Department of Chemistry and Biomolecular Sciences, Macquarie University , Sydney 2109, Australia.
Australian Proteome Analysis Facility, Macquarie University , Sydney 2109, Australia.
J Proteome Res. 2017 Oct 6;16(10):3917-3928. doi: 10.1021/acs.jproteome.7b00561. Epub 2017 Aug 31.
Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen known to inhabit hypoxic mucus plugs of cystic fibrosis (CF) patient lungs. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to low oxygen environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT-labeled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O < 1%) in media that mimic the nutrient components of the CF lung. Quantitated across all three strains were 3967 P. aeruginosa proteins, reflecting approximately 71% of predicted ORFs in PAO1 and representing the most comprehensive proteome of clinically relevant P. aeruginosa to date. Comparative analysis revealed 735, 640, and 364 proteins were altered by 2-fold or more when comparing low oxygen to aerobic growth in PAO1, PASS2, and PASS3, respectively. Strikingly, under hypoxic stress, all strains showed concurrent increased abundance of proteins required for both aerobic (cbb-1 and cbb-2 terminal oxidases) and anaerobic denitrification and arginine fermentation, with the two clinical isolates showing higher relative expression of proteins in these pathways. Additionally, functional annotation revealed that clinical strains portray a unique expression profile of replication, membrane biogenesis, and virulence proteins during hypoxia which may endow these bacteria with a survival advantage. These protein profiles illuminate the diversity of P. aeruginosa mechanisms to adapt to low oxygen and shows that CF isolates initiate a robust molecular response to persist under these conditions.
铜绿假单胞菌是一种普遍存在的革兰氏阴性病原体，已知栖息在囊性纤维化（CF）患者肺部缺氧的粘液塞中。尽管该病原体的高流行率和相关的患者死亡率很高，但使细菌适应低氧环境的蛋白质机制仍未完全阐明。我们通过对 TMT 标记肽进行 SWATH 质谱和数据依赖型 SPS-MS3 进行了研究，以分析在模拟 CF 肺中营养成分的培养基中，在低氧应激（O < 1%）下生长的两个铜绿假单胞菌 CF 分离株 PASS2 和 PASS3 以及实验室参考菌株 PAO1 的蛋白质组。在所有三种菌株中定量了 3967 种铜绿假单胞菌蛋白，反映了 PAO1 中大约 71%的预测 ORF，代表了迄今为止最全面的临床相关铜绿假单胞菌蛋白质组。比较分析显示，当比较 PAO1、PASS2 和 PASS3 中低氧与需氧生长时，分别有 735、640 和 364 种蛋白质的变化倍数为 2 倍或更多。引人注目的是，在低氧应激下，所有菌株都表现出同时增加了有氧（cbb-1 和 cbb-2 末端氧化酶）和厌氧反硝化和精氨酸发酵所需的蛋白质的丰度，两个临床分离株在这些途径中的蛋白质表达相对较高。此外，功能注释显示，临床菌株在低氧条件下描绘了复制、膜生物发生和毒力蛋白的独特表达谱，这可能使这些细菌具有生存优势。这些蛋白质谱阐明了铜绿假单胞菌适应低氧的机制多样性，并表明 CF 分离株在这些条件下启动了强大的分子反应以维持生存。
