Adhesive interactions within microbial consortia can be differentiated at the single-cell level through expansion microscopy

Abstract

SignificanceA single-cell understanding of microbe-microbe interactions is critical for unraveling the organization and dynamics of microbial communities. Through an unconventional application of expansion microscopy, we oppose the adhesive force holding microbes together by an expansion force pulling them apart, resulting in microbial separation dependent on the strength of microbial adhesion. Our new approach establishes a proof-of-principle for differentiating adhesive interactions within microbial consortia at the single-cell level.AbstractInvestigating 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-speciesStreptococcusbiofilms, and a three-species synthetic community (Fusobacterium nucleatum,Streptococcus mutans, andStreptococcus sanguinis) under conditions that facilitated interspecies coaggregation. Using adhesion mutants, we investigated the interplay betweenF. nucleatumouter membrane protein RadD and differentStreptococcusspecies. We also examined theSchaalia-TM7epibiont 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.