Probing the evolutionary robustness of two repurposed drugs targeting iron uptake in .

LAY SUMMARY

We probed the evolutionary robustness of two antivirulence drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine in the opportunistic pathogen . Using an experimental evolution approach in human serum, we showed that antivirulence treatments are not evolutionarily robust per se, but vary in their propensity to select for resistance.

BACKGROUND AND OBJECTIVES

Treatments that inhibit the expression or functioning of bacterial virulence factors hold great promise to be both effective and exert weaker selection for resistance than conventional antibiotics. However, the evolutionary robustness argument, based on the idea that antivirulence treatments disarm rather than kill pathogens, is controversial. Here, we probe the evolutionary robustness of two repurposed drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine of the opportunistic human pathogen .

METHODOLOGY

We subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an infection model. We screened evolved populations and clones for resistance phenotypes, including the restoration of growth and pyoverdine production, and the evolution of iron uptake by-passing mechanisms. We whole-genome sequenced evolved clones to identify the genetic basis of resistance.

RESULTS

We found that mutants resistant against antivirulence treatments readily arose, but their selective spreading varied between treatments. Flucytosine resistance quickly spread in all populations due to disruptive mutations in , a gene encoding an enzyme required for flucytosine activation. Conversely, resistance against gallium arose only sporadically, and was based on mutations in transcriptional regulators, upregulating pyocyanin production, a redox-active molecule promoting siderophore-independent iron acquisition. The spread of gallium resistance was presumably hampered because pyocyanin-mediated iron delivery benefits resistant and susceptible cells alike.

CONCLUSIONS AND IMPLICATIONS

Our work highlights that antivirulence treatments are not evolutionarily robust . Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.