Characterization of diet-linked amino acid pool influence on <i>Fusobacterium</i> spp. growth and metabolism

The genus <i>Fusobacterium</i> contains multiple proteolytic opportunistic pathogens that have been increasingly linked to colorectal cancer (CRC). "Oncomicrobes" such as these fusobacterial species within the gut microbiota may contribute to CRC onset and/or progression. Protein-rich diets may both directly increase CRC risk and enrich for proteolytic oncomicrobes, including <i>Fusobacterium</i> spp. Individual food substrates vary in amino acid content, and released amino acid content that is not absorbed in the small intestine may influence the growth of colonic proteolytic fermenters. Fusobacteria such as <i>Fusobacterium</i> spp. are known to preferentially metabolize certain amino acids. As such, some foods may better support the growth of these species within the colonic environment than others. To explore this, in this study, we created free amino acid pools (FAAPs) to represent proportions of amino acids in major proteins of three common dietary protein sources (soy, beef, and bovine milk). Growth curves were generated for 39 <i>Fusobacterium</i> spp. strains cultured in a dilute medium supplemented with each of the three FAAPs. Thereafter, amino acid use by 31 of the 39 <i>Fusobacterium</i> spp. strains in each FAAP treatment was assessed. FAAP supplementation increased growth metrics of all <i>Fusobacterium</i> spp. strains tested; however, the strains varied greatly in terms of the FAAP(s) generating the greatest increase in growth. Furthermore, the amino acid utilization strategy was highly variable between strains of <i>Fusobacterium</i> spp. Neither growth metrics nor amino acid utilization could be explained by species classification of <i>Fusobacterium</i> spp. strains. This report expands upon the previous knowledge of fusobacterial amino acid metabolism and indicates that proteolytic oncomicrobial activity should be assessed in the context of available protein sources.IMPORTANCE<i>Fusobacterium</i> spp. including <i>F. animalis</i>, <i>F. nucleatum</i>, <i>F. vincentii</i>, and <i>F. polymorphum</i> are common oral commensals with emerging importance in diseases across multiple body sites, including CRC. CRC lesions associated with fusobacteria tend to result in poorer prognosis and increased disease recurrence. While <i>Fusobacterium</i> spp. are thought to colonize after tumorigenesis, little is known about the factors that facilitate this colonization. Protein-rich diets yielding readily metabolized free amino acids within the colon may promote the growth of proteolytic fermenters such as fusobacteria. Here, we show that variable concentrations of free amino acids within pools that represent different dietary protein sources differentially influence fusobacterial growth, including CRC-relevant strains of <i>Fusobacterium</i> spp. This work highlights the high degree of variation in fusobacterial amino acid utilization patterns and suggests differing proportions of dietary amino acids that reach the colon could influence fusobacterial growth.