Recognition of microbial molecular patterns by Gram-negative bacteria-binding protein and stimulation of multiple immune mechanisms in Manduca sexta

Detection of pathogenic invaders is the first step of a successful defense response in multicellular organisms. Here we report a new member of the β1,3-glucan recognition protein (βGRP) family from the tobacco hornworm, named M. sexta Gram-negative bacteria-binding protein (GNBP). It is 61% identical in sequence to Bombyx mori GNBP but only 34-36% identical to M. sexta βGRP1 and βGRP2. The mRNA levels were drastically up-regulated in fat body and hemocytes of bacteria-injected larvae. We produced the GNBP in a baculovirus expression system and detected its specific binding to LPS and DAP-PGN from Escherichia coli. After incubating the purified GNBP with plasma of naïve larvae in the absence of a microbial elicitor, we detected a concentration-dependent increase in phenoloxidase (PO) activity. This was accompanied by proteolytic activation of hemolymph proteases (HPs) 6, 8, 14, 21, proPO-activating proteases, serine protease homologues (SPHs), PO, and spätzle. Using affinity chromatography, we found GNBP associated with immulectin-2, βGRP1, βGRP2, proHP14, proHP21, HP6, HP8, SPH1, SPH2, and proPO. The increase in proPO activation was more prominent when DAP-PGN was included in the GNBP-plasma mixture. Since DAP-PGN-elicited melanization in plasma was insignificant, GNBP and DAP-PGN interaction synergistically enhanced proPO activation in the plasma. Injection of the recombinant GNBP into hemocoel of naïve larvae induced the expression of antimicrobial peptide genes. These data suggest that M. sexta GNBP is a component of the surveillance mechanism and, by working together with other pattern recognition molecules and HP-related proteins, triggers defense responses, such as melanization and Toll pathway activation.