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首页> 外文期刊>RSC Advances >Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors
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Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

机译：细菌微阵列作为探索病原体表面表位和被宿主受体识别的敏感工具

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We describe a protocol for the generation and validation of bacteria microarrays and their application to the study of specific features of the pathogen's surface and interactions with host receptors. Bacteria were directly printed on nitrocellulose-coated glass slides, using either manual or robotic arrayers, and printing quality, immobilization efficiency and stability of the arrays were rigorously controlled by incorporating a fluorescent dye into the bacteria. A panel of wild type and mutant strains of the human pathogen Klebsiella pneumoniae, responsible for nosocomial and community-acquired infections, was selected as model bacteria, and SYTO-13 was used as dye. Fluorescence signals of the printed bacteria were found to exhibit a linear concentration-dependence in the range of 1 x 10(8) to 1 x 10(9) bacteria per ml. Similar results were obtained with Pseudomonas aeruginosa and Acinetobacter baumannii, two other human pathogens. Successful validation of the quality and applicability of the established microarrays was accomplished by testing the capacity of the bacteria array to detect recognition by anti-Klebsiella antibodies and by the complement subcomponent C1q, which binds K. pneumoniae in an antibody-independent manner. The biotin/AlexaFluor-647-streptavidin system was used for monitoring binding, yielding strain-and dose-dependent signals, distinctive for each protein. Furthermore, the potential of the bacteria microarray for investigating specific features, e.g. glycosylation patterns, of the cell surface was confirmed by examining the binding behaviour of a panel of plant lectins with diverse carbohydrate-binding specificities. This and other possible applications of the newly developed arrays, as e.g. screening/evaluation of compounds to identify inhibitors of host-pathogen interactions, make bacteria microarrays a useful and sensitive tool for both basic and applied research in microbiology, biomedicine and biotechnology.
机译：我们描述了一种协议，用于生成和验证细菌微阵列，并将其应用于研究病原体表面的特定特征以及与宿主受体的相互作用。使用手动或自动阵列仪将细菌直接印在涂有硝酸纤维素的载玻片上，并通过将荧光染料掺入细菌中来严格控制阵列的印刷质量，固定效率和稳定性。选择一组负责医院内和社区获得性感染的人类病原体肺炎克雷伯菌的野生型和突变株作为模型细菌，并将SYTO-13用作染料。发现印刷细菌的荧光信号在每毫升1 x 10（8）到1 x 10（9）细菌的范围内表现出线性的浓度依赖性。铜绿假单胞菌和鲍曼不动杆菌（两种其他人类病原体）也获得了相似的结果。通过测试细菌阵列检测抗克雷伯菌抗体和补体亚成分C1q（以抗体独立方式结合肺炎克雷伯氏菌）的能力，可以成功验证所建立微阵列的质量和适用性。生物素/ AlexaFluor-647-链霉亲和素系统用于监测结合，产生每种蛋白质都不同的应变和剂量依赖性信号。此外，细菌微阵列用于研究特定特征的潜力，例如通过检查一组具有各种碳水化合物结合特异性的植物凝集素的结合行为，证实了细胞表面的糖基化模式。新开发的阵列的这种和其他可能的应用，例如筛选/评估化合物以鉴定宿主-病原体相互作用的抑制剂，使细菌微阵列成为微生物学，生物医学和生物技术领域基础研究和应用研究的有用和敏感工具。

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