Antifungal activity of protamine

Abstract

Antimicrobial peptides (AMPs) are important innate defense molecules having wide spectrum of bioactivities like antibacterial, antifungal, antiparasitic and antiviral activities. The primary role of AMPs is to exert cytotoxicity on the invading pathogenic microorganisms and serve as immune modulators in higher organisms. Protamine is a polycationic peptide found in the nuclei of sperm in different vertebrate species has also antimicrobial activities. Protamine is thought to disrupt the cytoplasmic membrane caused by electrostatic interactions between the highly positively charged molecule and the negatively charged microbial cell surfaces. In addition to this, it is involved in wound healing activity and recruit leucocytes and modulators during inflammation. The present study proposes to investigate the anticandidal effects of protamine on clinical isolates of Candida spp. For experimental evaluation of anticandidal and antibiofilm activity, minimal inhibitory concentration (MIC) was determined against Candida albicans, Candida tropicalis and Candida krusei. Protamine inhibited the growth of all the tested Candida spp. pathogens in respective MIC of 16 μg ml-1, 32 μg ml-1 and 256 μg mg-1. After MIC determination, the mechanism of action was evaluated by assessing structural and cellular biochemical changes. The structural changes were examined by scanning electron microscopy (SEM) after treating C. albicans with protamine and SEM images clearly showed membrane rupture indicating that the peptide targets the membrane. Biochemical changes like induction of reactive oxygen species (ROS) inside the cells which is essential for cell death was detected by staining the cells with 2′,7′-Dichlorofluorescin diacetate which turns to green colour in response to oxidative metabolism by esterification reaction. Protamine increased ROS production in Candida cells. These results suggested that protamine could be a lead compound for preparation of biomaterials for anticandidal treatment.