Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis
Characterization of an Acinetobacter baumannii lptD Deletion Strain: Permeability Defects and Response to Inhibition of Lipopolysaccharide and Fatty Acid Biosynthesis
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DOI：
10.1128/jb.00639-15
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发表时间：
2016-02-01
影响因子：
3.2
通讯作者：
Dean, Charles R.
中科院分区：
文献类型：
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作者：
Bojkovic, Jade;Richie, Daryl L.;Dean, Charles R.
革兰氏阴性外膜(OM)上的脂质A通过LPX途径在细胞质中合成，并通过LPT途径转运到OM。部分鲍曼不动杆菌能耐受早期脂多糖途径基因如lpxC失活导致的脂多糖完全丧失。在这里，我们鉴定了一个缺失了lptD的突变体，该突变体编码一种OM蛋白，该蛋白介导完全合成的内毒素最终转移到OM。在所测试的生长条件下，缺乏lptD的细胞的生长缺陷与lpxC缺失突变体相似，但对疏水抗生素更敏感，表明内毒素易位受损对细胞通透性的影响比内毒素合成的丧失更显著。与此相一致的是，ATP渗漏和N-苯基-1-萘胺(NPN)荧光检测分别表明LptD缺失比LpxC缺失对内、外膜通透性的影响更严重。靶向液质联用分析表明，UDP-3-O-R-3-hydroxylauroyl-N-acetyl-alpha-D-glucosamine通过脂类IVA产生的脂多糖中间体的缺失导致了脂类IVA的蓄积。这表明，由于后来的内毒素途径步骤被阻断而导致的途径中间积累或错误定位影响了包膜的完整性。支持这一观点的是，化学抑制lptD缺失菌株中的脂质A前体酶，包括LpxC和FabB/F，部分挽救了生长和通透性缺陷。抑制内毒素的生物合成是很有吸引力的，因为这会影响细胞的活力和通透性。因此，更好地了解这一途径是很重要的，特别是在鲍曼不动杆菌ATCC 19606等菌株中，在这些菌株中，脂多糖的生物合成在体外并不是必需的。我们发现ATCC 19606在脂多糖最终易位到OM中的丢失(LPTD缺失)中也存活下来。有趣的是，这比内毒素生物合成(lpxC缺失)对细胞包膜完整性的损害更大，可能是由于有毒中间体的积累。支持这一点的是，化学抑制内毒素的生物合成部分逆转了这一通透性缺陷。这扩大了我们对脂多糖机制的理解，并提供了对这一重要途径上目标步骤的潜在相互关系的洞察。
Lipid A on the Gram-negative outer membrane (OM) is synthesized in the cytoplasm by the Lpx pathway and translocated to the OM by the Lpt pathway. Some Acinetobacter baumannii strains can tolerate the complete loss of lipopolysaccharide (LPS) resulting from the inactivation of early LPS pathway genes such as lpxC. Here, we characterized a mutant deleted for lptD, which encodes an OM protein that mediates the final translocation of fully synthesized LPS to the OM. Cells lacking lptD had a growth defect comparable to that of an lpxC deletion mutant under the growth conditions tested but were more sensitive to hydrophobic antibiotics, revealing a more significant impact on cell permeability from impaired LPS translocation than from the loss of LPS synthesis. Consistent with this, ATP leakage and N-phenyl-1-naphthylamine (NPN) fluorescence assays indicated a more severe impact of lptD deletion than of lpxC deletion on inner and outer membrane permeability, respectively. Targeted liquid chromatography-mass spectrometry (LCMS) analysis of LPS intermediates from UDP-3-O-R-3-hydroxylauroyl-N-acetyl-alpha-D-glucosamine through lipid IVA showed that the loss of LptD caused an accumulation of lipid IVA. This suggested that pathway intermediate accumulation or mislocalization caused by the blockage of later LPS pathway steps impacts envelope integrity. Supporting this notion, chemical inhibition of lipid A precursor enzymes, including LpxC and FabB/F, in the lptD deletion strain partially rescued growth and permeability defects.IMPORTANCENew antibiotics to treat Gram-negative bacterial infections are urgently needed. Inhibition of LPS biosynthesis is attractive because this would impact viability and cell permeability. Therefore, a better understanding of this pathway is important, especially in strains such as A. baumannii ATCC 19606, where LPS biosynthesis is not essential in vitro. We show that ATCC 19606 also survives the loss of the final translocation of LPS into the OM(lptD deletion). Intriguingly, this impaired cell envelope integrity more than the loss of LPS biosynthesis (lpxC deletion), presumably due to the accumulation of toxic intermediates. Supporting this, chemical inhibition of LPS biosynthesis partially reversed this permeability defect. This extends our understanding of the LPS machinery and provides insights into potential interrelationships of the target steps along this important pathway.