The ability of salivary peptides to inhibit diarrhea-causing bacteria from binding to the gut is newly discovered, and not well characterized. The innovative long-term goal is to realize the full potential of salivary components as both prophylactic and therapeutic agents against gastrointestinal and respiratory diseases. The objectives of this proposal are to define the efficacy of specific salivary components as pathogen inhibitors, and to determine the structure and mechanism by which one these peptides, Histatin-5, binds to pili that are external filaments on enterotoxigenic Escherichia coli (ETEC). This pilus/peptide interaction provides the dysfunction that inhibits bacterial binding, and knowing the mechanism of this dysfunction will lead to novel therapeutic approaches against ETEC and other pathogens. The central hypothesis is that enhanced utilization of the innate immune system to combat disease can be achieved through therapeutics developed from components of saliva. The rationale for this proposal is that completion of this research provides a path forward for utilizing salivary components to fight diarrheal diseases. In addition, determination of the mechanism of Histatin-5's action will permit creation of therapeutics with even greater efficacy. To achieve our goals, we will pursue the following two specific aims: 1. Define the capacity of selected salivary peptides to inhibit bacterial binding of enterotoxigenic Escherichia coli (ETEC) to target cells; 2. Determine the mechanism by which the salivary peptide, Histatin-5, inhibits bacterial binding via pili, an essential virulence factor of enterotoxigenic Escherichia coli (ETEC). These aims will be achieved using 1) bacterial adhesion studies on cell cultures and primary human intestinal cultures (“organoids”) and 2) structure determination at high resolution using electron cryomicroscopy and cryotomography (cryo-EM and cryo-ET). The proposed research is significant, because it will open a new avenue for development of therapeutics against diarrhea-causing bacteria. Unlike traditional antibiotics that broadly target enzymes involved in nucleic acid, protein, and cell wall synthesis, here, we explore a novel aspect of the host's innate immune system: the ability of salivary components to inhibit bacterial adherence to the host. The expected outcome of this research is a list of salivary components that can be exploited as therapeutics against diarrhea-causing pathogens, and the definition of the mechanism by which one component creates dysfunction of a critical virulence factor. The results will have an important positive impact immediately as a first step in defining and understanding the role of saliva in fighting GI disease, and long-term because they will lay the groundwork for bringing new researchers into this emerging field, and for development of saliva-based GI and respiratory therapeutics.

Project Narrative Every day each of us makes 1-2 liters of saliva that contains numerous antimicrobial components with demonstrated effectiveness against oral pathogens. We have discovered that at least one ingredient of saliva also helps prevent bacteria that cause diarrhea from sticking to cells in the gut. This proposal plans to figure out the mechanism of this antibacterial action, and to test other parts of saliva for new ways to prevent diarrhea diseases.