A genetic toolkit for the human gut bacterium Mediterraneibacter gnavus identifies capsular polysaccharides as a competitive colonization factor

Mediterraneibacter gnavus is a human symbiotic gut bacterium whose abundance often increases in patients with various diseases, such as active inflammatory bowel disease (IBD). However, the genetic factors governing its gut colonization and pathogenicity remain elusive due to the lack of genetic modification systems. In this study, we developed several genetic tools for M. gnavus, including a shuttle vector, an inducible promoter, fluorescent reporters, and systems for gene disruption and deletion. Using these genetic tools, we constructed mutants for six of the eight sortase-encoding genes in M. gnavus ATCC 29149 and identified those involved in the surface presentation of capsular polysaccharide (CPS) and superantigen-like proteins. We also identified a CPS biosynthetic gene cluster adjacent to the sortase gene and demonstrated that CPS production is crucial for competitive colonization in germ-free mouse intestines. Notably, CPS production was inversely correlated with inflammatory activity, and CPS cluster-positive strains were more prevalent in healthy individuals than in Crohn's disease patients. These findings suggest that CPS contributes to the modulation of inflammation and pathogenesis. This study highlights the potential of precise gene-modification systems to uncover genetic determinants of intestinal colonization and pathogenesis in gut bacteria.