Poster R11 - Analysis of microbial adhesion at the single-cell level through expansion microscopy

Introduction: 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 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.

Materials and

Methods: Gelation, digestion, expansion on biofilms
S. mutans UA159 biofilms and dual-species Streptococcus biofilms were fixed in 10% formalin for 1 hour, then glycine (100 mM) was added to quench the fixation for 5 min. After that, fixed biofilm samples were washed with 1×PBS twice before the gelation step. S. mutans UA159 biofilms were then stained with SYTO 9. 0.1 mg/ml Acryloyl X (AcX) in PBS was immediately added to the fixed biofilms samples overnight. After incubation with AcX, biofilms were washed with PBS twice, each time for 15 min. Then monomer solution was added to biofilms and gelation was allowed to occur for 1 hour at 37℃. Immediately afterwards, digestion was carried out in the presence of proteinase K (10 U/ml). After overnight-digestion, expansion was induced by adding water to the digested samples, and exchange with water for three times, each time for 20 min. Part of the expanded samples were then carefully mounted to a poly-L-lysine-coated cover glass, and a few drops of water were added to the top of the gel before image acquisition to prevent sample dehydration.

Gelation, expansion of three-member aggregates
Gelation in the presence of the monomer solution (Stock X) was immediately applied to the semi-dried three-member aggregates at 37℃ for 1 hour at a humidified chamber. Spectral imaging was then employed after gelation to acquire images before expansion. After imaging, water was directly added to the gels, and imaging after expansion.

Results, Conclusions, and Discussions: Our study demonstrates the utility of an unconventional application of expansion microscopy to the analysis of microbial biofilms. We show how heterotropic expansion can ‘decrowd’ dense biofilm communities, improve visualization of individual bacterial members, and enable differentiation of microbe-microbe adhesive interactions within microbial communities at the single-cell level.
The application is unconventional in that its purpose is to exploit the force driving expansion to assess microbial adhesion rather than to achieve super-resolution imaging. In conventional expansion microscopy, the biological matter is digested to enable uniform, isotropic expansion of the polymer gel. In contrast, in our application, the microbes remain intact while being entrapped in the polymer network. The space between the microbes is expanded while the space within the microbes is not, hence the term heterotropic expansion. Expansion of the swellable polymer gel generates a pulling force on the entrapped microbes. The result is a 'tug-of-war' competition between the expansion force and microbial adhesive forces. Weakly adherent cells will be pulled apart while strongly adherent cells will remain together. Thus, heterotropic expansion enables the differentiation of microbe-microbe interaction within microbial communities at the single-cell level.
Our approach for differentiating adhesive interactions within microbial consortia at the single-cell level has some limitations. First, microbial consortia often involve multiple bacterial species, where multiplexed fluorescence imaging is needed. Our study investigated adhesive interactions in a model microbial community consisting of three members. To investigate the feasibility of this single-cell functional platform within complex microbial communities, it will be necessary to evaluate the compatibility between heterotropic expansion microscopy and multiplexed fluorescence imaging. Second, our approach thus far achieved the differentiation of interspecies microbial adhesive strengths on a relative basis. However, the absolute binding strengths between microbes with their neighbors remain to be determined. For the quantification of microbial binding forces, a calibration method will be needed to quantify the gel expansion force. Finally, we need to take into consideration whether the expansion process affects the viability of microbes. Notwithstanding these limitations, our new approach establishes a proof-of-principle for differentiating adhesive interactions within microbial consortia at the single-cell level.

Acknowledgements (Optional): This work was supported by National Institutes of Health (NIH) grant T90 DE026110 (to P.-T.D.), Forsyth Pilot Grant FPILOT80 (to P.-T.D.), NIH Grant DE022586 (to J.L.M.W and G.G.B), and NIH Grant DE023810, DE030943 (to X.H). We thank Ms. Jennifer Gundrum for technical assistance, Dr. Jessica L. Mark Welch and Dr. S. Tabita Ramirez-Puebla for helpful discussions.