A combination of phenotypic responses and genetic adaptations enables Staphylococcus aureus to withstand inhibitory molecules secreted by Pseudomonas aeruginosa

Staphylococcus aureus and Pseudomonas aeruginosa frequently co-occur in infections, and there is evidence that their interactions can negatively affect disease outcomes. P. aeruginosa is known to be dominant, often compromising S. aureus through the secretion of inhibitory compounds. We previously demonstrated that S. aureus can become resistant to growth-inhibitory compounds during experimental evolution. While resistance arose rapidly, the underlying mechanisms were not obvious as only a few genetic mutations were associated with resistance, while ample phenotypic changes occurred. We thus hypothesize that resistance may result from a combination of phenotypic responses and genetic adaptation. Here, we tested this hypothesis using proteomics. We first focused on an evolved strain that acquired a single mutation in tcyA (encoding a transmembrane transporter unit) upon exposure to P. aeruginosa supernatant. We show that this mutation leads to a complete abolishment of transporter synthesis, which confers moderate protection against PQS and selenocystine, two toxic compounds produced by P. aeruginosa. However, this genetic effect was minor compared to the fundamental phenotypic changes observed at the proteome level when both ancestral and evolved S. aureus strains were exposed to P. aeruginosa supernatant. Major changes involved the downregulation of virulence factors, metabolic pathways, membrane transporters, and the upregulation of ROS scavengers and an efflux pump. Our results suggest that the observed multi-variate phenotypic response is a powerful adaptive strategy, offering instant protection against competitors in fluctuating environments and reducing the need for hard-wired genetic adaptations. ImportanceDifferent bacterial pathogens can co-occur in infections, where they interact with one another and influence disease severity. Previous research showed that pathogens can evolve and adapt to co-infecting species. Here, we show that evolution through genetic mutations and selection are not necessarily required to change pathogen behavior. Instead, we found that the human pathogen Staphylococcus aureus is able to plastically respond to the presence of Pseudomonas aeruginosa, a competing pathogen. Through proteomics and metabolomics, we demonstrate that S. aureus undergoes substantial proteomic alterations in response to P. aeruginosa by down-regulating virulence factor expression, changing metabolism, and mounting protective measures against toxic compounds. Our work highlights that pathogens possess sophisticated mechanisms to respond to competitors to secure growth and survival in polymicrobial infections. We predict such plastic responses to have significant impacts on infection outcomes.