Evaluating the Link Between Efflux Pump Expression and Motility Phenotypes in Pseudomonas aeruginosa Treated with Virulence Inhibitors

Antibiotic resistance continues to rise as a global health threat. Novel anti-virulence strategies diminish the drive for evolutionary pressure, but still hinder a pathogen's ability to infect a host. Treatment of the highly virulent Pseudomonas aeruginosa strain PA14 with virulence inhibitors (R-2 and R-6) elicited widely varying transcriptional profiles. Of interest, expression of a family of resistance-nodulation-division (RND) efflux pumps implicated in the intrinsic drug resistance of P. aeruginosa, was significantly altered by R-2 and R-6 treatment. While structurally similar, these inhibitors caused differential expression of various RND efflux pumps within the Mex family—R-2 treatment stimulated expression of mexEF-oprN while R-6 treatment led to increased mexAB-oprM expression. Further expansion into a small library of virulence inhibitors revealed chemical motifs that trigger increases in RND efflux pump expression. Additionally, activation of these efflux pumps suggests low accumulation of virulence inhibitors in WT PA14. Treatment of an efflux pump-deficient strain with R-2 or R-6 resulted in inhibition of several virulence factors, for example R-2 was found to abolish swimming motility. Collectively, treatment with either R-2 or R-6 gives rise to a convoluted transcriptomic response, confounded by the impact of efflux pump expression on the system. However, understanding the moieties that lead to high expression of the efflux pumps enables further rational design of novel virulence inhibitors that do not cause RND efflux pump activation. Overall design: PA14 diluted from overnight cultures were diluted into fresh LB media containing DMSO, R-2 or R-6 (200 uM) and grown to OD600 ~0.9 or ~1.2. Four biological replicates were grown for every treatment condition. RNA extracted and sequenced to evaluate differences in transcriptome profiles under each inhibitor at different growth phases.

抗生素耐药性持续攀升，已成为日益严峻的全球公共卫生威胁。新型抗毒力策略虽可弱化进化压力的驱动效应，却仍能有效阻断病原体感染宿主的能力。以毒力抑制剂R-2与R-6处理高毒力铜绿假单胞菌（Pseudomonas aeruginosa）菌株PA14后，观测到差异显著的转录组谱。值得关注的是，R-2与R-6处理显著改变了与铜绿假单胞菌固有耐药性相关的耐药结节分化（resistance-nodulation-division, RND）外排泵家族的表达水平。尽管二者结构相似，但这两种抑制剂可导致Mex家族不同RND外排泵出现差异表达：R-2处理可上调mexEF-oprN的表达，而R-6处理则促进mexAB-oprM的表达。进一步针对小型抗毒力抑制剂库的筛选发现，部分化学基团可诱导RND外排泵的表达上调。此外，这些外排泵的激活提示，野生型（wild type, WT）PA14菌株中抗毒力抑制剂的积累量较低。用R-2或R-6处理外排泵缺陷菌株后，可抑制多种毒力因子的活性；例如实验发现R-2可完全消除菌株的游泳运动能力。综合来看，R-2或R-6单独处理会引发复杂的转录组响应，而外排泵表达对系统的影响进一步加剧了这种复杂性。然而，明确可引发外排泵高表达的化学基团，可为后续合理设计不激活RND外排泵的新型抗毒力抑制剂提供指导。实验整体设计：将过夜培养的PA14菌株转接至含二甲基亚砜（DMSO）、R-2或R-6（200 μM）的新鲜LB培养基中，培养至光密度600nm（OD600）约0.9或1.2。每个处理组均设置4个生物学重复。提取RNA并进行转录组测序，以分析不同生长阶段下各抑制剂处理后的转录组谱差异。