Making a Pathogen? Evaluating the Impact of Protist Predation on the Evolution of Virulence in Serratia marcescens

Opportunistic pathogens are environmental microbes that are generally harmless and only occasionally cause disease. Unlike obligate pathogens, the growth and survival of opportunistic pathogens do not rely on host infection or transmission. Their versatile lifestyles make it challenging to decipher how and why virulence has evolved in opportunistic pathogens. The coincidental evolution hypothesis postulates that virulence results from exaptation or pleiotropy, i.e. traits evolved for adaptation to living in one environment that have a different function in another. In particular, adaptation to avoid or survive protist predation has been suggested to contribute to the evolution of bacterial virulence (the training ground hypothesis). Here, we used experimental evolution to determine how the selective pressure imposed by a protist predator impacts the virulence and fitness of a ubiquitous environmental opportunistic bacterial pathogen that has acquired multidrug resistance: Serratia marcescens. To this aim, we evolved S. marcescens in the presence or absence of generalist protist predator, Tetrahymena thermophila. After 60 d of evolution, we evaluated genotypic and phenotypic changes by comparing evolved S. marcescens with the ancestral strain. Whole-genome shotgun sequencing of the entire evolved populations and individual isolates revealed numerous cases of parallel evolution, many more than statistically expected by chance, in genes associated with virulence. Our phenotypic assays suggested that evolution in the presence of a predator maintained virulence, whereas evolution in the absence of a predator resulted in attenuated virulence. We also found a significant correlation between virulence, biofilm formation, growth, and grazing resistance. Overall, our results provide evidence that bacterial virulence and virulence-related traits are maintained by selective pressures imposed by protist predation.