Proteomic study of evolved Pseudomonas aeruginosa strains grown in Staphylococcus aureus- and Klebsiella pneumoniae-conditioned media.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes multiple infections. Co-infections of PA with Staphylococcus aureus (SA) and Klebsiella pneumoniae (KP) were frequently reported in severe community-acquired pneumonia. However, it is challenging to mimic such natural conditions and maintain a long-term stable community of these pathogens in the laboratory, thus limiting the study of PA's adaptation in the presence of SA and KP. In this study, we performed adaptive laboratory evolution (ALE) using a simplified supernatant-based culture model to explore the evolution of PA under the biochemical influences of SA and KP and elucidate the adaptation mechanism of the evolved PA strains using proteomics. After 15 growth cycles with the cell-free supernatants of SA (SASn) and KP (KPSn), PA displayed significant alterations in phenotypes, including motility, antibiotic sensitivity, toxicity, biofilm formation, pyocyanin production, relative fitness, and growth profile. Whole-genome sequencing revealed nonsynonymous mutations in the SASn- and KPSn-evolved PA strains in ampG, dipA, anmK, bifA, and rpoS genes, which were not observed in the evolved strains that were cultured in the absence of SA/KP supernatants. Notably, the supernatant-evolved mutants exhibited differential regulation of key pathways (type VI secretion system, biofilm formation, phenazine biosynthesis, translation, β-lactam resistance, and O-antigen biosynthesis) compared to the ancestral strain and the unmodified medium-evolved (UmMd-evolved) strain. Our results suggested that adaptive evolution in such supernatant-based pseudo-coculture models can be a viable strategy to gain insights into how PA adapts under the influence of other pathogens, which may have clinical implications in understanding and controlling co-infections.IMPORTANCEThrough the supernatant-based ALE approach, we examined the evolutionary adaptations of PA upon repetitive growth cycles in cell-free supernatants of SA and KP. Compared to the unmodified medium-evolved (UmMd-evolved) strain, the SA- and KP supernatant-evolved (SASn- and KPSn-evolved) strains acquired distinct mutations and exhibited different phenotypic and proteomic alterations. The SASn- and KPSn-evolved PA strains display elevated cytotoxicity and enhanced competitiveness against SA and KP compared to the ancestral strain. SASn- and KPSn-evolved PA strains displayed some similarities in terms of the proteomic profile, especially in the expression of type VI secretion system (T6SS). Both SASn- and KPSn-evolved PA strains positively and negatively regulated H2 and H3-Hcp secretion islands (HSIs) of T6SS, respectively, while the UmMd-evolved strain negatively regulated both H2 and H3-T6SS. These suggest the potential role of SA and KP in modulating the regulation of T6SS HSIs in PA.