Adhesion and inhibition assay of Mycoplasma genitalium and M-pneumoniae by immunofluorescence microscopy
Adhesion and inhibition assay of Mycoplasma genitalium and M-pneumoniae by immunofluorescence microscopy
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DOI：
10.1099/0022-1317-51-5-361
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发表时间：
2002-05-01
影响因子：
3
通讯作者：
Christiansen, G
中科院分区：
文献类型：
--
作者：
Svenstrup, HF;Nielsen, PK;Christiansen, G
对肺炎支原体及其亲缘关系密切的生殖支原体与Hep-2细胞的粘附性进行了研究。已知参与黏附的主要表面蛋白是肺炎支原体的PI及其同系物生殖支原体的MGPA。这两种蛋白也都是免疫优势蛋白。P116蛋白是肺炎支原体的另一种免疫优势蛋白。对这些免疫原蛋白的表面暴露和与宿主上皮细胞的粘连进行了研究。用免疫荧光显微镜(IFM)检测Hep-2细胞上肺炎支原体和生殖支原体的黏附情况。针对缺乏色氨酸终止密码子的表面蛋白片段制备单特异性抗体，用于黏附检测、表面暴露和黏附抑制IFM检测。制备了3株针对MGPA N-端、中部和C端覆盖区的单特异性抗体，2株针对P1覆盖区N-端和C-末端的单特异性抗体，1株针对P116大部分的单特异性抗体。只有P1和MGPA的C末端部分暴露在表面，用单特异性抗体封闭这些区域会抑制细胞的吸附。蛋白P116被证明是表面暴露的，并且是参与黏附的必要蛋白质，因为抗P116抗体阻止肺炎支原体独立于P1附着到Hep-2细胞。这项研究增加了对生殖支原体和肺炎支原体分子生物学的了解，并提供了一种研究这些支原体黏附蛋白的方法。
Adhesion of Mycoplasma pneumoniae and the closely related M. genitalium to HEp-2 cells was investigated. The main surface proteins known to be involved in adhesion are PI of M. pneumoniae and its homologue, MgPa, of M. genitalium. Both proteins are also immunodominant proteins. Protein P116 is another immunodominant protein of M. pneunmoniae. These immunogenic proteins were investigated for their surface exposure and involvement in adhesion to host epithelial cells. Immunofluorescence microscopy (IFM) was used to detect M. pneumoniae and M. genitalium adhering to HEp-2 cells. Monospecific antibodies were produced against fragments of the surface proteins lacking tryptophan stop codons and were used for adhesion detection, surface exposure and adhesion inhibition IFM assays. Three monospecific antibodies were made against MgPa covering regions in the N-terminal, the middle and the C-terminal part; two monospecific antibodies were produced against P1 covering regions of the N- and the C-terminal part and one monospecific antibody was made against most of P116. Only the C-terminal parts of P1 and MgPa were surface exposed and blocking of these regions with the monospecific antibody resulted in inhibition of cytadsorption. Protein P116 was shown to be surface exposed and an essential protein involved in adhesion because the anti-P116 antibody prevented attachment of M. pneumoniae to the HEp-2 cells independently of P1. This study adds to the understanding of the molecular biology of M. genitalium and M. pneumoniae and presents a method to study the proteins involved in adhesion of these mycoplasmas.