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 β-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.