Microbiota can protect their hosts from infection. The short timescales in which microbes can evolve presents the possibility that ‘protective microbes’ can take-over from the immune system of longer-lived hosts in the coevolutionary race against pathogens. Here, we found that coevolution between a protective bacterium (Enterococcus faecalis) and a virulent pathogen (Staphylococcus aureus) within an animal population (Caenorhabditis elegans) resulted in more disease suppression than when the protective bacterium adapted to uninfected hosts. At the same time, more protective E. faecalis populations became costlier to harbour and altered the expression of 134 host genes. Many of these genes appear to be related to the mechanism of protection, reactive oxygen species production. Crucially, more protective E. faecalis populations downregulated a key immune gene, sodh-1, known to be effective against S. aureus infection. These results suggest that a microbial line of defence is favoured by microbial coevolution and may cause hosts to plastically divest of their own immunity.

The evolution experiment consisted of two treatments: (i) S. aureus and E. faecalis were co- passaged within C. elegans (and so allowed to coevolve), and (ii) E. faecalis was passaged alone within C. elegans. Each treatment consisted of five replicate populations with 10 passages. 

Protective ability was assessed by calculating the proportion of dead worms in the population after 24h pathogen exposure, with and without E. faecalis co-colonisation. Cost was assessed by calculating the proportion of dead worms in the population after 24h exposure to E. faecalis. Both cost and protection experiments were repeated twice independently and the counts of total and dead worms were summed prior to statistical analysis.

E. faecalis-mediated suppression of S. aureus via reactive oxygen species production was assessed in vitro. We performed this experiment with either coevolved or evolved populations of E. faecalis against the ancestral stock of S. aureus. Bacteria were cultured overnight in THB shaking (200rpm) at 30°C. THB solution was made with 0.25M potassium phosphate buffer containing superoxide dismutase from bovine erythrocytes (Sigma-Aldrich) and catalase from bovine liver (Sigma-Aldrich), each at a concentration of 0.25mg ml-1. An enzyme-free THB solution served as control with only 0.25M potassium phosphate buffer. After standardising the bacteria (OD600 of 1.00), 3μl of each species was added to 194μl of THB and shaken at 30°C for 24h. Colony-forming units per ml (CFU/ml) of S. aureus were counted by plating dilutions onto MSA plates. This experiment was repeated two independent times and the results were averaged per replicate population.