Department of Chemistry, University of Toronto, Toronto, ON, Canada.
Department of Chemistry, University of Toronto, Toronto, ON, Canada.
Methods Enzymol. 2022;665:209-231. doi: 10.1016/bs.mie.2021.11.006. Epub 2021 Dec 7.
Bacterial biofilms consist of surface-attached communities that secrete polymeric substances to form a biofilm matrix, generating a local microenvironment which helps protect from external factors. One such matrix component produced by a diverse list of microorganisms is the polysaccharide poly-β-1,6-N-acetylglucosamine (PNAG). Dispersin B is a PNAG-specific glycosyl hydrolase, which by leveraging its unique specificity, can be used to design a macromolecular fluorescent PNAG binding probe. An active site mutant of Dispersin B was fused to a fluorescent protein, to generate a probe that bound PNAG but did not hydrolyze its polysaccharide target. The ease and versatility of this strategy has made it possible to study PNAG in the context of maturing biofilms, as the probe tends to sequester in regions of high PNAG density. In this chapter, typical workflows from probe construction to cell-binding and imaging experiments are described.
细菌生物膜由附着在表面的群落组成，它们分泌聚合物物质形成生物膜基质，产生有助于保护免受外部因素影响的局部微环境。由多种微生物产生的一种基质成分是多糖聚-β-1,6-N-乙酰葡糖胺（PNAG）。展青霉素 B 是一种 PNAG 特异性糖苷水解酶，通过利用其独特的特异性，可以用于设计高分子荧光 PNAG 结合探针。将展青霉素 B 的活性位点突变体融合到荧光蛋白上，生成一种探针，该探针可以结合 PNAG，但不水解其多糖靶标。这种策略的简便性和多功能性使得在成熟生物膜的背景下研究 PNAG 成为可能，因为探针往往会在 PNAG 密度高的区域被隔离。在本章中，描述了从探针构建到细胞结合和成像实验的典型工作流程。
