Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp

RESEARCH ARTICLE Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp Juhye Han1, Md. Anirban Jyoti1, Ho-Yeon Song1,2*, Woong Sik Jang2* 1 Department of Microbiology and Immunology, School of Medicine, Soonchunhyang University, Cheonan, Chungnam, 31151, South Korea, 2 Regional Innovation Center, Soonchunhyang University, Asan, Chungnam, 31538, South Korea * (WSJ); (H-YS) a11111 OPEN ACCESS Citation: Han J, Jyoti M.A, Song H-Y, Jang WS (2016) Antifungal Activity and Action Mechanism of Histatin 5-Halocidin Hybrid Peptides against Candida ssp. PLoS ONE 11(2): e0150196. doi:10.1371/ journal.pone.0150196 Editor: Anirban Bhunia, Bose Institute, INDIA Received: January 5, 2016 Accepted: February 10, 2016 Published: February 26, 2016 Copyright: © 2016 Han et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a grant from the National Research Foundation of Korea, Republic of Korea (NRF 2013R1A1A1075979) URLs: http:// www.nrf.re.kr/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abstract The candidacidal activity of histatin 5 is initiated through cell wall binding, followed by translocation and intracellular targeting, while the halocidin peptide exerts its activity by attacking the Candida cell membrane. To improve antimicrobial activities and to understand the killing mechanism of two peptides, six hybrid peptides were designed by conjugating histatin 5 and halocidin. A comparative approach was established to study the activity, salt tolerance, cell wall glucan binding assay, cytotoxicity, generation of ROS and killing kinetics. CD spectrometry was conducted to evaluate secondary structures of these hybrid peptides. Furthermore the cellular localization of hybrid peptides was investigated by confocal fluorescence microscopy. Of the six hybrid congeners, di-PH2, di-WP2 and HHP1 had stronger activities than other hybrid peptides against all tested Candida strains. The MIC values of these peptides were 1–2, 2–4 and 2–4 μg/ml, respectively. Moreover, none of the hybrid peptides was cytotoxic in the hemolytic assay and cell-based cytotoxicity assay. Confocal laser microscopy showed that di-PH2 and HHP1 were translocated into cytoplasm whereas di-WP2 was accumulated on surface of C. albicans to exert their candidacidal activity. All translocated peptides (Hst 5, P113, di-PH2) were capable of generating intracellular ROS except HHP1. Additionally, the KFH residues at C-terminal end of these peptides were assumed for core sequence for active translocation. 1.0 Introduction Antimicrobial peptides (AMP) are recognized as one of the primordial and evolutionary defense systems of the animal kingdom. AMPs are widely distributed in nature and therefore play pivotal roles in the innate immune system of many organisms [1–3]. AMPs, found in every tissue system also protect human beings continually against pathogenic invasion and diseases from viruses, bacteria, fungi, protozoa and other parasites, thus retaining their therapeutic potential as ‘peptide-antibiotic’ in humans as well [3, 4]. Moreover, AMPs for antibiotic applications are considered advantageous over conventional antibiotics as conventional PLOS ONE | DOI:10.1371/journal.pone.0150196 February 26, 2016 1 / 18 Histatin 5-Halocidin Hybrid Peptides with Amended Antifungal Activity antibiotics contribute to the emergence of resistant pathogens and fail to treat diseases from resistant pathogens afterward [5]. The therapeutic potential of a majority of AMPs are due to their ability to destabilize the cytoplasmic membrane of pathogens and the ability to kill multidrug resistant pathogens as well as rapid killing kinetics. Histatin 5 (Hst 5), one of the salivary histatins, is amphiphatic with α helices and inhibits fungus and bacteria. Of the three major human histatins (Hst 1, Hst 3 and Hst 5), Hst 5 is the most potent anticandidal peptide and kills both blastopore and germinated forms of Candida albicans [6]. Hst 5, a 24 amino acid residual peptide, is produced with multiple posttranslational proteolytic processing of Hst 3. A number of studies on the killing mechanism of Hst 5 showed that rather than a single target, multiple intracellular targets are orchestrated, leading to osmotic imbalance via the loss of potassium ions and triggering candidacidal activity [7, 8]. In addition, Helmerhorst et al. reported that histatin 5 induced the formation of reactive oxygen species (ROS) in C. albicans cells as well as in isolated mitochondria and that ROS levels were highly correlated with cell death [9]. The cellular uptake of Hst 5 involves one or all of the three uptake pathways: i) transporter mediated uptake, ii) direct transfer across the membrane and iii) receptor mediated endocytosis [10]. Also, it was shown that the structural conformation of Hst 5 is important for membrane binding and killing of C. albicans. At least 12 amino acids at the C terminus of Hst 5 form the functional domain required for candidacidal activity [11]. Based on the full length sequence of Hst 5, synthetic congeners of Hst 5 have been synthesized and investigated for more biological activities exceeding that of the parental Hst 5 [12]. One modified congener of Hst 5, P113, contains 12 amino acid residues from Hst 5 and was found to retain the anticandida activities. The mode of action for P113 was assumed to be similar to that of Hst 5 [13]. Halocidin is a heterodimeric peptide from tunicate Halocynthia aurantium with α-helical structure and is reported to exert a broad spectrum of antimicrobial activities in vitro [14, 15]. The heterodimer comprises two different subunits; one contains a total of 18 amino acid residues (WLNALLHHGLNCAKGVLA, 18Hc) and the other contains only 15 (ALLHHGLNCAK GVLA, 15Hc). Both subunits are linked covalently by an inter-disulfide bond. Previous studies reported that the dimeric congeners of halocidin are stronger antimicrobials than their monomeric forms [14, 15]. However, the target for candidacidal activity differs from that of Hst 5 and P113 because halocidin disrupts the cell membrane rather than intracellular translocating [15]. Interestingly, halocidin was found to retain its candidacidal activity under physiological salt conditions [15]. To design an antibiotic of peptide origin, several limitations have to be overcome. Conformational analysis and residual amino acid rearrangement of AMPs suggest that synthetic peptides with higher activity, high salt tolerance and low cytotoxicit (...truncated)