Structure–function analysis of PGRP‐S1 from the oriental armyworm, Mythimna separata

Abstract

Peptidoglycan recognition proteins (PGRPs) are well known for their abilities to recognize or hydrolyze peptidoglycan (PGN), one of the major bacterial cell wall components. However, much less is known about their antifungal activities. PGRP‐S1 was previously identified from a crop pest, Mythimna separata (Walker) (Lepidoptera: Noctuidae). PGRP‐S1 showed bacteriolytic activities against Gram‐positive and Gram‐negative bacteria. In this study, tissue expression analysis showed that PGRP‐S1 was mainly expressed in the midgut of naïve larvae. The induction analysis showed that it was significantly induced in the larval midgut 12 h post the injection of Beauveria bassiana conidia. To identify the key residues that are related to its microbicidal activities, the structure of PGPR‐S1 was predicted for structural comparison and molecular docking analysis. Six residues (H61, H62, Y97, H171, T175, and C179) were mutated to Ala individually by site‐directed mutagenesis. The recombinant wild‐type (WT) and mutant proteins were expressed and purified. The recombinant proteins bound to different polysaccharides, PGNs, and bacteria. H61A, Y97A, H171A, and C179A lost amidase activity. Accordingly, antibacterial assay and scanning electron microscopy confirmed that only H62A and T175A retained bacteriolytic activities. The germination of B. bassiana conidia was significantly inhibited by WT, H61A, Y97A, T175A, and C179A mutants. Electron microscopy showed that some conidia became ruptured after treatment. The growth of hyphae was inhibited by the WT, H61A, H62A, and T175A. In summary, our data showed that different residues of PGRP‐S1 are involved in the antibacterial and antifungal activities.

Molecular and Functional Characterization of Peptidoglycan Recognition Proteins OfPGRP-A and OfPGRP-B in Ostrinia furnacalis (Lepidoptera: Crambidae)

“…We predicted that both genes belong to the short form of PGRP, which is a secreted extracellular protein. For most PGRPs that have amidase activity, this activity is contained in the H–Y–H–T–C structure [ 25 ]. Sequence comparison showed that in the OfPGRP-A sequence, tyrosine is mutated to serine and cysteine is mutated to serine, suggesting that OfPGRP-A lacks amidase activity.…”

Identification of 35 C-Type Lectins in the Oriental Armyworm, Mythimna separata (Walker)

“…Understanding the molecular interactions between entomopathogens and insects may help to develop new strategies for pest management [ 24 ]. Although a few immune factors in M. separata have been identified, less is known about its CTL family [ 25 , 26 , 27 ]. This study aims to construct a transcriptome of M. separata larvae and identify CTLs.…”