UNLABELLED

Diet has strong impacts on the composition and function of the gut microbiota with implications for host health. Therefore, it is critical to identify the dietary components that support growth of specific microorganisms . We used protein-based stable isotope fingerprinting (Protein-SIF) to link microbial species in gut microbiota to their carbon sources by measuring each microbe's natural C content (δC) and matching it to the C content of available substrates. We fed gnotobiotic mice, inoculated with a 13 member microbiota, diets in which the C content of all components was known. We varied the source of protein, fiber or fat to observe C signature changes in microbial consumers of these substrates. We observed significant changes in the δC values and abundances of specific microbiota species, as well as host proteins, in response to changes in C signature or type of protein, fiber, and fat sources. Using this approach we were able to show that upon switching dietary source of protein, fiber, or fat (1) some microbial species continued to obtain their carbon from the same dietary component (e.g., protein); (2) some species switched their main substrate type (e.g., from protein to carbohydrates); and (3) some species might derive their carbon through foraging on host compounds. Our results demonstrate that Protein-SIF can be used to identify the dietary-derived substrates assimilated into proteins by microbes in the intestinal tract; this approach holds promise for the analysis of microbiome substrate usage in humans without the need of substrate labeling.

SIGNIFICANCE

The gut microbiota plays a critical role in the health of animals including humans, influencing metabolism, the immune system, and even behavior. Diet is one of the most significant factors in determining the function and composition of the gut microbiota, but our understanding of how specific dietary components directly impact individual microbes remains limited. We present the application of an approach that measures the carbon isotope "fingerprint" of proteins in biological samples. This fingerprint is similar to the fingerprint of the substrate used to make the proteins. We describe how we used this approach in mice to determine which dietary components specific intestinal microbes use as carbon sources to make their proteins. This approach can directly identify components of an animal's diet that are consumed by gut microbes.