Limosilactobacillus reuteri promotes melatonin release from human intestinal organoids via 5'ectonucleotidase activity

Strains of Limosilactobacillus reuteri have been used to prevent or treat various conditions; however, the mechanisms by which they exert beneficial effects are not completely understood. Infant colic is one example in which L. reuteri DSM 17938 reduces clinical symptoms. While the etiology of colic is unknown, abnormal melatonin levels in infants have been suggested as a possible contributor. L. reuteri DSM 17938 has been shown to produce adenosine from AMP via production of the extracellular enzyme 5'ectonucleotidase (5'NT). Adenosine is a potent signaling molecule that impacts several important aspects of host physiology, including the release of melatonin from the pineal gland in the brain. A second major source of melatonin production is enteroendocrine cells in the intestine. We hypothesized that the adenosine generated via the 5'NT activity of L. reuteri DSM 17938, would stimulate melatonin release from human intestinal organoids. Here, we characterized the growth conditions that impact L. reuteri DSM 17938 5'NT activity, including carbon source utilization and required metal cofactors. We found zinc to be an essential cofactor for 5'NT activity by L. reuteri and observed carbon utilization altered 5'NT activity levels. Stachyose and raffinose increased levels of 5'NT activity while sucrose decreased 5'NT activity. We demonstrated that L. reuteri DSM 17938 stimulates melatonin release from pediatric human intestinal organoids in a 5'NT-dependent manner. Surprisingly, adenosine was necessary, but not sufficient, for the induction of epithelial melatonin release, thereby suggesting that an additional secreted factor was also required. Furthermore, L. reuteri BG-R46®, an evolved strain of DSM 17938 that is known to express higher 5'NT activity, was shown to induce higher levels of melatonin secretion. Taken together, this work identifies zinc and carbon sources as key factors altering L. reuteri 5'NT activity levels and demonstrates that the L. reuteri strains stimulate intestinal melatonin release via 5'NT.