The Disulfide Bond of the Peptide Thanatin Is Dispensible for Its Antimicrobial Activity In Vivo and In Vitro

Abstract

b Thanatin (THA) displays potent antibiotic activity, especially against extended-spectrum-␤-lactamase (ESBL)-producing Escherichia coli both in vitro and in vivo, with minimal hemolytic toxicity and satisfactory stability in plasma. However, the high cost of thanatin significantly limits its development and clinical application. To reduce the cost of peptide synthesis, a formulation of cyclic thanatin (C-thanatin) called linear thanatin (L-thanatin) was synthesized and its activity was evaluated in vivo and in vitro. Results showed that C-thanatin and L-thanatin MICs did not differ against eight Gram-negative and two Gram-positive bacterial strains. Furthermore, the survival rates of ESBL-producing-E. coli-infected mice were consistent after C-thanatin or L-thanatin treatment at 5 or 10 mg/kg of body weight. Neither C-thanatin nor L-thanatin showed toxicity for human red blood cells (hRBCs) and human umbilical vein endothelial cells (HUVECs) at a concentration as high as 256 g/ml. Results of circular dichroism spectroscopy indicated that the secondary structure of L-thanatin is extremely similar to that of C-thanatin. Membrane permeabilization and depolarization assays showed that C-thanatin and L-thanatin have similar abilities to permeabilize the outer and inner membranes and to induce membrane depolarization in ESBL-producing E. coli. However, neither of them caused significant HUVEC membrane permeability. These findings indicate that the two peptides have similar effects on bacterial cell membranes and that the disulfide bond in thanatin is not essential for its antimicrobial activities in vivo and in vitro. L-thanatin is thus a promising low-cost peptide candidate for treating ESBL-producing E. coli infections. T hanatin (THA) is a cationic antimicrobial peptide that was first discovered in the hemipteran insect Podisus maculiventris.This peptide is composed of 21 amino acids (GSKKPVPIIYCNR RTGKCQRM), with an intramolecular disulfide bridge between the two cysteines at positions 11 and 18. Our previous studies revealed that thanatin exhibits potent antibiotic activity, especially against extended-spectrum-␤-lactamase (ESBL)-producing Esc herichia coli, both in vitro and in vivo, with a low inherent ability to induce microbial resistance, minimal hemolytic toxicity, and high stability in plasma (1, 2).Despite their promise for clinical benefit, the high cost of producing peptide drugs such as thanatin significantly limits their development and clinical application. Compared with conventional antibiotics, the costs of cationic antimicrobial peptides are always prohibitively high for large-scale production (3). Intrachain disulfide bonds exist in many naturally occurring peptides and play important roles in biological activities. However, synthesis of peptide drugs with one or more disulfide bridges is complicated and expensive (4).Previous studies found that the disulfide bonds of protegrin-1 are not essential for its antimicrobial and hemolytic activities in vitro (5). To reduce the cost of thanatin synthes...

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