Binding properties and crystallization of crustinPm1 and crustinPm7 from tha black tiger shrimp penaeus monodon

Abstract

Crustin is one of several antimicrobial peptides (AMPs) in innate immune system of crustacean. In previous study, crustinPm1 strongly inhibited gram-positive bacteria, whilst crustinPm7 acted against both gram-positive and gram-negative bacteria. The aim of this study is to investigate (1) Binding properties of rcrustinPm1 and rcrustinPm7 to bacterial cells and cell wall components, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) (2) Induction of bacterial agglutination by rcrustinPms (3) Inner membrane permeabilization (4) Effects of rcrustinPms on bacteria cells and (5) Crystallization of rcrustinPms. In this study, the recombinant crustinPm1 and crustinPm7 were produced in Escherichia coli and purified under denaturing condition. The purified rcrustinPms were then renatured and used in antimicrobial activity test. Binding study suggested that both crustin isoforms can bind to both gram-positive and gram-negative bacteria, as well as cell wall components LTA and LPS. CrustinPms can induce bacterial agglutination in some strains of bacteria. They can also change inner membrane permeability of E. coli strain MG1655, resulting in cytoplasmic -galactosidase release. Scanning Electron Microscopy (SEM) revealed physical change on cell surface of Staphylococcus aureus, Vibrio harveyi and E. coli treated with rcrustinPm7. Recombinant crustinPm1; however, can cause physical changes on cell surface of S. aureus and E. coli only. Crystallization experiments were carried out. Recombinant crustinPm1 was crystallized in 0.2 M Lithium citrate tribasic tetrahydrate, 20% PEG 3350, pH 8.4. However, no crytallization condition for rcrustinPm7 was found.