Molecular and functional characterization of ApPGRP from Anatolica polita in the immune response to Escherichia coli.

Peptidoglycan recognition proteins (PGRPs) widely distributed in invertebrates and vertebrates are pattern-recognition molecules in innate immunity. In the present study, a novel short PGRP gene, designated as ApPGRP was identified from Anatolica polita. The deduced amino acid sequence of ApPGRP is composed of 196 residues and contains a conserved PGRP domain at the C-terminus. The phylogenetic tree showed that ApPGRP shared high homology of amino acid sequence with TcPGRP2 from Tribolium castaneum. The recombinant protein of ApPGRP exhibited binding activity toward Escherichia coli and DAP-type PGN from E. coli. Quantitative real time PCR (RT-qPCR) analysis indicated that the relative expression level of ApPGRP was up-regulated significantly after E. coli and DAP-type PGN challenge in the fifth instar larvae of A. polita. Moreover, the expressions of three antimicrobial peptides (AMPs) (ApAttacin 1, ApAttacin 2 and ApColeoptericin) were significantly increased after E. coli and DAP-type PGN challenge. RNA interference (RNAi) experiments showed that the expressions of three AMPs in the larvae of A. polita were significantly decreased after injection of ApPGRP dsRNA. Furthermore, the expressions of three AMPs in the larvae injected were still significantly decreased after E. coli challenge compared to the control samples without dsRNA injection. The predicted 3D structure showed that the ApPGRP could form the protein core of five β-sheet and three ɑ-helices, which would be involved in specific PGN recognition. These results suggested that ApPGRP may play an important role in the immune response to E. coli infection and function as a receptor for antimicrobial peptide gene induction in Anatolica polita.