Sinonasal T2R-mediated nitric oxide production in response to Bacillus cereus

Background: Upper airway epithelial cells produce bactericidal nitric oxide (NO) in response to both gram-positive and gram-negative bacteria. Our previous work demonstrated that T2R38, a bitter taste receptor (T2R) expressed in airway epithelium, produces NO in response to quorum-sensing molecules secreted by Pseudomonas aeruginosa. We also demonstrated that Staphylococci products elicit an NO response when using a T2R-independent pathway. When screening additional human pathogens for epithelial T2R activation, we found that the gram-positive aerobe Bacillus cereus secretes a T2R agonist that yields NO production. Objective: The objective of this study was to characterize the activating B. cereus product(s) and to describe the epithelial cell signaling pathway involved. Methods: Sinonasal air-liquid interface cultures were treated with B. cereus conditioned medium (CM), and NO production was measured by using 4-amino-5-methylamino-2',7' -difluorofluorescein fluorescence imaging. Ciliary beat frequency (CBF) was assessed in response to B. cereus CM. Pharmacologic studies that use inhibitors of the T2R-signaling pathway were used to determine if the production of NO was mediated by a T2R. Purification studies were performed to analyze the physical properties of the activating product(s) contained in the CM. Results: A product(s) secreted by B. cereus induced NO production and increased CBF. The response varied markedly between individual patients and involved two important components of bitter taste signaling, phospholipase C isoform beta-2 and the transient receptor potential melastatin isoform 5 ion channel. Conclusions: This study demonstrated that a B. cereus product(s) elicited an NO-mediated innate defense response in upper airway epithelium that seemed to be partially mediated by a T2R signaling pathway. The active product that elicited the NO response was likely a small nonpeptide compound, but further purification is required for identification. Patient variation in the NO response to B. cereus products could potentially be due to genetic differences in T2Rs.