Mechanism of Interaction of Human Parotid Secretory Protein - Derived Antimicrobial Peptides: A Biophysical Insight

Despite the success in conventional antibiotic development, antibiotic resistance and development of superbugs has become a global public health problem with much deeper impact on our society and economy. Naturally occurring antimicrobial peptides (AMPs) are an organism's built-in defense that combat microbial infections in healthy individuals. AMPs and their synthetic analogs have attracted attention worldwide as potential therapeutics against multi-drug resistant bacterial strains. Human parotid secretory protein (hPSP) is a major salivary protein expressed in the oral cavity of humans. GL13K, GL13NH2 and GL13D/N are short thirteen amino acid long cationic AMPs derived from the hPSP and found to exhibit antimicrobial and/or anti-inflammatory effects. It is essential to identify the membrane and peptide properties that allow GL13 peptides identify and target bacterial membranes over eukaryotic membranes, to reveal the mechanistic details for the mechanism of action of GL13 peptides. Liposomes and supported lipid bilayers comprised of 1, 2-dioleoylphosphatidylcholine and 1, 2-dioleoylphosphatidylglycerol were used as models for eukaryotic and bacterial membranes respectively to study the effect of electrostatic forces. Membranes containing cholesterol were used to study the effect of membrane packing density. A research methodology was adopted in which we first investigated the peptide binding and secondary structure transformation upon interaction with membrane using isothermal titration calorimetry and circular dichroism. Later we identified the impact of this interaction on the membrane integrity, whether it causes membrane fusion and/or membrane lysis using carboxyfluorescein leakage assays, dynamic light scattering, dual polarization interferometry and atomic force microscopy. The activity and selectivity of GL13 peptides are due to a fine balance between their cationic and amphipathic nature. Increased amphipathicity or structuring of the peptide is found to be associated with a loss of specificity. Cholesterol was found to significantly attenuate the membrane lytic activity and enhance the selectivity towards bacterial model membranes. GL13K and GL13NH2 were found to act by the carpet mechanism where GL13K caused localized micellization accompanied by the loss of lipid molecules and GL13NH2 acted mainly by causing transient destabilization of the membrane without loss of lipid molecules from the membrane. GL13D/N seemed to act by a combination of various mechanisms such as the carpet and toroidal pore mechanisms. GL13K is proposed to be the best candidate for therapeutic applications as antimicrobial.