The biomass of the human microbiome is estimated to account for 1-3% of total host body weight. This bulk quantity includes an abundance of microbial metabolites and macromolecules that are sensed by and alter host physiology. In fact, recent studies have highlighted how microbes within this community have evolved surface macromolecules with unique inflammatory responses, redefining our understanding of bacterial sensing and signaling within the immune system. Despite these advances, the technology available to track these molecules in host cells and tissue remains primitive. Thus, there is correspondingly little known about the spatial and temporal distribution of the microbial products that signal through host receptors within the tissue. Since most of the mammalian microbiome resides in the intestine, deciphering how and when these immunomodulatory bacterial molecules are obtained by the host is especially important towards understanding the progression of colorectal cancer; a disease in which the innate immune sensing system fundamentally fails, leading to a highly inflammatory state and a tumorigenic environment.
As an answer to these technical restrictions, we have developed an interdisciplinary approach utilizing bioorthogonal click chemistry (i.e. reactions performed in living organisms) to label and track specific surface macromolecules of commensal bacteria. By incorporating non-natural sugars and amino acids into bacterial cell surface molecules, we are able to fluorescently tag the lipopolysaccharide (LPS), capsular polysaccharide (CPS) and peptidoglycan (PGN) of a wide variety of live intestinal bacteria including gram positive and gram negative species. Combining this cutting-edge chemistry with modern methods in immunology and microscopy has allowed us to simultaneously track these Toll-like receptor ligands (LPS, CPS) and NOD-like receptor ligands (PGN) of the living commensal bacteria within the host tissue. Furthermore, we present data demonstrating that we can successfully label the PGN of endogenous bacteria within the murine host, which expands our methodology to track bacterial PGN within an established, living community.
Utilizing this approach, we are able to trace the differentially labeled surface molecules into specific host immune cells within the intestine and isolate these cells based on bacterial ligand uptake. Additionally, we are using two-photon intravital microscopy to image commensals and these surface molecules within the small intestine and colon of the living murine host. We are currently applying this system to investigate whether certain cell types associate with distinct commensal molecules and observe changes associated with disease progression in mouse models of colitis and colitis-associated colorectal cancer. We hope these tools will find broad use in the field to decipher the role of specific bacterial surface molecules in host response.
J.E.H. is supported by a Cancer Research Institute Irvington postdoctoral fellowship.
Citation Format: Jason E. Hudak, David Alvarez, Ashwin Skelly, Ulrich H. von Andrian, Dennis L. Kasper. Bioorthogonal chemical labeling of specific immunomodulatory surface molecules in live commensals [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR07.
- ©2016 American Association for Cancer Research.