Omwaprin

Two cationic antimicrobial peptides (AMP) were designed based on the snake venom peptide, omwaprin, hypothesized to be shorter, cost effective and potent. Omw1 and omw2 demonstrated significant broad-spectrum antimicrobial activity against standard and clinical strains at a MIC ranging from 15.625 to 250 µg/ml for omw1 and from 31.3 to 500 µg/ml for omw2. Time-kill kinetics revealed that omw1 caused complete lysis of E. coli ATCC 25922 at 1× MIC and S. aureus ATCC 25923 at 2× MIC after 40 and 60 min of incubation, respectively. Membranolytic activity of the peptides was assessed by propidium iodide stain, where red fluorescence was observed in cells treated with the peptides compared to untreated cells. Notable morphological changes were observed in the microbes treated with peptides, as revealed by scanning electron micrographs. Omw1 and omw2 were also potent to inhibit the formation as well as dispersal of matured biofilms at 1/2× MIC against clinical strain, C. albicans. Further, minimal hemolytic activity demonstrated by both the peptides at microbicidal concentration against human erythrocytes proves that the designed peptides were less toxic and potent antimicrobial agents which could be considered for further studies with animal models to affirm its efficiency.

We have isolated and characterized omwaprin, a 50-amino-acid cationic protein from the venom of inland taipan (Oxyuranus microlepidotus). It is a new member of the waprin family of snake venom proteins. A synthetic gene was designed and constructed for expressing the recombinant protein in Escherichia coli. Recombinant omwaprin was used for carrying out functional analyses. The protein is non-toxic to Swiss albino mice at doses of up to 10 mg/kg when administered intraperitoneally. However, it shows selective and dose-dependant antibacterial activity against Gram-positive bacteria. The minimum inhibitory doses were in the range 2-10 microg for selected species of bacteria in radial diffusion assays. The antibacterial activity is salt-tolerant up to 350 mM NaCl. However, omwaprin lost its antibacterial activity upon reduction and alkylation of its cysteine residues, or upon deletion of six N-terminal amino acid residues, four of which are positively charged. These observations indicate that the three-dimensional structure constrained by four disulfide bonds and the N-terminal residues are essential for its activity. The mechanism of action is via membrane disruption, as shown by scanning electron microscopy. Importantly, omwaprin lacks haemolytic activity on human erythrocytes. This demonstrates the specificity of omwaprin for bacterial membranes. Unlike other reported WAP (whey acidic protein) domain-containing antibacterial proteins, including elafin, EPPIN (epididymal proteinase inhibitor), SWAM1 and SWAM2 [single WAP (whey acidic protein) motif proteins 1 and 2] and SLPI (secretory leucocyte proteinase inhibitor), omwaprin shows species-specific activity on the Gram-positive bacteria tested.

The 50-residue snake venom protein L-omwaprin and its enantiomer D-omwaprin were prepared by total chemical synthesis. Radial diffusion assays were performed against Bacillus megaterium and Bacillus anthracis; both L- and D-omwaprin showed antibacterial activity against B. megaterium. The native protein enantiomer, made of L-amino acids, failed to crystallize readily. However, when a racemic mixture containing equal amounts of L- and D-omwaprin was used, diffraction quality crystals were obtained. The racemic protein sample crystallized in the centrosymmetric space group P2(1)/c and its structure was determined at atomic resolution (1.33 A) by a combination of Patterson and direct methods based on the strong scattering from the sulfur atoms in the eight cysteine residues per protein. Racemic crystallography once again proved to be a valuable method for obtaining crystals of recalcitrant proteins and for determining high-resolution X-ray structures by direct methods.