Molecular mechanisms of hypervirulence in antibiotic-resistant Pseudomonas aeruginosa

项目摘要/摘要 抗生素耐药性是一个主要的医疗问题，因为它使治疗无效，导致慢性 感染和死亡。铜绿假单胞菌(PSA)因其变异和成为 在慢性感染期间产生抗生素耐药性。进化论预测，这些突变将导致 适合性缺陷，包括生长速度下降，降低了耐药生物体的生存能力 当抗生素治疗结束时，与野生型敏感兄弟姐妹的竞争。此外，健身成本可能会 还可以通过降低抗药性种群的复制速度来降低毒力，从而克服致死 通过宿主的免疫反应。事实上，这些预测经常是相互矛盾的：具有抗生素耐药性的PSA 在长达数十年的慢性感染期间，很容易与易受感染的兄弟姐妹隔离，最近我们 发现增加外排泵表达的抗生素耐药突变也会增加PSA 即使在没有抗生素的情况下也是致命的。然而，使耐药细菌能够竞争的条件 对敏感细菌的耐药性尚不清楚，耐药性突变增加毒力的机制尚不清楚 都不清楚。这一建议的中心假设是外排泵组件的突变导致 抗生素耐药菌株的毒力增加。这项建议的三个具体目标是 1)确定群体感应在氨曲南耐药PSA毒力增强中的作用， 2)确定氨曲南抗性突变体相对于野生型PSA的适合性；3)鉴定新的 氨曲南耐药PSA的治疗靶点。在我们之前的工作中，超强毒力抗性PSA 菌株具有通过增加mexAB-oprM抗生素外排泵表达而起作用的耐药突变。 除了输出抗生素的作用外，MexAB-OprM还分泌一种PSA群体感应分子，该分子 参与了几个毒力因子的调节。在初步实验中，超强毒力抗性PSA 细菌群体感应介导的群集性和生物被膜表型增加 调节毒力因子鼠李糖脂。在本提案的第一个目标中，我们将测试以下假设 耐药PSA的毒性是由于群体感应调节的鼠李糖脂产量增加所致。在第二个 目的，我们将检验这样一个假设，即特定的营养物质、压力或宿主细胞使耐药细菌能够 与易受感染的细菌竞争。在第三个目标中，我们将测试靶向治疗针对 超强毒力抗性PSA将降低毒力并提高感染期间的清除能力，我们将使用 随机转座子突变结合下一代测序寻找新药靶点 在耐抗生素的PSA中。这些研究将帮助我们了解抗药性细菌是如何产生的。 以及我们如何利用这些知识并将其转化为迫切需要的 抗生素耐药细菌的治疗方法。

PROJECT SUMMARY/ABSTRACT Antibiotic resistance is a major healthcare problem because it renders treatment ineffective, leading to chronic infections and death. Pseudomonas aeruginosa (PSA) is notorious for its capacity to mutate and become antibiotic resistant during chronic infections. Evolutionary theory predicts that these mutations will lead to fitness defects, including decreased growth rates that reduce the resistant organism’s ability to survive competition with wild-type susceptible siblings when antibiotic treatment ends. Moreover, fitness costs could also reduce virulence by reducing the resistant population’s ability to replicate fast enough to overcome killing by the host immune response. In reality, these predictions are frequently contradicted: antibiotic resistant PSA are readily isolated alongside susceptible siblings during decades-long chronic infections, and recently we discovered that antibiotic resistance mutations that increase efflux pump expression can also increase PSA virulence even in the absence of antibiotics. However, the conditions that enable resistant bacteria to compete with susceptible bacteria are unknown and the mechanisms by which resistance mutations increase virulence are unclear. The central hypothesis of this proposal is that mutation of efflux pump components leads to increased virulence in antibiotic resistant strains. The three specific aims of this proposal are to 1) determine the role of quorum sensing in enhanced virulence of aztreonam resistant PSA, 2) determine fitness of aztreonam resistant mutants relative to wild-type PSA, and 3) identify novel therapeutic targets in aztreonam-resistant PSA. In our previous work, both hypervirulent resistant PSA strains had resistance mutations that functioned by increasing mexAB-oprM antibiotic efflux pump expression. In addition to its role exporting antibiotics, MexAB-OprM also secretes a PSA quorum sensing molecule which is involved in regulating several virulence factors. In preliminary experiments, hypervirulent resistant PSA increased swarming and biofilm phenotypes, which are both mediated by the bacterial quorum sensing regulated virulence factor rhamnolipid. In the first aim of this proposal we will test the hypothesis that increased virulence of resistant PSA is due to increased quorum sensing regulated rhamnolipid production. In the second aim, we will test the hypothesis that specific nutrients, stresses, or host cells enable resistant bacteria to compete with susceptible bacteria. In the third aim, we will test the hypothesis that targeted therapies against hypervirulent resistant PSA will reduce virulence and improve clearance during infection, and we will use random transposon mutagenesis in combination with next-generation sequencing to identify new drug targets in antibiotic resistant PSA. These studies will help us understand how antibiotic resistant bacteria can arise during infection, as well as how we can leverage this knowledge and translate it into desperately needed therapies for antibiotic resistant bacteria.