Chemical ecology: Bacterial phenazines as toxins and paracrine signals in Candida albicans

Candida albicans can co-exist with bacteria within biofilms, a microenvironment wherein several kinds of interactions can occur. It has been shown that the nature of these interspecies interactions can determine the fate of the microbial populations within these communities. C. albicans and Pseudomonas aeruginosa exhibit an antagonistic relationship where bacterial phenazines may play a crucial role. In this study, we identify and characterize the unique molecular properties that make Pseudomonad methylphenaziniums such as 5MPCA, and its analog PMS, a potent toxin against C. albicans in close proximity. We demonstrate that the reducing environment within colony biofilms facilitate methylphenazinium uptake by fungal cells. These molecules are concentrated intracellularly and their accumulation is due to covalent binding to cellular amines, a process that yields red species. The generation of reactive oxygen species, together with the effects induced by modification of cellular macromolecules, represent the main mechanisms by which methylphenaziniums efficiently kill C. albicans. Notably, the well-characterized P. aeruginosa phenazine toxin PYO exhibited less toxicity than 5MPCA and was incapable of binding to cellular amines. Our data indicate that the chemical reactivity of 5MPCA contributes to its concentration within fungal cells, where it retains toxic activity particularly in environments such as biofilms. Furthermore, this study demonstrates a novel role for phenazines as paracrine modulators of community behavior in C. albicans when used at sub-inhibitory concentrations. Our work indicates that by preventing fungal yeast-to-filament transition, PMS and PYO inhibit the development of wrinkled colony biofilms. This process involved phenazine interactions with C. albicans respiratory chain, which correlated with decreased colony oxygen tension and lower energy yield. We demonstrate that sublethal phenazine exposure shifted carbon metabolism towards a fermentative growth, thus promoting secretion of acids decreasing the extracellular pH. Importantly, this low pH was sufficient to suppress C. albicans fungal morphological transition. A genetic screen of C. albicans mutants pointed two candidates that maybe mediate the phenazine-induced response, the kinase Ssn3 and its cyclin pair Ssn8. Together, our data reveal a novel role for phenazines not only as toxins but also as paracrine signals that modulate the physiology of the opportunistic human pathogen C. albicans.