Project Summary Our gut contains about 100 trillion commensal bacteria that collectively contribute to nutrient absorption and maturation of the immune system, as well as play a central role in protecting the host from enteric bacterial infections. However, many enteric bacterial pathogens have developed strategies to colonize the intestinal mucosa and cause diseases. Clinically significant enteric bacteria, such as Salmonella, Shigella, Yersinia, and pathogenic E. coli, are a major public health concern due to their pathogenic capacities to cause severe diarrheal and extraintestinal diseases with potentially fatal consequences, and their ease of transmission through contaminated food and water. These bacteria have developed common strategies to specifically target and invade a relatively small number of follicle-associated epithelial (FAE) cells known as Microfold (M) cells to induce inflammation. Contamination with extremely low doses, sometimes with only a few pathogens, can cause severe enteritis and/or disseminated infections. It remains poorly understood how so few bacterial pathogens, which are typically surrounded by millions (if not billions) of commensal microbes, find a way to their targeted portal of entry—the low abundance M cells of the FAE. Previously, we have demonstrated the existence of endogenous bioelectric fields in the tracheal mucus epithelium of the rhesus monkey and, and for the first time, detected Salmonella infection-generated electric fields (IGEF) in mouse cecum FAE. These bioelectrical signals play critical roles during embryonic development, tissue regeneration and wound healing, as well as in disseminated infections as we demonstrate in our most recent work. By applying electric fields mimicking IGEF we have shown that commensal E. coli migrate to the anode and pathogenic Salmonella migrate to the cathode, exclusively and simultaneously. In this exploratory R21, we propose a novel mechanism of bioelectrical control in pathogenic bacterial targeting. Our central hypothesis is that an active epithelial “battery” exists around the FAE, which is intrinsically exploited by bacterial pathogens for invasive targeting. We will test our hypothesis through the following specific aims: 1) Spatially define and characterize bioelectrical activities at gut epithelia. Using advanced electrophysiological techniques, we will measure and pharmacologically manipulate ionic current density, trans-epithelial potential, and transmembrane potential in FAE and surrounding villus epithelium in an ex vivo mouse cecum model. Successful completion will establish the first bioelectricity profile of intestinal epithelium. 2) Dissect the mechanisms of bioelectricity at gut epithelia in bacterial invasive targeting. Our working hypothesis is that enteric pathogens utilize local bioelectricity to strategically target the FAE depending on the surface electrical properties of the bacteria. This will be tested genetically and affirmed in vitro and...