Virulence evolution in the opportunistic bacterial pathogen Pseudomonas aeruginosa

Bacterial opportunistic pathogens are feared for their difficult-to-treat nosocomial infections and for causing morbidity in immunocompromised patients. Here, we study how such a versatile opportunist, Pseudomonas aeruginosa, adapts to conditions inside and outside its model host Caenorhabditis elegans, and use phenotypic and genotypic screens to identify the mechanistic basis of virulence evolution. We found that virulence significantly dropped in unstructured environments both in the presence and absence of the host, but remained unchanged in spatially structured environments. The observed virulence decline was driven by a substantial reduction in the production of multiple virulence factors, including siderophores, toxins, and proteases. Because these virulence factors are secreted, we argue that the spread of non-producers is at least partially due to cheating, where mutants exploit the shareable virulence factors produced by the wildtype. Whole-genome sequencing of evolved clones revealed positive selection and parallel evolution across replicates, and showed an accumulation of mutations in regulator genes, controlling the expression of these virulence factors. Our study identifies the spatial structure of the non-host environment as a key driver of virulence evolution in an opportunistic pathogen, and indicates that disrupting spatial structure in chronic infections could steer pathogen evolution towards lower virulence.