Bacterial persisters, a dormant and multidrug tolerant subpopulation that are able to resuscitate after antibiotic treatment, have recently received considerable attention as a major cause of relapse of various infectious diseases in the clinic. However, because of their low abundance and inherent transience, it is extremely difficult to study them by proteomics. Here we developed a magnetic-beads-based separation approach to enrich Escherichia coli persisters and then subjected them to shotgun proteomics. Rifampin pretreatment was employed to increase persister formation, and the resulting cells were exposed to a high concentration of ampicillin (10× MIC) to remove nonpersisters. The survivors were analyzed by spectral counting-based quantitative proteomics. On average, 710 proteins were identified at a false discovery rate of 0.01 for enriched E. coli persisters. By spectral counting-based quantification, 105 proteins (70 down-regulated, 35 up-regulated) were shown to be differentially expressed compared with normal cells. A comparison of the differentially expressed proteins between the magnetic beads-enriched persisters and nonenriched persisters (a mixture of persisters and intact dead cells) shows only around half (∼58%) overlap and different protein–protein interaction networks. This suggest that persister enrichment is important to eliminate the cumulative effect of dead cells that will obscure the proteome of persisters. As expected, proteins involved in carbohydrate metabolism, fatty acid and amino acid biosynthesis, and bacterial chemotaxis were found to be down-regulated in the persisters. Interestingly, membrane proteins including some transport proteins were up-regulated, indicating that they might be important for the drug tolerance of persisters. Knockout of the pal gene expressing peptidoglycan-associated lipoprotein, one of the most up-regulated proteins detected in persisters, led to 10-fold reduced persister formation under ampicillin treatment.

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利福平预处理对大肠杆菌的特异性富集和蛋白质组学分析

细菌持久性细菌是一种休眠的，耐多药的亚群，在抗生素治疗后能够复苏。作为临床上各种传染病复发的主要原因，细菌持久性最近受到了广泛的关注。但是，由于它们的丰度低和固有的瞬时性，用蛋白质组学研究它们非常困难。在这里，我们开发了一种基于磁珠的分离方法来富集大肠杆菌坚持者，然后让他们接受shot弹枪蛋白质组学研究。利福平预处理可用于增加持久性形成，并将产生的细胞暴露于高浓度的氨苄西林（10x MIC）中，以去除非持久性物质。通过基于光谱计数的定量蛋白质组学分析幸存者。平均而言，对于富集大肠埃希氏菌，以错误发现率为0.01鉴定出710种蛋白质坚持者。通过基于光谱计数的定量，与正常细胞相比，105种蛋白质（70种下调，35种上调）显示差异表达。富含磁珠的持留者和未富集的持留者（持留者和完整的死细胞的混合物）之间差异表达的蛋白质的比较显示，只有大约一半（〜58％）的重叠和不同的蛋白质-蛋白质相互作用网络。这表明持久性物质富集对于消除会掩盖持久性蛋白质组的死细胞的累积效应很重要。正如预期的那样，与碳水化合物代谢，脂肪酸和氨基酸生物合成以及细菌趋化性有关的蛋白质在持久性物质中被下调。有趣的是，包括一些转运蛋白在内的膜蛋白被上调，表明它们对于持久性药物的耐药性可能很重要。淘汰赛表达肽聚糖相关脂蛋白的pal基因是持久性蛋白中检测到的上调程度最高的蛋白之一，在氨苄青霉素治疗下导致持久性蛋白形成减少了10倍。