A G-protein coupled receptor is involved in the DUOX pathway in Tribolium castaneum

Activation of the dual oxidase (DUOX) pathway is an important intestinal defense mechanism against enteric infection triggering the formation of radical oxygen species by stimulating DUOX enzyme activity and/or gene expression. In insects, several studies have suggested that uracil released by pathogenic bacteria functions as a major trigger molecule for the activation of DUOX, which leads to the formation of antimicrobial hypochlorous acid (HOCl). While the recognition of pathogen-associated molecular patterns of microbes by pattern recognition receptors is well understood, the detection of uracil is still elusive. It has been postulated that a G-protein coupled receptor (GPCR) binds the pyrimidine uracil, which activates PLCβ signalling and further downstream events. So far, no pyrimidinergic receptor has been identified in insects, particularly none that binds uracil nucleotides or sugar derivatives. To identify potential candidates for insect pyrimidine receptors, we used a human P2Y₄ receptor as a template to screen the Tribolium castaneum reference proteome. Four promising receptor candidates were identified, of which two were analyzed using RNA interference to determine their influence on uracil-induced TcDUOX expression, HOCl formation and development in control larvae and larvae that were challenged with the enteric pathogen Bacillus thuringiensis. Silencing TcGPCR41 resulted in a loss of uracil-induced TcDUOX expression and HOCl formation. Furthermore, the development of challenged larvae was affected in a manner like that observed in a TcDUOX knockdown. We conclude that the identified receptor may play a role in the uracil-dependent activation of the DUOX-pathways.