The Envelope Proteome Changes Driven by RamA Overproduction in Klebsiella pneumoniae that Enhance Acquired β-Lactam Resistance
The Envelope Proteome Changes Driven by RamA Overproduction in Klebsiella pneumoniae that Enhance Acquired β-Lactam Resistance
复制标题
肺炎克雷伯菌中 RamA 过量产生导致的包膜蛋白质组变化增强了获得性 β-内酰胺耐药性
DOI：
10.1101/133918
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
M. Avison
中科院分区：
文献类型：
--
作者：
Juan;Wan Ahmad Kamil Wan Nur Ismah;Yuiko Takebayashi;J. Findlay;T. Schneiders;K. Heesom;M. Avison
目的 在肺炎克雷伯菌中，RamA 的过量产生导致包膜通透性降低和抗菌药物敏感性降低，但很少观察到临床相关的耐药性。在这里，我们测试了 RamA 过量生产是否可以增强肺炎克雷伯菌的获得性 β-内酰胺耐药机制，并确定了在低渗透压和高渗透压培养基中生长期间 RamA 过量生产时观察到的包膜蛋白丰度变化。方法 使用荧光染料累积测定法估计包膜通透性。使用纸盘测试来测量抗生素敏感性。使用 LC-MS/MS 蛋白质组学对总包膜蛋白产量进行定量，并通过实时 RT-PCR 定量转录水平。结果 RamA 过量产生增强了 β-内酰胺酶介导的 β-内酰胺抗性，在某些情况下显着增强，但不改变 β-内酰胺酶的产生。它增加了外排泵的产生并减少了 OmpK35 孔蛋白的产生，尽管 micF 过度表达表明 OmpK35 的减少对包膜通透性几乎没有影响。对肺炎克雷伯菌血流分离株的调查显示，四分之三的碳青霉烯酶生产菌株、1/21 的 CTX-M 生产菌株和 2/19 不携带 CTX-M 或碳青霉烯酶的菌株中 ramA 过度表达。结论 虽然 RamA 本身并不是肺炎克雷伯菌抗生素耐药性的关键介质，但它对于增强获得性 β-内酰胺酶介导的 β-内酰胺耐药性谱系具有潜在重要意义。 LC-MS/MS 蛋白质组学分析表明，这种增强主要是通过激活外排泵产生来实现的。
OBJECTIVES In Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA over-production can enhance acquired β-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes seen upon RamA overproduction during growth in low and high osmolarity media. METHODS Envelope permeability was estimated using a fluorescent dye accumulation assay. Antibiotic susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels quantified by Real Time RT-PCR. RESULTS RamA overproduction enhanced β-lactamase mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF over-expression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 out of 4 carbapenemase producers, 1/21 CTX-M producers and 2/19 strains not carrying CTX-M or carbapenemases. CONCLUSIONS Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.
影响因子：
5.2
作者：
Bratu, Simona;Landman, David;Quale, John
通讯作者：
Quale, John
影响因子：
5.2
作者：
Coldham, Nick G.;Webber, Mark;Piddock, Laura J. V.
通讯作者：
Piddock, Laura J. V.