LAY SUMMARY We probed the evolutionary robustness of two antivirulence drugs, gallium and flucytosine, targeting the iron-scavenging pyoverdine in the opportunistic pathogen Pseudomonas aeruginosa. 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 Pseudomonas aeruginosa. METHODOLOGY We subjected replicated cultures of bacteria to two concentrations of each drug for 20 consecutive days in human serum as an ex vivo 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 upp, 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 per se. Instead, evolutionary robustness is a relative measure, with specific treatments occupying different positions on a continuous scale.

中文翻译：

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探讨两种针对铜绿假单胞菌铁吸收的再利用药物的进化稳健性。

初步总结 我们探讨了两种抗毒药物镓和氟胞嘧啶的进化稳健性，其目标是机会性病原体铜绿假单胞菌中的铁清除性pyoverdine。通过在人血清中进行实验进化方法，我们发现抗毒治疗本身在进化上并不稳健，但选择耐药性的倾向各不相同。背景和目标 抑制细菌毒力因子表达或功能的治疗方法有望有效，但与传统抗生素相比，其耐药性选择较弱。然而，基于抗毒治疗解除而不是杀死病原体这一观点的进化稳健性论点是有争议的。在这里，我们探讨了两种重新利用的药物（镓和氟胞嘧啶）的进化鲁棒性，它们针对机会性人类病原体铜绿假单胞菌的铁清除能力。方法学 我们将细菌的复制培养物连续 20 天置于人血清中的每种药物的两种浓度下，作为离体感染模型。我们筛选了进化群体和克隆的抗性表型，包括生长和pyoverdine生产的恢复，以及铁吸收旁路机制的进化。我们对进化的克隆进行全基因组测序，以确定抗性的遗传基础。结果我们发现，对抗病毒治疗具有抗性的突变体很容易出现，但它们的选择性传播在治疗之间存在差异。由于 upp 的破坏性突变，氟胞嘧啶耐药性在所有人群中迅速蔓延，upp 是编码氟胞嘧啶激活所需酶的基因。相反，对镓的耐药性只是偶尔出现，并且是基于转录调节因子的突变，上调绿脓素的产生，绿脓素是一种氧化还原活性分子，促进不依赖于铁载体的铁获取。镓耐药性的传播可能受到阻碍，因为绿脓素介导的铁输送对耐药细胞和易感细胞都有好处。结论和意义我们的工作强调抗毒治疗本身在进化上并不稳健。相反，进化稳健性是一种相对衡量标准，特定的处理方法在连续范围内占据不同的位置。