Research On The Heterologous Expression, Antimicrobial Mechanism And Structure-activity Relationship Of Plectasin

Plectasin with potent activity against Gram-positive bacteria but no hemolytic activity, the first defensin-type antimicrobial peptide isolated from a saprophytic fungus, was an inoffensive antibiotic with therapeutic potential.The Escherichia coli (E. coli) fusion expression, characterization, functions, the disulfide structure-activity relationship and anti-Staphylococcus aureus (S. aureus) mechanism of plectasin were studied in this paper. The main results are as follows:1) In this study, we present the high-level expression of Cys-rich plectasin in E. coli by the integration of three key strategies: codon usage bias, fusion partner and on-column cleavage. The expression level of the fusion protein Trx-plectasin accounted for 53.6% of cellular protein, and about 58.5% of the target proteins were in a soluble form. The soluble fusion protein was easily purified to near homogeneity by affinity chromatography using hexahistidine tag. Recombinant plectasin was effectively obtained by on-column cleavage of the fusion protein with factor Xa. The molecular mass of recombinant plectasin determined by MALDI-TOF (matrix assisted laser desorption ionization-time-of-flight) is equal to its theoretical molecular weight. High purity plectasin was achieved by gel filtration chromatography and RP-HPLC.2) The characterization and functions of plectasin was studied from folding, oxidation and reduction, heat treatment, ion-effect tests and minimal inhibitory assays and hemolytic assays. Recombinant plectasin with three cysteine bridges can be properly refolded in TGE buffer or in 0.01% acetic acid with 5 M Arg. Oxidation, reduction and heat treatment of plectasin showed that the destruction of the plectasin disulfide bonds affect the stability of its conformation and therefore lost its antimicrobial activity. Antimicrobial activity assays showed that plectasin was active in vitro against Gram-positive bacteria, but showed no or limited activities against Gram-negative bacteria and fungi. The effects of the different valent cations on the anti-S. aureus activity of plectasin were different. Monovalent cation had no effect on the antimicrobial activity of plectasin. However, divalent cations had a stronger negative effect on the antimicrobial activity of plectasin. These results also indicate that the antimicrobial activity of plectasin was more sensitive to calcium ions than magnesium ions. Hemolysis assays demonstrated that plectasin was not hemolytic for rabbit erythrocytes.3) For test the role of the disulfide array in the antimicrobial activity of plectasin, we have adopted a site-directed mutagenesis approach to generate seven paired Ala for Cys amino acid substitutions corresponding to plectasin with one, two or three disulfide bridges. With bioinformatics tools, the parameters of the primary and secondary structure of mutations that disrupted disulfide bonds were predicted. Plectasin and its mutations were expressed in Pichia pastoris expression system. Paired Ala for Cys amino acid substitutions in plectasin were tested for effects on antimicrobial activity. The results showed that the disulfide bonds have different effects on antimicrobial activity. The greatest effect on the antimicrobial activity of plectasin was the disulfide bond of C1-C4, the next effect was C3-C6, and the least effect was C2-C5. 4) Through the ion-effect test, morphology observation, and gel retardation experiments, the anti-S. aureus mechanism of plectasin was preliminaryly inferenced as follows:Plectasin can not bond to the plasmid DNA. The mode of action with cell wall is not a simple electrostatic attraction, and mainly through the specificity interaction with the divalent cations binding place in the cell wall of S. aureus, such as teichoic acid (TA) and lipoteichoic acid, competitively replace divalent cations, as Ca2 + and Mg2 +, which plays a bridge and charge neutralization role. 1) The plectasin may disrupt the cell wall and increase the permeability of the cell wall, resulting in pore formation and leakage of contents, and eventually led to the death of a whole cell lysis; 2) The plectasin may also be displace and activate autolysins leading to uncontrolled degradation of the muramidase layer and often spontaneous lysis of the cytoplasmic membrane.In short, the successful expression of a disulfide-rich plectasin gene in E. coli in this study would provide an efficient and facile platform for the production or study of disulfide-rich AMPs. This study confirmed that the destruction of the plectasin disulfide bonds affect the stability of its conformation and therefore lost its antimicrobial activity, and the disulfide bonds have different effects on antimicrobial activity. This study also inferenced that the anti-S. aureus mechanism of plectasin was through the specificity interaction with the divalent cations binding place in the cell wall of S. aureus. It help to clarify the antimirobial mechanism of plectasin. These results would provide a theoretical basis for the development of new prevention drug products with anti-Gram-positive bacteria activity, and promote the applications of plectasin on the control of streptococcus disease.