Bioengineering and functional characterization of arenicin shortened analogs with enhanced antibacterial activity and cell selectivity

Abstract

New bioengineering approaches are required for development of more active and less toxic antimicrobial peptides. In this study we used β-hairpin antimicrobial peptide arenicin-1 as a template for design of more potent antimicrobials. In particular, six shortened 17-residue analogs were obtained by recombinant expression in Escherichia coli. Besides, we have introduced the second disulfide bridge by analogy with the structure of tachyplesins. As a result, a number of analogs with enhanced activity and cell selectivity were developed. In comparison with arenicin-1, which acts on cell membranes with low selectivity, the most potent and promising its analog termed ALP1 possessed two-fold higher antibacterial activity and did not affect viability of mammalian cells at concentration up to 50 μM. The therapeutic index of ALP1 against both Gram-positive and Gram-negative bacteria was significantly increased compared with that of arenicin-1 while the mechanism of action remained the same. Like arenicin-1, the analog rapidly disrupt membranes of both stationary and exponential phase bacterial cells and effectively kills multidrug-resistant Gram-negative bacteria. Furthermore, ALP1 was shown to bind DNA in vitro at a ratio of 1:1 (w/w). The circular dichroism spectra demonstrated that secondary structures of the shortened analogs were similar to that of arenicin-1 in water solution, but significantly differed in membrane-mimicking environments. This work shows that a strand length is one of the key parameters affecting cell selectivity of β-hairpin antimicrobial peptides.

Effect of N- and C-Terminal Modifications on Cytotoxic Properties of Antimicrobial Peptide Tachyplesin I

“…The cytotoxic effects were evaluated 48 h after peptide addition by MTT testing [8]. The results were processed statistically using GraphPad PRISM 6.0 software.…”

“…For evaluation of the non-specific effect of tachyplesin I on human erythrocytes, hemolytic test was performed [8]. The data obtained in triplicates in two independent experiments are presented as the M±SD.…”

Dimerization of the antimicrobial peptide arenicin plays a key role in the cytotoxicity but not in the antibacterial activity

“…In contrast, Ar-1[V8R] caused 50% cell death at concentrations of >100 µM in the presence of FBS and of 52 µM in the absence of FBS. It was reported that the decreased peptide cytotoxicity in the presence of FBS could be caused by antimicrobial peptides binding to serum components [10]. Notably, Ar-1[V8R] was not bound to erythrocytes at 25 µM (Fig.S5), while the wildtype arenicin-1 caused significant hemolysis at the same concentration.…”

“…In particular, the suspension of hRBS with peptide was incubated in U-bottom polypropylene microplate (Corning) for 24 h at 37°C under stirring at 900 rpm. The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay was used to determine cytotoxicity of the peptides on human embryonic fibroblasts (HEF) as described previously [10]. The experiment was performed in DMEM/F12 medium both with and without 10% fetal bovine serum (FBS, Gibco) for 24 h. The quantitative data were presented as an average means of the values measured in triplicate experiments with standard deviations.…”

“…To examine an ability of the peptides to permeabilize the cytoplasmic membrane of Gram-negative bacteria, a colorimetric assay with the use of o-nitrophenyl-β-D-galactoside (ONPG, AppliChem) and E. coli ML-35p strain was performed as previously described [10] with some modifications. The assay was performed in 96-well flat-bottom microplates, the were performed in triplicate, and the curve patterns were similar for each peptide in all three series.…”