Pattern recognition, hemolymph protease-14 activation, and enhancement of lysozyme-mediated bacteria killing by soluble peptidoglycan recognition proteins in Manduca sexta

Peptidoglycan recognition proteins (PGRPs) detect invading bacteria during insect immune responses, and some can damage bacterial cell walls. We previously produced M. sexta PGRPs 1–5, 12, and 13, and demonstrated that the PGRP repertoire in hemolymph preferentially detects meso-diaminopimelic acid-peptidoglycans (DAP-PGs). In this study, we found that adding DAP-PGs and PGRPs to larval hemolymph significantly enhanced prophenoloxidase activation beyond the sum of their individual effects. Lys-PG of Micrococcus luteus and PGRP4/5 also displayed the synergy, but Lys-PG of Staphyloccous aureus did not. Structural modeling and ligand docking supported the preferrential binding of DAP-PGs over Lys-PGs. DAP/Lys-PG, PGRP3s/3f/5/13N, and microbe binding protein activated hemolymph protease-14 (HP14), suggesting that these PGRPs initiate the serine protease system in the same way as PGRP1. Using fluorescein-labeled M. luteus peptidoglycan as a substrate, we detected increases in fluorescence signal caused by PGRP2, 4, 13N, 12e, and 3f, suggesting that these PGRPs have amidase activity for hydrolyzing peptidoglycan, which was enhanced by Zn²⁺ and decreased by EDTA. Spatial locations of the catalytic residues, Zn²⁺ ion, and scissile bond in the models of PGRP-peptidoglycan complexes explained some of the activity differences. PGRP2 and PGRP4 had the highest specific activity. Only PGRP4 (60 μg/ml) decreased Bacillus megaterium colony-forming units (CFU) compared to controls, whereas other PGRPs did not affect CFU numbers. A mixture of PGRP1−5 or 3s (2 μg/ml) and Manduca lysozyme (20 μg/ml) significantly reduced CFU compared to lysozyme alone, even for PGRPs without amidase activity. Scanning electron microscopy revealed that lysozyme caused structural damage to the bacterial cell walls, and when combined with PGRP2, this effect was enhanced. In summary, the soluble PGRPs preferentially recognize DAP-PGs, stimulate melanization via HP14, and enhance bacterial killing by lysozyme. Mechanisms for the amidase-independent bacterial killing by PGRPs and lysozyme require further exploration.