Bacterial bioactive metabolites can facilitate cooperative interactions within Aspergillus fumigatus–Pseudomonas aeruginosa mixed biofilms by promoting fungal polysaccharide production

Pathogens are often present alongside other microbial species in chronic infections. The mold Aspergillus fumigatus and bacteria Pseudomonas aeruginosa are both prevalent opportunistic pathogens in cystic fibrosis (CF) patients, and their interactions play a role in the pathogenesis of chronic airway disease. In this study, their interactions were investigated during the formation of A. fumigatus–P. aeruginosa mixed biofilms. P. aeruginosa is capable of differentially modulating mixed biofilm formation, shifting from suppressing fungal growth to promoting robust mixed biofilm formation as the concentration of P. aeruginosa decreases. Bioactive secondary metabolites derived from P. aeruginosa , such as phenazines and rhamnolipids, have dual roles on fungal development and mixed biofilm formation. At high concentrations, these metabolites inhibit fungal growth, whereas at low concentrations, they promote the production of the fungal polysaccharide GAG; this, in turn, modulates mixed biofilm formation and enhances both bacterial biomass and drug resistance in the mixed biofilms. Both phenazines and rhamnolipids can induce the production of mitochondria-derived reactive oxygen species (ROS), which in turn act as signaling molecules to enhance GAG production. Finally, mitogen-activated protein kinase A (MpkA) was identified to mediate the response to bacterial metabolite-induced ROS and the enhancement of GAG production. Bacterial bioactive metabolites facilitate cooperative interactions with fungi via a conserved stress response pathway. IMPORTANCE The secondary metabolites of Pseudomonas aeruginosa , including phenazines and rhamnolipids, are traditionally regarded as toxins that inhibit the growth of competing microorganisms. Here, it is demonstrated that low doses of phenazines and rhamnolipids trigger the pathogenic fungus Aspergillus fumigatus to produce polysaccharide GAG, which in turn promotes robust A. fumigatus–P. aeruginosa mixed biofilm formation and subsequently enhances bacterial biomass and drug resistance. Mitogen-activated protein kinase A (MpkA) plays a crucial role in this process. Collectively, these bioactive metabolites play a previously unrecognized role in the interactions of human fungal pathogens. This provides a foundation for understanding interspecies signaling mechanisms in clinical biofilms.