Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli
Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli
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DOI：
10.1073/pnas.1521168113
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发表时间：
2016-03-15
影响因子：
11.1
通讯作者：
George, John
中科院分区：
文献类型：
--
作者：
Emiola, Akintunde;Andrews, Steven S.;George, John
革兰氏阴性菌的外膜由内小叶中的磷脂和外小叶中的脂多糖（LPS）组成。LPS是一种引起人体强烈免疫应答的内毒素，其生物合成部分通过蛋白酶FtsH（EC 3.4.24.-）降解LpxC（EC3.5.1.108）和WaaA（EC 2.4.99.12/13）酶来调节。由于这两种分子的合成途径都很复杂，除了以严格的比例产生外，我们还开发了一种计算模型来询问所涉及的调控机制。我们的模型研究结果表明，LpxK（EC 2.7.1.130）的催化活性似乎取决于不饱和脂肪酸的浓度。这在生物学上是重要的，因为它有助于维持LPS/磷脂稳态。磷脂和LPS生物合成途径之间的进一步串扰揭示了实验观察，LpxC是另外调节的一种身份不明的蛋白酶，其活性是独立的脂质A二糖浓度（FtsH介导的LpxC调节的反馈源），但可以在体外诱导棕榈酸。进一步的实验分析为WaaA调节的基本原理提供了证据。waaA的过表达导致膜提取物中3-脱氧-D-甘露-辛-2-酮糖酸（Kdo）糖的水平增加，而Kdo和庚糖水平在LPS中没有升高。这意味着不受控制的WaaA的产生不会增加LPS的产生速率，而是使脂质A前体再糖基化。总的来说，这项工作的结果提供了以前未确定的见解大肠杆菌外膜的复杂的生物发生。
The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production ofWaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.