Unveiling the distant interaction of staphylococcus aureus with pseudomonas aeruginosa: a novel analysis of long-term and short-term responses on the proteome level

Staphylococcus aureus and Pseudomonas aeruginosa frequently co-occur in infections, and there is evidence that their interactions negatively affect disease outcome. P. aeruginosa is known to inhibit S. aureus through the secretion of inhibitory compounds and therefore P. aeruginosa is thought to be the dominant species in this interaction. However, S. aureus may adapt to inhibitory compounds in cases of prolonged co-occurrence in chronic infections.

We performed a 30-day evolution experiment with S. aureus exposed to P. aeruginosa supernatant containing growth-inhibitory compounds. While afterwards various phenotypic changes were observed, only a few genetic mutations were identified after whole genome sequencing. Based on these findings we hypothesize that resistance results from a combination of phenotypic responses and genetic adaptations. We tested this hypothesis using label-free quantitative proteomics and targeted metabolomics for one particular evolved S. aureus clone and its ancestor grown in either only normal media or exposed to growth-inhibitory compounds secreted from P. aeruginosa.

As predicted, we found that S. aureus shows fundamental phenotypic changes at the proteome level when exposed to P. aeruginosa supernatant regardless of whether S. aureus had evolved in the supernatant or not. Using the prolfqua R package and a linear model with two factors, we identified differentially expressed proteins associated with both genotype and growth-condition effects. Major changes involved the downregulation of virulence factors and a deregulation of multiple membrane transporters. At the genetic level, only a single mutation in tcyA encoding a transmembrane transporter was observed. We further show that this mutation leads to a non-functional protein and therefore a complete loss of function and confers protection against selenocysteine, a toxic compound secreted by P. aeruginosa.

Our findings suggest a two-stage response of S. aureus to P. aeruginosa inhibition: A phenotypic response securing instant survival and growth, followed by genetic adaptation reducing the negative effects of toxic compounds in the long-term. This two-stage model could explain how pathogens adapt to one another and how co-existence in polymicrobial infections can arise.