Sivaranjani Murugesan, Hansen Elizabeth G, Perera Sumudu R, Flores Pamela A, Tükel Çagla, White Aaron P
Vaccine and Infectious Disease Organization, Saskatoon, Saskatchewan, Canada.
Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Bio Protoc. 2022 May 20;12(10):e4419. doi: 10.21769/BioProtoc.4419.
Microbiologists are learning to appreciate the importance of "functional amyloids" that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell communication. Bacterial functional amyloids are also beginning to be appreciated in the context of host-pathogen interactions, where there is evidence that they can trigger the innate immune system and are recognized as non-self-molecular patterns. The characteristic three-dimensional fold of amyloids renders them similar across the bacterial kingdom and into the eukaryotic world, where amyloid proteins can be undesirable and have pathological consequences. The bacterial protein curli, produced by pathogenic and strains, was one of the first functional amyloids discovered. Curli have since been well characterized in terms of function, and we are just starting to scratch the surface about their potential impact on eukaryotic hosts. In this manuscript, we present step-by-step protocols with pictures showing how to purify these bacterial surface structures. We have described the purification process from , acknowledging that the same method can be applied to . In addition, we describe methods for detection of curli within animal tissues (, GI tract) and discuss purifying curli intermediates in a mutant strain as they are more cytotoxic than mature curli fibrils. Some of these methods were first described elsewhere, but we wanted to assemble them together in more detail to make it easier for researchers who want to purify curli for use in biological experiments. Our aim is to provide methods that are useful for specialists and non-specialists as bacterial amyloids become of increasing importance.
微生物学家开始认识到“功能性淀粉样蛋白”的重要性，许多细菌物种都会产生这种蛋白，其影响超出了微生物界。细菌利用这些结构连接在一起，推测是为了提高生存能力、抵御恶劣环境，或许还能影响细胞间通讯。在宿主与病原体的相互作用中，细菌功能性淀粉样蛋白也开始受到关注，有证据表明它们能触发先天免疫系统，并被识别为非自身分子模式。淀粉样蛋白独特的三维折叠结构使其在细菌界乃至真核生物界都具有相似性，而在真核生物中，淀粉样蛋白可能是有害的，并会产生病理后果。由致病性大肠杆菌菌株产生的细菌蛋白卷曲纤维（curli）是最早发现的功能性淀粉样蛋白之一。此后，卷曲纤维在功能方面得到了充分的表征，而我们才刚刚开始探究它们对真核宿主的潜在影响。在本论文中，我们提供了详细的步骤方案，并配有图片，展示了如何纯化这些细菌表面结构。我们描述了从大肠杆菌中进行纯化的过程，同时承认相同的方法也可应用于其他菌株。此外，我们还介绍了在动物组织（如胃肠道）中检测卷曲纤维的方法，并讨论了在一种突变菌株中纯化卷曲纤维中间体的方法，因为它们比成熟的卷曲纤维原纤维更具细胞毒性。其中一些方法最初在其他地方有过描述，但我们希望将它们更详细地整合在一起，以便那些想要纯化卷曲纤维用于生物学实验的研究人员更容易操作。我们的目标是提供对专业人员和非专业人员都有用的方法，因为细菌淀粉样蛋白正变得越来越重要。
