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单核细胞增生李斯特菌膜囊泡的制备及生物活性
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国家自然科学基金(31571931);遗传调控与整合生物学湖北省重点实验室开放课题(GRIB201709);中央高校基本科研业务费专项基金(CCNU18TS024)


Isolation and activity of Listeria monocytogenes-derived membrane vesicles
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    摘要:

    【背景】细胞外囊泡(Extracellular Vesicles,EVs)是一种在自然界中普遍存在的包含生物学活性物质的囊泡状结构,其中包括革兰氏阳性菌分泌的膜囊泡(Membrane Vesicles,MVs)。近年来,单核细胞增生李斯特菌(Listeria monocytogenes,Lm)作为一种能产MVs的革兰氏阳性致病菌引起了众多科研人员的关注,尽管如此,对其MVs的了解仍然有限。【目的】建立健全Lm-MVs制备方法,同时深入了解其生物活性。【方法】以Lm野生菌株EGDe及其毒力突变株(EGDe?prfA、EGDe?prfA+pERL3-prfA*)为研究对象,利用超滤浓缩法和Optiprep密度梯度离心法提取Lm的MVs,并对这两种提取MVs的方法进行比较。此外,通过检测不同MVs对菌株生物被膜的影响,以及不同MVs的溶血活性和对棉铃虫的感染毒性来探究MVs的生物活性。【结果】相较于超滤浓缩法,Optiprep密度梯度离心法提取的Lm-MVs产率高、电镜观测效果好,但该方法操作相对复杂,耗时较长;不同毒力的Lm均可向外分泌直径20?200 nm的MVs,而且MVs的形态结构与菌株的毒力无明显关系;但MVs可以影响菌株生物被膜的形成,具有一定的溶血活性,并能导致棉铃虫幼虫死亡或者降低其存活率和化蛹率,而且其毒性与菌株本身毒性正相关。【结论】Lm分泌的MVs极有可能直接参与了Lm对宿主的致病,并在细菌-细菌之间以及细菌-宿主相互作用中扮演了重要角色。研究结果将对进一步研究革兰氏阳性菌MVs的形成和功能以及Lm的致病机制具有重要意义。

    Abstract:

    [Background] Extracellular vesicles are lipid bound vesicles harboring specific subsets of bioactive proteins, lipids, nucleic acids, and metabolites, secreted by all living cells into the extracellular space. They have been named as membrane vesicles (MVs) in Gram-positive bacteria. Recently, vesicle production is described in Listeria monocytogenes, a Gram-positive intracellular foodborne pathogen that causes listeriosis in human as well as in animals. However, the knowledge about the impact of L. monocytogenes-derived MVs on host health and disease is still limited. [Objective] To establish and improve the isolation method to extract MVs from L. monocytogenes, and to explore its roles of biological activities. [Methods] Two methods, the ultrafiltration concentration method and Optiprep density gradient centrifugation method, were used to extract MVs from the wild strain EGDe and its virulence mutants (EGDe?prfA, EGDe?prfA+pERL3-prfA*) and compared. The impact of different virulent L. monocytogenes-derived membrane vesicles on the bacterial biofilm formation and pathogenesis to the insect infection model was investigated. [Results] Comparing with the ultrafiltration concentration method, the MVs isolated by the Optiprep density gradient centrifugation have got higher yield and presented more uniform and clear image under transmission electron microscope, but it’s relatively complicated and took a long time; All different virulent L. monocytogenes strains can secrete membrane vesicles with 20?200 nm in diameter, and no obvious differences in their shapes and sizes. However, these vesicles inhibited biofilm formation of L. monocytogenes, harbored a certain hemolytic activity, reduced the pupation and survival rate of the Helicoverpa armigera larvae, and even caused them death. The toxicity of MVs to the larvae showed a clear link with the virulence of strains they originated. [Conclusion] L. monocytogenes-derived membrane vesicles have got versatile roles, and likely participate in bacteria-bacteria and bacteria-host interactions. These results may be of great significance for the further study of the formation and function of Gram-positive bacteria-derived MVs and the pathogenic mechanism of L. monocytogenes.

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杨丽玉,杨诗怡,林巍,刘赛南,罗勤. 单核细胞增生李斯特菌膜囊泡的制备及生物活性[J]. 微生物学通报, 2021, 48(1): 145-155

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  • 在线发布日期: 2021-01-07
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