School of Molecular Bioscience, The University of Sydney, 2006 Australia.
J Proteome Res. 2012 Feb 3;11(2):776-95. doi: 10.1021/pr200659h. Epub 2011 Dec 13.
Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R.
铜绿假单胞菌是一种机会性病原体，是囊性纤维化（CF）患者发病和死亡的主要原因。虽然大多数 CF 患者被认为是从环境中获得铜绿假单胞菌，但在世界各地的 CF 诊所已经发现了人与人之间可传播的菌株，而可传播性的分子基础仍知之甚少。我们采用互补的蛋白质组学方法来描述澳大利亚流行株 1（AES-1R）的可传播性急性分离株、毒力较强的烧伤/伤口分离株 PA14 以及实验室相关、毒力较弱的菌株 PAO1 的蛋白质图谱，这些菌株在模仿 CF 肺部环境的人工培养基中生长时，与在标准实验室培养基中生长相比，其蛋白质丰度发生了变化。AES-1R 中上调的蛋白质包括参与甲硫氨酸和 S-腺苷甲硫氨酸生物合成以及苯并嗪合成的蛋白质。通过测量毒力因子绿脓菌素的培养上清液水平来验证蛋白质组学数据，绿脓菌素是苯并嗪途径的最终产物。与 PA14 相比，当在模仿 CF 肺部的条件下生长时，AES-1R 和 PAO1 释放出更高水平的细胞外绿脓菌素。铁螯合铁载体吡咯并喹啉啉（PchDEFGH 和 FptA）生物合成相关的蛋白质在 AES-1R 中也大量上调，且上调幅度明显高于 PAO1，而在 PA14 中则下调。这些蛋白质变化导致细胞外铁摄取能力增强，具体表现为 AES-1R 培养上清液中的吡咯并喹啉啉水平升高，这分别通过铬天青 S 测定法和荧光法检测到。吡咯并喹啉啉基因（pchDFG 和 fptA）的转录分析表明，它们在人工痰培养基中生长的早期（18 h）高度表达，但在微菌落生长（72 h）建立后恢复到基础水平，这与在 CF 肺部观察到的情况一致。这进一步证明了铁螯合吡咯并喹啉啉可能在与 AES-1R 相关的可传播 CF 感染的早期阶段发挥作用。
