The intestinal microbiota is required for immune system development and function. Studies using germ-free animals demonstrated that not all bacterial species in the gut equally promote immunity. In rabbits, intestinal bacteria polyclonally stimulate B cell proliferation and the formation of B cell follicles in the gut-associated lymphoid tissues (GALT) as well as somatic diversification of immunoglobulin (Ig) genes, but not all bacteria are equally stimulatory. Previous data indicated that Bacillus subtilis is a strong inducer of GALT development, but the mechanisms by which B. subtilis induces the formation of B cell follicles in rabbit GALT remain largely unknown.

To determine a mechanism by which Bacillus species promote GALT development, I focused on B. subtilis and an intestinal-related bacterium, Bacillus anthracis, and demonstrated that B. anthracis is sufficient to induce the formation of B cell follicles in GALT. Interestingly, the B cell receptor (BCR) and surface Ig, IgM, bound to B. anthracis spores, and I identified an ∼30kD molecule(s) from B. anthracis spores that binds IgM. Additionally, using a single-chain antibody, I was able to isolate Bacillus spores from the appendix lumen, suggesting that a molecule(s) on the surface of spores may bind to B cells through the BCR to promote GALT development.

I stained spores with single-chain antibody fragments and antibodies of different isotypes and specificities, and determined that IgM bound to the surface of spores through a superantigen-like binding site. Additionally, spores bound to the surface of B cells, and upon binding, stimulated calcium flux, suggesting that spores can directly activate B cells. Introduction of a B. anthracis mutant, lacking the outermost spore surface, into germ-free appendices resulted in fewer follicles of proliferating B cells than observed in response to wild type B. anthracis, suggesting that the surface of B. anthracis spores contributes to B cell proliferation in vivo. Taken together, my data suggest that one mechanism by which the intestinal microbiota promotes GALT development is that intestinal Bacillus spores stimulate B cell proliferation and follicle formation through a superantigen-like mechanism.