Schurwanz Nicol, Jacobs Enno, Dumke Roger
Dresden University of Technology, Medical Faculty Carl Gustav Carus, Institute of Medical Microbiology and Hygiene, Fetscherstrasse 74, D-01307 Dresden, Germany.
Infect Immun. 2009 Nov;77(11):5007-15. doi: 10.1128/IAI.00268-09. Epub 2009 Aug 10.
The cell wall-less bacterium Mycoplasma pneumoniae is one of the most common agents of respiratory tract diseases in humans. Adhesin-mediated binding of the bacteria to host cells is a crucial step in colonization and subsequent pathogenesis. For the first time, we expressed 16 recombinant proteins covering almost the whole major adhesin P1 and the adherence-associated protein P30 to characterize these proteins immunologically and functionally. We describe a new in vitro assay using several human cell lines in combination with fluorescence-activated cell sorting analysis to screen antisera raised against the recombinant proteins quantitatively for adherence inhibition activity. The protein derived from the nearly C-terminal part of the P1 adhesin (amino acids [aa] 1288 to 1518) and the protein P30 (aa 17 to 274) especially showed prominent immunoreactivity with sera from M. pneumoniae-immunized guinea pigs as well as with M. pneumoniae-positive patient sera. We demonstrate that the same protein regions are involved in mediating cytadherence since antibodies against these adhesin regions decrease mycoplasma adhesion to human cells significantly. For further vaccine studies, we optimized the immunogenic and adherence-mediating properties of the antigen by combining both the P1 and the P30 regions in a novel chimeric protein. Antibodies against this protein show an increased reduction of M. pneumoniae adherence to human bronchial epithelial cells by 95%, which is comparable to results with polyspecific anti-M. pneumoniae animal serum. Our strategy results in a promising defined antigen candidate for reducing or even preventing M. pneumoniae colonization of the respiratory tract in future vaccination studies.
无细胞壁细菌肺炎支原体是人类呼吸道疾病最常见的病原体之一。细菌通过黏附素与宿主细胞结合是定植及后续发病机制中的关键步骤。我们首次表达了16种重组蛋白，这些重组蛋白几乎覆盖了整个主要黏附素P1和黏附相关蛋白P30，以便从免疫学和功能上对这些蛋白进行表征。我们描述了一种新的体外试验，该试验使用几种人类细胞系并结合荧光激活细胞分选分析，以定量筛选针对重组蛋白产生的抗血清的黏附抑制活性。源自P1黏附素近C末端部分（氨基酸[aa]1288至1518）的蛋白和P30蛋白（aa 17至274）尤其与肺炎支原体免疫豚鼠的血清以及肺炎支原体阳性患者血清显示出显著的免疫反应性。我们证明相同的蛋白区域参与介导细胞黏附，因为针对这些黏附素区域的抗体可显著降低支原体对人类细胞的黏附。为了进一步开展疫苗研究，我们通过将P1和P30区域组合在一种新型嵌合蛋白中来优化抗原的免疫原性和黏附介导特性。针对该蛋白的抗体使肺炎支原体对人支气管上皮细胞的黏附减少增加了95%，这与多特异性抗肺炎支原体动物血清的结果相当。我们的策略为未来疫苗研究提供了一种有前景的确定抗原候选物，用于减少甚至预防肺炎支原体在呼吸道的定植。
