The host and its commensal microbiota generally maintain a peaceful coexistence; however, a few bacteria disrupt immune homeostasis and cause disease. For example, attaching and effacing (A/E) enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC) cause diarrheal diseases that affect more than 2 billion people and kill 1.5 million children annually. Commensal bacteria can protect the host from disease caused by enteric pathogens; however, the mechanisms by which this occurs are poorly understood. We recently found that a single oral dose of commensal Bacillus subtilis or Bacillus formis spores can protect mice from disease caused by the A/E pathogen, Citrobacter rodentium. In contrast, a closely related commensal Bacillus licheniformis, and two B. subtilis mutants, hag and espH, did not protect the host. Because the single dose of protective Bacillus spp. is given 24 hr prior to pathogenic C. rodentium, protection is not likely due to initiation of an adaptive immune response, but is more likely due to an innate anti-inflammatory immune response. Alternatively, Bacillus spp. could protect from disease by directly interfering with localization of C. rodentium to the intestinal epithelium. In Aim 1 we will use in vivo bioluminescence imaging (IBIS) to determine if protective Bacillus spp. alter the colonization site of pathogenic C. rodentium. One of the non-protective B. subtilis mutants is deficient in flagellin, a ligand for TLR5, and another is deficient in exopolysaccharide, presumed ligand for TLR2, and in Aim 2 we will use TLR agonists and TLR knockout mice to determine if protection is mediated through TLR. Similarly, we will use Nod-like receptor (NLR) agonists and knockout mice to determine if protection is mediated through NLR. In Aim 3, we will test the possibility that B. subtilis mediates protection through T regulatory cells. PUBLIC HEALTH RELEVANCE: We investigate a means of protecting against enteric pathogens that cause diarrheal disease by a single oral dose of spores from a commensal-like (beneficial) bacterium. The experiments are designed to determine the mechanism by which this protection occurs. We investigate the possibilities that the commensal bacterium interferes with colonization of the enteric pathogen, or stimulates an anti-inflammatory response in the host.