Ballok Alicia E, Filkins Laura M, Bomberger Jennifer M, Stanton Bruce A, O'Toole George A
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA.
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA
J Bacteriol. 2014 Oct;196(20):3633-42. doi: 10.1128/JB.01760-14. Epub 2014 Aug 11.
Pseudomonas aeruginosa produces outer membrane vesicles (OMVs) that contain a number of secreted bacterial proteins, including phospholipases, alkaline phosphatase, and the CFTR inhibitory factor (Cif). Previously, Cif, an epoxide hydrolase, was shown to be regulated at the transcriptional level by epoxides, which serve as ligands of the repressor, CifR. Here, we tested whether epoxides have an effect on Cif levels in OMVs. We showed that growth of P. aeruginosa in the presence of specific epoxides but not a hydrolysis product increased Cif packaging into OMVs in a CifR-independent fashion. The outer membrane protein, OprF, was also increased under these conditions, but alkaline phosphatase activity was not significantly altered. Additionally, we demonstrated that OMV shape and density were affected by epoxide treatment, with two distinct vesicle fractions present when cells were treated with epibromohydrin (EBH), a model epoxide. Vesicles isolated from the two density fractions exhibited different protein profiles in Western blotting and silver staining. We have shown that a variety of clinically or host-relevant treatments, including antibiotics, also alter the proteins packaged in OMVs. Proteomic analysis of purified OMVs followed by an analysis of transposon mutant OMVs yielded mutants with altered vesicle packaging. Finally, epithelial cell cytotoxicity was reduced in the vesicles formed in the presence of EBH, suggesting that this epoxide alters the function of the OMVs. Our data support a model whereby clinically or host-relevant signals mediate differential packaging of virulence factors in OMVs, which results in functional consequences for host-pathogen interactions.
铜绿假单胞菌产生外膜囊泡（OMV），其中包含多种分泌的细菌蛋白，包括磷脂酶、碱性磷酸酶和囊性纤维化跨膜传导调节因子抑制因子（Cif）。此前研究表明，作为环氧化物水解酶的Cif在转录水平上受环氧化物调控，环氧化物作为阻遏物CifR的配体。在此，我们测试了环氧化物是否会对外膜囊泡中Cif的水平产生影响。我们发现，在特定环氧化物而非其水解产物存在的情况下，铜绿假单胞菌的生长会以不依赖CifR的方式增加Cif在外膜囊泡中的包装。在此条件下，外膜蛋白OprF也会增加，但碱性磷酸酶活性没有显著变化。此外，我们证明环氧化物处理会影响外膜囊泡的形状和密度，在用典型环氧化物表溴醇（EBH）处理细胞时会出现两种不同的囊泡组分。从这两个密度组分中分离出的囊泡在蛋白质印迹和银染中呈现出不同的蛋白质谱。我们已经表明，包括抗生素在内的多种临床或与宿主相关的处理也会改变外膜囊泡中包装的蛋白质。对纯化的外膜囊泡进行蛋白质组学分析，随后对转座子突变体的外膜囊泡进行分析，得到了囊泡包装发生改变的突变体。最后，在存在EBH的情况下形成的囊泡对上皮细胞的细胞毒性降低，这表明这种环氧化物改变了外膜囊泡的功能。我们的数据支持一种模型，即临床或与宿主相关的信号介导毒力因子在外膜囊泡中的差异包装，这会对宿主 - 病原体相互作用产生功能影响。
