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

Area of Science:

• Insect immunology
• Molecular biology
• Biochemistry

Background:

• Peptidoglycan recognition proteins (PGRPs) are key immune molecules recognizing bacterial peptidoglycan.
• While known for antibacterial functions, the antifungal roles of PGRPs, particularly PGRP-S1 from Mythimna separata, are less understood.
• PGRP-S1 has demonstrated bacteriolytic activity against both Gram-positive and Gram-negative bacteria.

Purpose of the Study:

• To investigate the antifungal activity of Mythimna separata PGRP-S1 (MsPGRP-S1).
• To identify critical amino acid residues responsible for MsPGRP-S1's microbicidal activities.
• To elucidate the specific roles of MsPGRP-S1 in defense against fungal pathogens like Beauveria bassiana.

Main Methods:

• Tissue expression analysis of MsPGRP-S1 in naive and infected larvae.
• Structural prediction and molecular docking of MsPGRP-S1.
• Site-directed mutagenesis of key residues (H61, H62, Y97, H171, T175, C179) followed by protein expression and purification.
• Assays for amidase activity, polysaccharide binding, antibacterial activity, and antifungal activity (conidial germination, hyphal growth inhibition).
• Scanning electron microscopy and transmission electron microscopy to visualize microbial damage.

Main Results:

• MsPGRP-S1 was primarily expressed in the larval midgut and significantly induced after Beauveria bassiana infection.
• Mutations H61A, Y97A, H171A, and C179A abolished amidase activity, while H62A and T175A retained bacteriolytic function.
• MsPGRP-S1 and specific mutants (H62A, T175A) exhibited antibacterial activity.
• Several MsPGRP-S1 variants (WT, H61A, Y97A, T175A, C179A) inhibited Beauveria bassiana conidial germination, and WT, H61A, H62A, T175A inhibited hyphal growth.
• Microscopic analysis revealed ruptured conidia and inhibited hyphal development.

Conclusions:

• Different amino acid residues within MsPGRP-S1 contribute distinctly to its antibacterial and antifungal activities.
• MsPGRP-S1 plays a significant role in the insect's immune response against both bacterial and fungal pathogens.
• Understanding these structure-function relationships can inform strategies for pest and disease management in agriculture.