Linking system-wide impacts of RNA polymerase mutations to the fitness cost of rifampin resistance in Pseudomonas aeruginosa

Fitness costs play a key role in the evolutionary dynamics of antibiotic resistance in bacteria by generating selection against resistance in the absence of antibiotics. Although the genetic basis of antibiotic resistance is well understood, the precise molecular mechanisms linking the genetic basis of resistance to its fitness cost remain poorly characterized. Here, we examine how the system-wide impacts of mutations in the RNA polymerase (RNAP) gene rpoB shape the fitness cost of rifampin resistance in Pseudomonas aeruginosa. Rifampin resistance mutations reduce transcriptional efficiency, and this explains 76% of the variation in fitness among rpoB mutants. The pleiotropic consequence of rpoB mutations is that mutants show altered relative transcript levels of essential genes. We find no evidence that global transcriptional responses have an impact on the fitness cost of rifampin resistance as revealed by transcriptome sequencing (RNA-Seq). Global changes in the transcriptional profiles of rpoB mutants compared to the transcriptional profile of the rifampin-sensitive ancestral strain are subtle, demonstrating that the transcriptional regulatory network of P. aeruginosa is robust to the decreased transcriptional efficiency associated with rpoB mutations. On a smaller scale, we find that rifampin resistance mutations increase the expression of RNAP due to decreased termination at an attenuator upstream from rpoB, and we argue that this helps to minimize the cost of rifampin resistance by buffering against reduced RNAP activity. In summary, our study shows that it is possible to dissect the molecular mechanisms underpinning variation in the cost of rifampin resistance and highlights the importance of genome-wide buffering of relative transcript levels in providing robustness against resistance mutations.

IMPORTANCE Antibiotic resistance mutations carry fitness costs. Relative to the characteristics of their antibiotic-sensitive ancestors, resistant mutants show reduced growth rates and competitive abilities. Fitness cost plays an important role in the evolution of antibiotic resistance in the absence of antibiotics; however, the molecular mechanisms underlying these fitness costs is not well understood. We applied a systems-level approach to dissect the molecular underpinnings of the fitness costs associated with rifampin resistance in P. aeruginosa and showed that most of the variation in fitness cost can be explained by the direct effect of resistance mutations on the enzymatic activity of the mutated gene. Pleiotropic changes in transcriptional profiles are subtle at a genome-wide scale, suggesting that the gene regulatory network of P. aeruginosa is robust in the face of the direct effects of resistance mutations.

适合度代价在细菌抗生素抗性的进化动力学中发挥关键作用，其通过在无抗生素环境下产生对抗性抗性的选择压力。尽管对抗生素抗性的遗传基础已被充分阐明，但将抗性遗传基础与其适合度代价相关联的精确分子机制，仍未得到充分表征。在此，我们探究了RNA聚合酶（RNA polymerase, RNAP）基因rpoB的突变所产生的全系统效应，如何塑造铜绿假单胞菌（Pseudomonas aeruginosa）利福平（rifampin）抗性的适合度代价。利福平抗性突变会降低转录效率，这一现象可解释rpoB突变体中76%的适合度变异。rpoB突变存在多效性：突变体的必需基因相对转录水平发生改变。我们通过转录组测序（RNA-Seq）分析发现，并无证据表明全局转录反应会对利福平抗性的适合度代价产生影响。相较于利福平敏感的祖先菌株，rpoB突变体的转录组全局变化较为微弱，这表明铜绿假单胞菌的转录调控网络对rpoB突变相关的转录效率降低具有鲁棒性。在更精细的尺度上，我们发现利福平抗性突变会通过削弱rpoB上游衰减子（attenuator）的转录终止作用，提升RNA聚合酶的表达量；我们认为，这一机制可通过缓冲RNA聚合酶活性降低带来的影响，帮助最小化利福平抗性的适合度代价。总而言之，本研究表明，我们可以解析驱动利福平抗性适合度代价变异的分子机制，并强调了全基因组相对转录水平缓冲在为抗性突变提供鲁棒性方面的重要性。

重要性 抗生素抗性突变携带适合度代价。相较于其抗生素敏感的祖先菌株，抗性突变体的生长速率与竞争能力均出现下降。适合度代价在无抗生素环境下的抗生素抗性进化中发挥重要作用；然而，这些适合度代价背后的分子机制仍未得到充分阐释。我们采用系统级别的研究方法，解析了铜绿假单胞菌利福平抗性相关适合度代价的分子基础，并证明了大部分适合度代价变异可由抗性突变对突变基因酶活性的直接效应所解释。转录组层面的多效性变化在全基因组尺度上较为微弱，这表明铜绿假单胞菌的基因调控网络可在抗性突变的直接影响下保持鲁棒性。