Elsevier

Peptides

Volume 32, Issue 7, July 2011, Pages 1488-1495
Peptides

Inhibitory effects and mechanisms of physiological conditions on the activity of enantiomeric forms of an α-helical antibacterial peptide against bacteria

https://doi.org/10.1016/j.peptides.2011.05.023Get rights and content

Abstract

Enantiomeric amphipathic α-helical antibacterial peptides were synthesized and their biophysical and biological properties under different physiological conditions were studied. In the absence of physiological factors, the l- and d-peptides exhibited similar antimicrobial activities against a broad spectrum of bacteria, even against clinical isolates with resistance to traditional antibiotics. However, in the presence of NaCl, CaCl2 or human serum albumin (HSA) at physiological concentrations, the enantiomers revealed bacterium-species dependent attenuations in antibacterial activity. In the presence of salts the electrostatic interaction between the peptides and the biomembrane was inhibited. Salts, especially CaCl2, weakened the ability of the peptides to permeabilize the outer membrane of Gram-negative bacteria, as determined by a 1-N-phenylnaphthylamine uptake assay. HSA exhibited variable inhibitory effects on the activity of the peptides when incubated with different bacterial strains. The peptides showed different binding association abilities to HSA at different molar ratios, regardless of their chirality, resulting in reduced peptide biological activity. The d-peptide performed better than its l-enantiomer in all conditions tested because of its resistance to proteolysis, and may therefore represent a promising candidate for development as a therapeutic agent.

Highlights

► In the presence of NaCl, CaCl2 or human serum albumin at physiological concentrations, inhibitory effects of physiological conditions on antimicrobial activity of enantiomeric peptides have been investigated. ► The electrostatic interaction between peptides and biomembrane was inhibited in the presence of salts. ► Salts especially CaCl2 weakened the ability of peptides to permeabilize the outer membrane of Gram-negative bacteria. ► Peptide showed different binding association abilities to HSA at different molar ratios, which is irrelevant to the peptide chirality and caused the reduction of peptide antimicrobial activity. ► d-peptide showed better performance than its l-enantiomer in all tested conditions owing to its resistance against proteolysis, hence, may be a promising candidate as therapeutic in clinical practices.

Introduction

Cationic antimicrobial peptides (AMPs) have many useful biological properties, including broad-spectrum antimicrobial activity, fast action and slow resistance development. They are therefore promising lead compounds for new antibiotics [5]. The most significant feature of AMPs is their potent bactericidal activity against multi-drug-resistant bacteria, which is thought to be due to differences in their mechanism of action compared to traditional antibiotics [25]. Although the mechanism of action of AMPs has not been clarified, it is believed that most AMPs kill bacteria by membrane disruption and/or by interaction with cytoplasmic anionic molecules, resulting in lethal interference with intracellular processes [20], [29]. In previous studies, we have demonstrated that an amphipathic α-helical peptide disrupts bacterial membranes by a detergent-like mechanism [7], [8]. In general, AMPs exhibit excellent antimicrobial activity in vitro [39]. However, their use in systemic applications may be limited due to a lack of activity under physiological conditions. The presence of salts (monovalent and divalent cations), anionic proteins (human serum albumin, HSA) and proteases in body fluids is known to hinder their activity [6], [14]. This is one of the main obstacles preventing the widespread adoption of AMPs for systemic clinical use [15], [38].

Several studies have examined AMPs composed of all d-amino acids to determine whether there are chirality-dependent receptors for the peptides on the surface of microbes [3], [11], [35]. Most studies have shown that all-d-amino acid peptides have similar biological activities in vitro to their all-l-enantiomers [4], [9], [10], [11], [35]. This suggests that the antimicrobial mechanism of these peptides does not involve stereoselective interactions with chiral enzymes, lipids or protein receptors. All d peptides generally show higher therapeutic potential than their l-enantiomers, due to their higher stability toward proteolytic degradation in vivo [22], [34].

In a previous study, a 26-residue amphipathic α-helical antimicrobial peptide V13K (referred to as LP in this study) was obtained by replacing a hydrophobic valine residue with a hydrophilic lysine residue in the center of the hydrophobic face of peptide V681 [8], an analog of a cecropin A (1–8) and melittin B (1–18) hybrid [40], [41] peptide. LP exhibited an enhanced peptide therapeutic index, with excellent antibacterial activity against various Gram-negative and Gram-positive bacteria and negligible hemolytic activity against human red blood cells in vitro [8], [19]. To evaluate the effects of physiological conditions on the antibacterial activity of LP and its d-enantiomer (referred to as DP), we examined the effects of NaCl, CaCl2, HSA and proteases on the biological activities of the peptides in this study. The mechanisms of these effects were also studied. A series of resistant bacterial clinical isolates were used to compare the biological activities of the l- and d-enantiomeric peptides. This activity-mechanism study of l- and d-antimicrobial peptides provides important information toward the development of new antimicrobial therapeutics.

Section snippets

Materials and bacterial strains

Rink amide 4-methylbenzhydrylamine resin (0.8 mmol/g) and all N-α-Fmoc protected amino acids were purchased from GL Biochem (Shanghai, China). The coupling reagents for peptide synthesis O-benzotriazole-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), N,N′-diisopropylethylamine (DIEA), 1-N-phenylnaphthylamine (NPN), polymyxin B sulfate, human serum albumin (HSA) and trifluoroacetic acid (TFA) were purchased from Sigma (St. Louis, MO, USA).

Peptides and their secondary structure

Fig. 1 shows the amino acid sequence, helical wheel and helical net representations of peptide LP. It can be seen that LP is an amphipathic α-helical peptide with a lysine at the center of its non-polar face (position 13). DP shares the same amino acid sequence as its l-enantiomer, but is composed of all d-amino acids and exhibits a left-handed helix in contrast to the right-handed helix of LP.

To determine the secondary structure of these peptides in different environments, CD spectra were

Discussion

Enantiomers are non-superimposable mirror images of one another. In the present study, we have shown that LP and DP enantiomers have the same RP-HPLC retention behaviors and similar secondary structure both in an aqueous and a hydrophobic environment. Although in aqueous medium both peptide enantiomers showed negligible secondary structure, in a hydrophobic environment they formed α-helical amphipathic structures to the same extent. This characteristic is critical to the disruption of

Conflict of interest

The authors have no potential conflicts of interest to disclose.

Acknowledgements

This work was supported by a Grant from the Doctorate Program of New Teachers, given by the Ministry of Education of China 200801831064 (Y.C.), a Natural Science Foundation of Jilin Province China Grant 201015103 (Y.C.), a grant from the Scientific Frontier and Interscience Innovation Program of Jilin University 200903095 (Y.C.), the Youth Foundation of Jilin Province 20100126 (Y.B.H.), and a Basic Scientific Research Grant from Jilin University (Y.B.H.).

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