da Silva R A G, Churchward C P, Karlyshev A V, Eleftheriadou O, Snabaitis A K, Longman M R, Ryan A, Griffin R
School of Life Sciences, Pharmacy and Chemistry, Kingston University, Kingston upon Thames, UK.
Br J Pharmacol. 2017 Jul;174(14):2247-2260. doi: 10.1111/bph.13660. Epub 2016 Dec 21.
The level of cell surface expression of the meningococcal vaccine antigen, Factor H binding protein (FHbp) varies between and within strains and this limits the breadth of strains that can be targeted by FHbp-based vaccines. The molecular pathway controlling expression of FHbp at the cell surface, including its lipidation, sorting to the outer membrane and export, and the potential regulation of this pathway have not been investigated until now. This knowledge will aid our evaluation of FHbp vaccines.
A meningococcal transposon library was screened by whole cell immuno-dot blotting using an anti-FHbp antibody to identify a mutant with reduced binding and the disrupted gene was determined.
In a mutant with markedly reduced binding, the transposon was located in the lnt gene which encodes apolipoprotein N-acyl transferase, Lnt, responsible for the addition of the third fatty acid to apolipoproteins prior to their sorting to the outer membrane. We provide data indicating that in the Lnt mutant, FHbp is diacylated and its expression within the cell is reduced 10 fold, partly due to inhibition of transcription. Furthermore the Lnt mutant showed 64 fold and 16 fold increase in susceptibility to rifampicin and ciprofloxacin respectively.
We speculate that the inefficient sorting of diacylated FHbp in the meningococcus results in its accumulation in the periplasm inducing an envelope stress response to down-regulate its expression. We propose Lnt as a potential novel drug target for combination therapy with antibiotics.
This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.
脑膜炎球菌疫苗抗原H因子结合蛋白（FHbp）的细胞表面表达水平在不同菌株间以及同一菌株内存在差异，这限制了基于FHbp的疫苗所能针对的菌株范围。迄今为止，尚未对控制FHbp在细胞表面表达的分子途径进行研究，包括其脂化、分选至外膜及输出过程，以及该途径的潜在调控机制。这些知识将有助于我们评估FHbp疫苗。
利用抗FHbp抗体通过全细胞免疫斑点印迹法筛选脑膜炎球菌转座子文库，以鉴定结合能力降低的突变体，并确定被破坏的基因。
在一个结合能力显著降低的突变体中，转座子位于lnt基因中，该基因编码载脂蛋白N-酰基转移酶Lnt，负责在载脂蛋白分选至外膜之前添加第三个脂肪酸。我们提供的数据表明，在Lnt突变体中，FHbp是二酰化的，其在细胞内的表达降低了10倍，部分原因是转录受到抑制。此外，Lnt突变体对利福平的敏感性增加了64倍，对环丙沙星的敏感性增加了16倍。
我们推测，脑膜炎球菌中二酰化FHbp的分选效率低下导致其在周质中积累，从而引发包膜应激反应以下调其表达。我们提出Lnt作为与抗生素联合治疗的潜在新药物靶点。
本文是关于微生物药物代谢与抗生素耐药性主题部分的一部分。若要查看本部分的其他文章，请访问http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc。
