中文摘要：本研究建立了一种稳定有效的标记支原体细胞的荧光分析方法。对支原体中Tn4001- mini转座子进行修饰，以实现mCherry、mKO2和mNeonGreen的持续高效表达。应用这些方法诱导遗传距离较远的牛支原体和丝状支原体亚种相应的荧光蛋白表达，并以此作为两种支原体的染色体标签。荧光克隆的分析结果表明，2种支原体中观察到绿色和红色两种荧光克隆，且均无明显的细胞毒性。采用流式细胞仪分析2种支原体的荧光表达量，结果表明这些方法可广泛应用于支原体各分支。巨噬细胞感染试验表明，mNeonGreen表达系可用于监测支原体感染和细胞侵染。应用流式细胞仪检测和定量分析吞噬细胞内荧光支原体的含量，并通过共焦显微镜进行验证能实现对感染细胞细胞质内单个支原体的鉴定。研究建立的荧光分析方法适用于宿主-病原互作分析，且在体内外分析支原体功能方面具有不可估量的应用前景。
外文摘要：Fluorescence expression tools for stable and innocuous whole mycoplasma cell labelling have been developed. A Tn4001-derivative mini-transposon affording unmarked, stable mutagenesis in mycoplasmas was modified to allow the constitutive, high-level expression of mCherry, mKO2 and mNeonGreen. These tools were used to introduce the respective fluorescent proteins as chromosomal tags in the phylogenetically distant species Mycoplasma mycoides subsp. mycoides and Mycoplasma bovis. The production, selection and characterisation of fluorescent clones were straightforward and resulted in the unprecedented observation of red and green fluorescent mycoplasma colonies in the two species, with no apparent cytotoxicity. Equivalent fluorescence expression levels were quantified by flow cytometry in both species, suggesting that these tools can be broadly applied in mycoplasmas. A macrophage infection assay was performed to assess the usefulness of mNeonGreen-expressing strains for monitoring mycoplasma infections, and notably cell invasion. The presence of fluorescent mycoplasmas inside live phagocytic cells was detected and quantified by flow cytometry and corroborated by confocal microscopy, which allowed the identification of individual mycoplasmas in the cytoplasm of infected cells. The fluorescence expression tools developed in this study are suitable for host-pathogen interaction studies and offer innumerable perspectives for the functional analysis of mycoplasmas both in vitro and in vivo.
外文关键词：Mycoplasma；Fluorescence；Whole cell labelling；Host-pathogen interactions；Flow cytometry；Confocal microscopy
作者：Bonnefois, T； Vernerey, MS；Rodrigues, V等
作者单位：法国农业发展研究中心
期刊名称：JOURNAL OF BIOTECHNOLOGY
期刊影响因子：1.403
出版年份：2016
出版刊次：10
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