Refinement of the Fusion Tag PagP For Efficient Inclusion Body-Targeted Expression of Recombinant Antimicrobial Peptides in Escherichia Coli

The methods developed for efficient insoluble protein production are less well explored. Our data demonstrated that PagP, an E. coli outer membrane protein with high β-sheet content, could function as an efficient fusion partner for inclusion body-targeted expression of antimicrobial peptide Magainin II, Metchnikowin and Andropin. The primary structure of a given polypeptide determines to a large extent its propensity to aggregate. The aggregation “hot spots” (HSs) in PagP was subsequently analyzed with the web-based software AGGRESCAN, leading to identification of the C-terminal region with high dense distribution of HSs. The absolute yields of recombinant antimicrobial peptide Metchnikowin and Andropin could be increased significantly when expressed in fusion with this version of PagP. Moreover, a Proline-rich region was found in the β-strands of PagP. Substitution for these prolines by residues with high β-sheet propensity and hydrophobicity significantly improved its ability to form aggregates, and greatly increased the yield of the recombinant passenger peptides. Fewer examples have been presented to separate the recombinant target proteins expressed in fusion inclusion bodies. Here, we reported an artificial linker peptide NHT with three motifs, by which separation and purification of the authentic recombinant antimicrobial peptides could be implemented.

Specific and rapid reverse assaying protocol for detection and antimicrobial susceptibility testing of Pseudomonas aeruginosa based on dual molecular recognition

The worldwide emergence and spread of antimicrobial resistance is accelerated by irrational administration and use of empiric antibiotics. A key point to the crisis is a lack of rapid diagnostic protocols for antimicrobial susceptibility testing (AST), which is crucial for a timely and rational antibiotic prescription. Here, a recombinant bacteriophage tail fiber protein (TFP) was functionalized on magnetic particles to specifically capture Pseudomonas aeruginosa, while fluorescein isothiocyanate-labeled-magainin II was utilized as the indicator. For solving the magnetic particles’ blocking effects, a reverse assaying protocol based on TFP recognition was developed to investigate the feasibility of detection and AST of P. aeruginosa. P. aeruginosa can be rapidly, sensitively and specifically detected within 1.5 h with a linear range of 1.0 × 102 to 1.0 × 106 colony forming units (CFU)⋅mL−1 and a detection limit of 3.3 × 10 CFU⋅mL−1. Subsequently, AST results, which were consistent with broth dilution results, can be obtained within 3.5 h. Due to the high specificity of the TFP, AST can actually be conducted without the need for bacterial isolation and identification. Based on the proof-of-principle work, the detection and AST of other pathogens can be extended by expressing the TFPs of their bacteriophages.

Dimerization of cell-penetrating buforin II enhances antimicrobial properties

Antimicrobial peptides (AMPs) that selectively permeabilize bacterial membranes are promising alternatives to conventional antibiotics. Dimerization of AMP is considered an attractive strategy to enhance antimicrobial and membrane-lytic activity, but it also increases undesired hemolytic and cytotoxic activity. Here, we prepared Lys-linked homodimers of membrane-permeabilizing magainin II and cell-penetrating buforin II. Dimerization did not significantly alter conformational behavior, but it had a substantial impact on antimicrobial properties. We found that while the magainin II dimer showed increased antimicrobial and cytotoxic effects, the buforin II dimer conferred much greater antibacterial potency without exhibiting cytotoxic activity. Interestingly, the buforin II dimer was highly effective against several antibiotic-resistant bacterial isolates. Membrane permeabilization experiments indicated that the magainin II dimer rapidly disrupted both anionic and zwitterionic membranes, whereas the buforin II dimer selectively disrupted anionic membranes. Like the monomeric form, the buforin II dimer was efficiently translocated across lipid bilayers. Therefore, our results suggest that the dimerization of cell-penetrating buforin II not only disrupts the bacterial membrane, but also translocates it across the membrane to target intracellular components, resulting in effective antimicrobial activity. We propose that dimerization of intracellular targeting AMPs may present a superior strategy for therapeutic control of pathogenic bacteria.

Specific and Rapid Reverse Assaying Protocol for Detection and Antimicrobial Susceptibility Testing of Pseudomonas Aeruginosa based on Bacteriophage Tail Fiber Protein and Magainin II Recognition

The worldwide emergence and spread of antimicrobial resistance are accelerated by irrational administration and use of empiric antibiotic. A key point to the crisis is lack of rapid diagnostic protocol to antimicrobial susceptibility testing (AST) for timely and rational antibiotic prescription. Here, bacteriophage tail fiber protein (TFP) recombined in Escherichia coli expression system was functionalized on magnetic particles (MPs) to specifically capture P. aeruginosa, and FITC-labeled-magainin II was utilized as the indicator. For solving the MPs’ blocking effects, a reverse assaying protocol (RAP) based on TFP recognition was investigated the feasibility of detection and AST of P. aeruginosa. P. aeruginosa detection can be rapidly, sensitively and specifically detected within 1.5 h with a linear range of 1.0 × 102 to 1.0 × 106 CFU⋅mL− 1 and a detection limit of 3.3 × 10 CFU⋅mL− 1. Subsequently, the results of AST which was consistent in the results of broth dilution can be obtained within 3.5 h. Due to the high specificity of TFP, the AST can actually be conducted without the requirement of bacterial isolation and identification by this RAP. Based on the proof-of-principle work, the detection and AST of other pathogens can be extended by expressing the TFP of their bacteriophages.