Dong Pu-Ting, Shi Wenyuan, He Xuesong, Borisy Gary G
Department of Microbiology, The American Dental Association Forsyth Institute, Cambridge, MA 02142, USA.
bioRxiv. 2024 Jun 25:2024.06.25.600639. doi: 10.1101/2024.06.25.600639.
Investigating microbe-microbe interactions at the single-cell level is critical to unraveling the ecology and dynamics of microbial communities. In many situations, microbes assemble themselves into densely packed multi-species biofilms. The density and complexity pose acute difficulties for visualizing individual cells and analyzing their interactions. Here, we address this problem through an unconventional application of expansion microscopy, which allows for the 'decrowding' of individual bacterial cells within a multispecies community. Expansion microscopy generally has been carried out under isotropic expansion conditions and used as a resolution-enhancing method. In our variation of expansion microscopy, we carry out expansion under heterotropic conditions; that is, we expand the space between bacterial cells but not the space within individual cells. The separation of individual bacterial cells from each other reflects the competition between the expansion force pulling them apart and the adhesion force holding them together. We employed heterotropic expansion microscopy to study the relative strength of adhesion in model biofilm communities. These included mono and dual-species biofilms, and a three-species synthetic community (, , and ) under conditions that facilitated interspecies coaggregation. Using adhesion mutants, we investigated the interplay between outer membrane protein RadD and different species. We also examined the epibiont association. Quantitative proximity analysis was used to evaluate the separation of individual microbial members. Our study demonstrates that heterotropic expansion microscopy can 'decrowd' dense biofilm communities, improve visualization of individual bacterial members, and enable analysis of microbe-microbe adhesive interactions at the single-cell level.
在单细胞水平上研究微生物与微生物之间的相互作用对于揭示微生物群落的生态和动态至关重要。在许多情况下，微生物会自行组装成密集的多物种生物膜。这种密度和复杂性给可视化单个细胞并分析它们之间的相互作用带来了极大的困难。在这里，我们通过一种非传统的扩展显微镜应用来解决这个问题，这种方法可以使多物种群落中的单个细菌细胞“疏解”。扩展显微镜通常是在各向同性扩展条件下进行的，并用作一种提高分辨率的方法。在我们改进的扩展显微镜方法中，我们在非各向同性条件下进行扩展；也就是说，我们扩展细菌细胞之间的空间，但不扩展单个细胞内部的空间。单个细菌细胞彼此分离反映了将它们拉开的扩展力与将它们聚集在一起的粘附力之间的竞争。我们采用非各向同性扩展显微镜来研究模型生物膜群落中粘附的相对强度。这些包括单物种和双物种生物膜，以及在促进种间共聚集的条件下的三物种合成群落（、和）。利用粘附突变体，我们研究了外膜蛋白RadD与不同物种之间的相互作用。我们还研究了体表共生体的关联。定量接近度分析用于评估单个微生物成员之间的分离情况。我们的研究表明，非各向同性扩展显微镜可以“疏解”密集的生物膜群落，改善单个细菌成员的可视化，并能够在单细胞水平上分析微生物与微生物之间的粘附相互作用。
