Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant
Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant
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DOI：
10.1111/hel.12196
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发表时间：
2015-08-01
期刊：
影响因子：
4.4
通讯作者：
Ferrero, Richard L.
中科院分区：
文献类型：
--
作者：
Turner, Lorinda;Praszkier, Judyta;Ferrero, Richard L.
背景多项研究已经确定了 tol-pal 基因簇在大肠杆菌细菌细胞膜完整性和外膜囊泡 (OMV) 形成中的重要性。相比之下，Tol-Pal 蛋白在病原生物（包括 Epsilonproteobacteria 的功能）中的功能即使有明确的定义，仍然很差。本研究的目的是表征 Tol-Pal 系统的两个关键成分 TolB 和 Pal 在致病菌幽门螺杆菌 OMV 形成中的作用。方法 生成幽门螺杆菌 tolB、pal 和 tolBpal 突变体以及互补菌株，并通过扫描电子显微镜和酶联技术评估形态和 OMV 产生的变化。 分别进行免疫测定（ELISA）。分别通过质谱和白细胞介素-8 (IL-8) ELISA 对 OMV 刺激细胞的培养上清液测定 OMV 的蛋白质含量和促炎特性。结果幽门螺杆菌 tolB 和 pal 细菌表现出异常的细胞形态和/或鞭毛生物合成。重要的是，H.pylori tolB（而非pal）的破坏导致了OMV产量的显着增加。来自幽门螺杆菌 tolB 和 pal 细菌的 OMV 含有在野生型 (WT) OMV 中观察到的许多主要外膜和毒力蛋白。有趣的是，与 WT OMV 相比，tolB、pal 和 tolBpal OMV 诱导宿主细胞产生显着更高水平的 IL-8。结论这项工作表明，TolB 和 Pal 对于幽门螺杆菌中的膜完整性很重要。此外，它还展示了如何操纵幽门螺杆菌 tolB-pal 基因来开发用于疫苗目的的高泡菌株。
BackgroundMultiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori.MethodsH.pylori tolB, pal and tolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively.ResultsH.pylori tolB and pal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H.pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H.pylori tolB and pal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, tolB, pal and tolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs.ConclusionsThis work demonstrates that TolB and Pal are important for membrane integrity in H.pylori. Moreover, it shows how H.pylori tolB-pal genes may be manipulated to develop hypervesiculating strains for vaccine purposes.