The oral microbiome plays a crucial role not only in oral health but also in systemic health. Dysfunctional oral
microbiomes have been linked to various chronic human diseases. However, understanding how the
organization of the oral microbiome is connected to its function remains a major challenge. Next-generation DNA
sequencing has revolutionized the study of oral microbial communities by providing comprehensive information
about their composition. CLASI-FISH has contributed to our understanding by revealing the spatial organization
of the microbiome. While imaging can inform us about which microbes are in proximity to each other at the
micron scale, it cannot alone differentiate whether this spatial proximity is due to functional interactions or chance
occurrences. To overcome this limitation, our proposed study aims to investigate microbe-microbe adhesion as
a functional attribute of their interactions. We will employ a combination of expansion microscopy (ExM) and
multiplex FISH imaging, which will provide a unique opportunity to globally analyze microbial interactions at the
micron level. This approach will allow us to explore the functional aspects of microbial communities beyond their
spatial arrangement. Furthermore, the presence of thick biofilms or aggregates within their ecological niches
presents significant challenges for single-cell dissection and imaging, often leading to difficulties or ambiguities
in taxonomic assignment. Our proposed study will address this concern by quantifying the relative microbial
adhesion forces at the single-cell level. This will enhance our understanding of the forces governing microbial
interactions within biofilms and contribute to unraveling the complexity of the oral microbiome. Overall, this study
aims to investigate the functional attributes of the oral microbiome at the micron scale, bridging the gap between
microbial organization and function. By employing advanced microscopy techniques and quantifying microbial
adhesion forces, we hope to gain valuable insights into the intricate dynamics of the oral microbiome and its
implications for human health. There are three interconnected Aims: 1) To combine multiplex FISH imaging with
Expansion Microscopy. Here, the focus will be investigating the feasibility of combining multiplex peptide nucleic
acid (PNA) FISH imaging with ExM on an in vitro polymicrobial biofilm. 2) To quantify relative microbe-microbe
adhesion force. Here, expansion forces within the gel matrices will be calibrated through force-reference bead
strategy. Following PNA FISH hybridization on the polymicrobial biofilm, the microbial adhesion forces within the
biofilm will be quantified by comparison with gel expansion forces. 3) To evaluate species-level adhesion forces
within natural human oral biofilms. Here, we will validate our adhesion force measurement approach using a
diverse range of in vitro coaggregation pairs with established binding phenotypes. Then species-level adhesive
forces within human natural oral biofilms will be obtained. This independence award (PA-20-188) will foster my
independent research growth and allow me to accomplish my long-term career goal of becoming a productive,
independent translational scientist in the field of oral microbiology.
