A colorimetric assay for rapid screening of antimicrobial peptides

Abstract

The increased resistance of various bacteria toward available antibiotic drugs has initiated intensive research efforts into identifying new sources of antimicrobial substances. Short antibiotic peptides (10-30 residues) are prevalent in nature as part of the intrinsic defense mechanisms of most organisms and have been proposed as a blueprint for the design of novel antimicrobial agents. Antimicrobial peptides are generally believed to kill bacteria through membrane permeabilization and extensive pore-formation. Assays providing rapid and easy evaluation of interactions between antimicrobial membrane peptides and lipid bilayers could significantly improve screening for substances with effective antibacterial properties, as well as contribute to the elucidation of structural and functional properties of antimicrobial peptides. Here we describe a colorimetric sensor in which particles composed of phospholipids and polymerized polydiacetylene (PDA) lipids were shown to exhibit striking color changes upon interactions with antimicrobial membrane peptides. The color changes in the system occur because of the structural perturbation of the lipids following their interactions with antimicrobial peptides. The assay was also sensitive to the antibacterial properties of structurally and functionally related peptide analogs.

Biomimetic Model Membrane Systems Serve as Increasingly Valuable in Vitro Tools

“…When mixed with PDA, lipids tend to form bilayers in interspersed, segregated phases, creating a convenient model system of the lipid bilayer. Kolushiva et al used 3:1:6 PE/PG/PDA and 3:1:6 PE/CL/PDA vesicles to create a simple screening assay to study the interaction of AMPs with the membrane (Kolusheva et al, 2000). Using colorimetric analyses, the insertion of AMPs into the membrane was found to depend on bilayer composition, with higher insertion into the PG-containing system, consistent with observed biological data for levels of antimicrobial activity.…”

“…Not all lipid systems used as bacterial models reflect the natural composition of a bacterial membrane, yet these models have been used to provide valuable information in regards to the behaviour and influence of AMPs on the membrane. Synthetic polydiacetylene (PDA) has been used in combination with lipids in bacterial membrane mimetics because of its colorimetric properties (Kolusheva et al, 2000). PDA monomers are similar in structure to lipids, and vesicles exhibit colorimetric blue-red transitions when exposed to several environmental perturbations, including changes in surface pressure.…”

Simple LED spectrophotometer for analysis of color information

“…The soluble solid content, oil content, acidity, and other physiological properties of various fruits and vegetables have been detected in food industry [10]. Cell viability was tested with a colorimetric method using a reductive coloring reagent such as tetrazoliumsalt, alamarBlue and PrestoBlue in biotechnology [11,12]. Spectrometry can be used in the analysis of pollutants in water and the ocean by measuring the Chemical Oxygen Demand (COD) of organic matter using hydrogen peroxide (H2O2) and potassium permanganate (KMnO4) [13,14].…”

Biomimetic lipid/polymer colorimetric membranes

“…Our results have demonstrated that lipid/PDA systems similarly exhibit unique bio-chromatic transitions attributed to specific interfacial perturbation of the incorporated lipid domains. Such interactions indirectly affect the conjugated network of the adjacent PDA framework, thereby inducing colorimetric transitions (2)(3)(4)(5)(6)(7).…”

“…Such membrane properties have been investigated by a variety of analytical and structural methods (1). To better detect and analyze diverse membrane processes, we have recently developed a novel colorimetric lipid assay (2)(3)(4)(5)(6)(7). This assay has been used to examine various aspects of membrane biology, including enzymatic cleavage of lipids (2), screening of antimicrobial peptides (3), peptide-membrane interactions (4), ion sensing (5), antibody-epitope recognition (6), as well as assessing the activities of membrane penetrationenhancers (7).…”