Peptide 1018 disrupts swarming and the swarming-associated, anr-mediated metabolic program of Pseudomonas aeruginosa. Peptide 1018 disrupts swarming and the swarming-associated, anr-mediated metabolic program of Pseudomonas aeruginosa

Abstract: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen found ubiquitously in the environment. Responsible for considerable human morbidity and mortality, particularly in nosocomial infections and individuals with cystic fibrosis, P. aeruginosa can adapt to surface growth by undergoing swarming motility. In P. aeruginosa, swarming motility is a rapid multicellular movement that occurs on soft surfaces of appropriate viscosity with amino acids as a nitrogen source. Here we tested the small synthetic host defense peptide, innate defense regulator 1018, and found that it inhibited swarming motility at concentrations as low as 0.75 μg/ml, well below the MIC for planktonic cells. A screen of the PA14 transposon insertion mutant library revealed twenty-nine mutants that demonstrated partial tolerance to 1018 under swarming conditions. Two of these mutants, in the genes anr and rhlB (a regulator of anaerobic metabolism and protein involved in rhamnolipid production, respectively), were complemented to restore susceptibility to 1018. RNA-Seq of peptide-treated cells under swarming conditions revealed the dysregulation of 1,190 genes compared to the untreated swarm front, and 67% of these genes were similarly dysregulated at the untreated swarm centre. In contrast, expression of 70 Anr-regulated genes was upregulated by peptide treatment, and 45 genes showed differential or opposite regulation of expression in peptide-treated and swarm centre cells. Many transcriptional regulators required for swarming were dysregulated in peptide-treated cells, indicative of a mechanism by which 1018 may inhibit swarming motility. Overall, this study illustrates a use for peptide 1018 in inhibiting swarming surface motility, an important bacterial adaptation. Overall design: RNA-Seq of P. aeruginosa inoculated on swarming agar centre and edge cells in the absence or presence of host-defense peptide IDR-1018 (0.75 ug/ml). Thus, three samples with two replicates each.

摘要：铜绿假单胞菌（Pseudomonas aeruginosa）是一种在环境中广泛分布的革兰氏阴性机会致病菌。该菌可引发严重的人类发病与死亡，尤其多见于医院获得性感染及囊性纤维化患者，且可通过群集运动（swarming motility）适应表面生长。
在铜绿假单胞菌中，群集运动是一种快速的多细胞运动过程，发生于黏度适宜且以氨基酸为氮源的软质表面上。
本研究测试了小型合成宿主防御肽——固有防御调节因子1018（innate defense regulator 1018），发现其最低可在0.75 μg/ml的浓度下即抑制群集运动，该浓度远低于浮游态细胞的最低抑菌浓度（MIC）。
通过对PA14转座子插入突变体文库进行筛选，我们获得了29株在群集运动条件下对1018具有部分耐受性的突变体。
其中两株突变体分别涉及anr基因（厌氧代谢调节因子）与rhlB基因（参与鼠李糖脂合成的蛋白），通过互补实验可恢复其对1018的敏感性。
对群集运动条件下经肽处理的细胞进行RNA测序（RNA-Seq）分析发现，与未处理的群集前沿细胞相比，共有1190个基因表达失调，其中67%的基因在未处理的群集中心细胞中也呈现相似的表达失调趋势。
与之相反，经肽处理后，70个受Anr调控的基因表达上调；另有45个基因在肽处理组与群集中心细胞组中的表达调控存在差异甚至完全相反。
许多参与群集运动必需的转录调控因子在肽处理组细胞中出现表达失调，这提示了1018抑制群集运动的潜在作用机制。
综上，本研究证实了肽1018可用于抑制细菌的群集表面运动——这是细菌的一种重要适应性行为。
实验整体设计：将铜绿假单胞菌接种于群集运动琼脂平板，分别收集其中心与边缘细胞，设置添加与不添加宿主防御肽IDR-1018（0.75 μg/ml）两组对照，共包含3组样本，每组设置2次生物学重复。