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  • Maturation of an Antimicrobial Peptide Inhibits Aeromonas hydrophila Infection in Crayfish.

Maturation of an Antimicrobial Peptide Inhibits Aeromonas hydrophila Infection in Crayfish.

Journal of immunology (Baltimore, Md. : 1950) (2019-12-20)
Bao-Rui Zhao, Yi Zheng, Jie Gao, Xian-Wei Wang
ABSTRACT

Rapid synthesis and release of active antimicrobial peptides (AMPs) is an important strategy in innate immune. Processing of the precursor into the active form is a common posttranslational modification of AMPs in mammals. However, in invertebrates, the mechanism of AMP maturation is largely unknown. In the current study, to our knowledge, a novel potential AMP, designated as PcnAMP, was identified because of its significant induction by bacterial infection in the red swamp crayfish (Procambarus clarkii). PcnAMP was cleaved into a short fragment postinfection. Using the purified native peptide, this cleavage was found to be mediated by trypsin after synthesis. Proteolysis produced an N-terminal peptide that exerted the antibacterial function. Although the N-terminal peptide did not show significant similarity to any other sequences, it was predicted to have an overall helical structure and high amphipathicity, both of which are typical features of many AMPs. The N-terminal active peptide exhibited a wide spectrum of antimicrobial activity. Atomic force microscope imaging and flow cytometry analysis showed that treatment with the active form of PcnAMP led to the collapse of the bacterial cell wall and permeabilization of the bacterial cell membrane. Thus, this study provided a new candidate for therapeutic agent development, and revealed new insights into the maturation of AMPs in invertebrates.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Freund′s Adjuvant, Complete, cell suspension
Sigma-Aldrich
Trypsin from bovine pancreas, Type I, ~10,000 BAEE units/mg protein
Sigma-Aldrich
Freund′s Adjuvant, Incomplete, liquid
Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), lyophilized powder
Sigma-Aldrich
3,3′,5,5′-Tetramethylbenzidine, ≥99%
Sigma-Aldrich
IPTG, ≥99% (TLC), ≤0.1% Dioxane
Millipore
Ni-NTA His•Bind® Resin