Department of Pediatrics, Morsani College of Medicine, Children's Research Institute, University of South Florida, Saint Petersburg, FL, USA.
Biology and Evolution of Marine Organisms (BEOM), Stazione Zoologica Anton Dohrn, Naples, Italy.
Methods Mol Biol. 2022;2421:127-140. doi: 10.1007/978-1-0716-1944-5_9.
The formation of biofilms is critical for the successful and stable colonization of mucosal surfaces by microbes, which often build three-dimensional environments by exuding exopolysaccharides and other macromolecules such as proteins, lipids, and even DNA. It is not just bacteria, but fungi such as yeast, that form these adherent interacting communities. Historically, biofilms have been studied in the context of pathogenesis, but only recently it has been recognized that important relationships among members of host-associated microbiomes are maintained within the context of biofilms. Host immune responses impact biofilm formation in various ways; for example, it is likely that formation of stable biofilms by non-pathogens improves barrier defenses by not just filling available niche spaces but also by helping to ward off pathogens directly. Recently, it was found that soluble immune effector molecules such as immunoglobulin A (IgA) in mammals serve essential roles in modulating complex biofilm communities in ways that benefit the host. Additional lines of evidence from other secreted immune effectors, such as the variable region-containing chitin-binding proteins (VCBPs) in protochordates, now suggest that this phenomenon is much more widespread than previously recognized. The activity of these immune molecules also likely serves roles beyond those of simple defense strategies; rather, they may be improving the outcome of symbiotic interactions benefiting the host. Thus, traditional immune assays that are aimed at studying the function of secreted immune effectors, such as agglutination assays, should take into account the possibility that the first observation may not be the last if the microbes under study are not directly killed. Here, we describe a series of simple approaches to characterize biofilm formation when bacteria (or yeast) are cultured in the presence of a secreted immune effector. To model this approach, we use microbes isolated from the gut of Ciona robusta, each grown in the presence or absence of VCBPs. The approaches defined here are amenable to diverse model systems and their microbes.
生物膜的形成对于微生物在黏膜表面成功和稳定定植至关重要，它们通常通过分泌胞外多糖和其他大分子物质（如蛋白质、脂质，甚至 DNA）来构建三维环境。不仅是细菌，还有酵母等真菌，也会形成这些附着的相互作用的群落。从历史上看，生物膜一直是在发病机制的背景下进行研究的，但直到最近才认识到，宿主相关微生物组的成员之间的重要关系是在生物膜的背景下维持的。宿主免疫反应以各种方式影响生物膜的形成；例如，非病原体形成稳定的生物膜不仅通过填补可用的生态位空间，而且通过直接帮助抵御病原体，可能改善屏障防御。最近发现，哺乳动物中的可溶性免疫效应分子，如免疫球蛋白 A (IgA)，在调节复杂生物膜群落方面发挥着至关重要的作用，从而使宿主受益。来自其他分泌免疫效应物的额外证据，如原索动物中的可变区结合壳多糖结合蛋白 (VCBP)，现在表明这种现象比以前认识到的更为普遍。这些免疫分子的活性也可能不仅在简单的防御策略方面发挥作用；相反，它们可能通过改善对宿主有益的共生相互作用的结果来发挥作用。因此，旨在研究分泌免疫效应物功能的传统免疫测定，例如凝集测定，应该考虑到如果研究的微生物没有被直接杀死，那么第一个观察结果可能不是最后一个。在这里，我们描述了一系列简单的方法来描述当细菌（或酵母）在分泌免疫效应物存在的情况下培养时生物膜的形成。为了模拟这种方法，我们使用从 Ciona robusta 肠道中分离的微生物，每种微生物在存在或不存在 VCBP 的情况下生长。这里定义的方法适用于各种模型系统及其微生物。
