms. The inhibition of filamentation and colony wrinkling was not solely due to lowered extracellular pH induced by fermentation. Compared to smooth, unstructured colonies, wrinkled colony biofilms had higher oxygen concentrations within the colony, and wrinkled regions of these colonies had higher levels of respiration. Together, our data suggest that the structure of the fungal biofilm promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by bacterial molecules such as phenazines or compounds with similar activities disrupts these pathways. These findings may suggest new ways to limit fungal biofilms in the context of disease. IMPORTANCE Many of the infections caused by Candida albicans, a major human opportunistic fungal pathogen, involve both morphological transitions and the formation of surface-associated biofilms. Through the study of C. albicans interactions with the bacterium Pseudomonas aeruginosa, which often coinfects with C. albicans, we have found that P. aeruginosa-produced phenazines modulate C. albicans metabolism and, through these metabolic effects, impact cellular morphology, cell-cell interactions, and biofilm formation. We suggest that the structure of C. albicans biofilms promotes access to oxygen and enhances respiratory metabolism and that the perturbation of respiration by phenazines inhibits biofilm development. Our findings not only provide insight into interactions between these species but also provide valuable insights into novel pathways that could lead to the development of new therapies to treat C. albicans infections. Received 18 November 2012 Accepted 9 January 2013 Published 29 January 2013 Citation Morales DK, Grahl N, Okegbe C, Dietrich LEP, Jacobs NJ, Hogan DA. 2013. Control of Candida albicans metabolism and biofilm formation by Pseudomonas aeruginosa phenazines. mBio 4(1):e00526-12. doi:10.1128/mBio.00526-12. Editor Judith Berman, University of Minnesota, GCD Copyright © 2013 Morales et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Deborah A. Hogan, dhogan@dartmouth.edu. Candida albicans, a commensal resident of mucosal surfaces, can overgrow under favorable conditions, leading to a wide range of diseases, referred to as candidiasis (1). C. albicans infections often involve more than one cellular morphology; yeast, pseudohyphal, and hyphal growth forms have all been associated with virulence (2). It has been proposed that filaments are responsible for tissue penetration, whereas yeasts play an important role in early dissemination and in less invasive disease infections (3). C. albicans infections are also associated with its formation of biofilms, which are dense single-species and mixed-species populations adhering to one another through the action of surface adhesins and extracellular polymers. Biofilms on implanted medical devices can serve as a source for infection, and dense C. albicans populations within the host may have properties in common with biofilms, such as high levels of drug resistance and resistance to host immune defenses (4, 5). Importantly, biofilm formation involves filamentation and adhesin-mediated aggregation of cells (6, 7). Many environmental cues impact fungal morphology and biofilm formation. Many conditions, such as hypoxia, elevated extracellular pH, N-acetylglucosamine (GlcNAc), amino acids, low concentrations of glucose, body temperature, and elevated CO2 have been associated with C. albicans filamentation (8, 9). There have been a number of studies that also indicate that metabolic status can influence the decision to form hyphae (10–16). There are still many questions regarding how the fungus integrates these RESEARCH ARTICLE January/February 2013 Volume 4 Issue 1 e00526-12 ® mbio.asm. org 1 different signals and whether there are important links between known inducers of hyphal growth, biofilm formation, and metabolism. C. albicans and bacteria can coexist within the host (17–19), where the nature of the interspecies interactions can determine the fate of the microbial populations (20) and thus probably the outcome of polymicrobial diseases. C. albicans and Pseudomonas aeruginosa, two species commonly found together in mixedspecies, biofilm-related infections (21), interact in many different ways through physical association, killing by secreted factors and signaling events that modulate virulence properties (22–29). P. aeruginosa adheres to and forms biofilms on C. albicans filaments (23) and ultimately causes the death of the fungal hypha. A P. aeruginosa quorum-sensing molecule also induces a transition to yeast growth, and yeasts are more resistant to killing by P. aeruginosa (23). Redox-active phenazines produced by P. aeruginosa play a key role in controlling C. albicans when the two species are in close proximity (24, 29). We reported that C. albicans growth was antagonized by two P. aeruginosa phenazines, pyocyanin (PYO) and its biosynthetic precursor 5-methylphenazinium-1-carboxylate (5MPCA), as well as by a synthetic methylphenazinium analog phenazine methosulfate (PMS), which we have shown to be a surrogate for studying 5MPCA’s antifungal activity (24, 29) (see Fig. S1 in the supplemental material for chemical structures). Phenazines play important roles in bacterial interactions with fungi in which high concentrations of phenazines inhibit fungal growth (30). Here, we demonstrate a new role for bacterial phenazines as modulators of C. albicans metabolism and community behavior. In the presence of phenazine-producing P. aeruginosa or low concentrations of purified phenazines, the fungus grew but no longer developed wrinkled colonies or robust biofilms on plastic surfaces. Phenazines markedly inhibited the yeast-to-filament transition. Consistent with published data indicating that phenazines can impact mitochondrial activity (31, 32), their presence led to increased production of fermentation products and decreased respiratory activity at concentrations that are found in clinical samples (33). Methylene blue (MB), which can also interact with the electron transport chain (ETC) (34, 35), inhibited the development of wrinkled C. albicans colony biofilms. These studies also revealed a link between wrinkles in colony biofilms and respiratory activity, and this finding was supported by the detection of higher oxygen concentrations in wrinkled colonies than in smooth and flat colonies comprised of yeast. Together, our data revealed a previously unidentified role for phenazines as modulators of C. albicans metabolism and, through these effects, on cellular morphology, colony development, and biofilm formation on solid surfaces. Moreover, this work also provides new insights into the links between metabolism, morphogenesis, and community behaviors such as biofilm formation in C. albicans, and this may represent an emerging theme among the microbes.