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

Abstract

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 multispecies 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 Streptococcus biofilms and a three-species synthetic community (Fusobacterium nucleatum, Streptococcus mutans, and Streptococcus sanguinis) under conditions that facilitated interspecies coaggregation. Using adhesion mutants, we investigated the interplay between F. nucleatum outer membrane protein RadD and different Streptococcus species. We also examined the Schaalia-TM7 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.

Keywords: expansion microscopy; human microbiome; microbial adhesion; polymicrobial communities; single-cell level.

Fig. 1.

The diagram illustrates application of expansion microscopy for the differentiation of microbe–microbe adhesive interactions within a microbial community. Strongly adhesive bacteria (green and yellow) are not pulled apart during expansion while weakly adhesive bacteria (blue vs green and yellow) are pulled apart.

Fig. 2.

The expansion procedure separates bacteria away from each other in a monospecies biofilm. A representative image of monospecies biofilm before expansion (A) and after expansion (B). Monospecies S. mutans (SYTO nine-labeled; green) biofilms were cultured with fluorescent beads (magenta) serving as an internal standard for nonadhesive objects. Images obtained with 63× oil immersion objective (NA = 1.4).

Fig. 3.

The expansion procedure allows visualization of individual bacteria within a dual-species biofilm. A representative image along with a high-magnification image callout of dual-species biofilm before (A) and after expansion (B). Dual-member Streptococcus biofilms were cultured with fluorescent beads serving as an internal standard. Color annotation: fluorescent beads (magenta), mCherry-encoding S. mutans (yellow), and GFP-encoding S. sanguinis (cyan).

Fig. 4.

Linear dipole analysis quantifies spatial organization. Proximity analysis of fluorescent beads before and after expansion (A). Linear dipole analysis of images in Fig. 3 shows within-taxon autocorrelation (B and C); intertaxon correlation between S. mutans and S. sanguinis (D). Data: Mean (solid line) with 95% CI (shaded area). A pair correlation value of 1 is highlighted by orange dashed lines in the figure panels. Pairwise correlations were calculated using 500,000 random dipoles per field of view.

Fig. 5.

Expansion microscopy differentiates the interaction between F. nucleatum wild type with two Streptococcus species. (A). Spectral imaging (after linear unmixing) of F. nucleatum–S. mutans–S. sanguinis aggregates before expansion under a 63× oil immersion objective (NA = 1.4). (Scale bar, 10 µm.) (B). Spectral imaging (after linear unmixing) of F. nucleatum–S. mutans–S. sanguinis aggregates after expansion. 20× objective (NA = 0.8). (Scale bar, 50 µm.) (C). Orthoslice views of F. nucleatum–S. mutans–S. sanguinis aggregates in panel (B) in the axial and lateral directions. (D and E). High-magnification views (D) and single-cell image callouts (E) of merged view of F. nucleatum wild type with S. mutans and S. sanguinis. (F and G). Proximity analysis of image in (D) between F. nucleatum and different Streptococcus species aggregates after expansion. A pair correlation value of 1 is highlighted by orange dashed lines in the figure panels. Pairwise correlations were calculated using 500,000 random dipoles per field of view. Color annotation: F. nucleatum (white), S. mutans (red), and S. sanguinis (green).

Fig. 6.

Expansion microscopy differentiates the interaction between F. nucleatum ΔradD with two Streptococcus species. (A). Coaggregation assay comparison between F. nucleatum wild type and F. nucleatum ΔradD with two different Streptococcus species. Images obtained by phase microscopy; insert shows the aggregation in test tube with heatmap to help visualize flocculant precipitate. (B). Spectral imaging (after linear unmixing) of F. nucleatum ΔradD–S. mutans–S. sanguinis aggregates after expansion under a 20× objective (NA = 0.8). (Scale bar, 50 µm.) (C and D). High-magnification views (C) and single-cell image callouts (D) of merged view of F. nucleatum ΔradD with S. mutans and S. sanguinis. (E and F). Proximity analysis of image in (C) between F. nucleatum ΔradD and different Streptococcus species aggregates after expansion. A pair correlation value of 1 is highlighted by orange dashed lines in the figure panels. Pairwise correlations were calculated using 500,000 random dipoles per field of view. Color annotation: F. nucleatum ΔradD (white), S. mutans (red), and S. sanguinis (green).

Fig. 7.

Expansion force does not pull apart the epibiont interaction pair: Saccharibacteria N. lyticus strain TM7x and S. odontolytica (XH001). Confocal fluorescence imaging of SYTO 9-labeled coculture XH001–TM7x complexes before (A) and after expansion (B). Two regions of interest (ROI), dashed white boxes, are shown at high magnification (Inserts). TM7x cells are highlighted by white arrows.