Antimicrobial and antimycotic peptides are small cationic and amphipathic molecules, generally with fewer than 50 amino acids. These ubiquitous peptides have been isolated from prokaryotes and eukaryotes in the plant, bacterial, fungal, and animal kingdoms [1, 2]. Nature has strategically placed antimicrobial and antifungal peptides as a first line of defence between the host organism and its surrounding environment, because these peptides are able to inhibit quickly a wide spectrum of infectious microbes without significant toxicity to the host organism. When insects are infected within a short period they secrete an array of cationic peptides to combat the invading organism [3]. Although antimicrobial peptides (AMP) are the primary means of combating organisms in lower forms of life, these peptides have an adjunct role in the immune system of phylogenetically more advanced organisms.

There is a large array of antifungal proteins with different structures. In addition to the well-known glucanases [4], chitinases [5], thaumatin-like proteins [6], defensins [7] and ribosome-inactivating proteins [8], there is a diversity of other antifungal proteins such as lipid transfer proteins [9] and protease inhibitors [10].

Both fungi and humans are eukaryotes and at the molecular level, their cells are similar. This makes it more difficult to find or design drugs that target fungi without affecting human cells. Consequently many antifungal drugs cause side effects. Some of these side effects can be life threatening if the drugs are not used properly. Despite chemical therapies, serious fungal infections remain difficult to treat, and resistance to the available drugs is emerging [11]. Antifungals work by exploiting differences between mammalian and fungal cells to kill the fungal organism without dangerous effects on the host. A common theme with most of these wide-spectrum AMPs is that they lyse the cell membranes of the pathogens without harming the host targets. Despite this non-specific mechanism, many of these peptides do not lyse mammalian membranes at concentrations that can inhibit the pathogen [12].

In the last decades, the incidence of fungal infections by pathogenic C. albicans and other related human opportunistic yeast species has increased dramatically due to the rise in the number of immunocompromised patients. Several Candida species especially C. albicans normally inhabit the oral cavity, respiratory and intestinal tracts, and vaginal cavity of humans and animals. In recent years, there has been a marked increase in the incidence of treatment failures in candidiasis patients receiving long-term antifungal therapy, which has posed a serious problem in its successful use in chemotherapy. Candida cells acquire multidrug resistance (MDR) during the course of the treatment [13].

Many bacterial strains, and particularly their enzymes, that perform catalysis efficiently at low temperatures are used in a number of biotechnology applications [14]. Enterococci, as part of the natural intestinal flora of humans and animals, are known to play an important role in maintaining microbial balance [15, 16]. Many different enterocins have been described from Enterococcus faecalis and E. faecium. Some of these peptides showed activity against Escherichia coli[17] and Salmonella pullorum[18].

Since the literature on bacterial antifungal proteins is rather scanty compared with that on bacterial bacteriocins, there is a pressing need to explore and isolate from new sources potential bacteria capable of producing novel AMPs and to characterise them for further applications. In the present study, we report the purification and characterisation of an antifungal protein produced by E. faecalis, that shows broad-spectrum activity against the indicator organisms, multidrug resistant C. albicans with negligible haemolytic activity.

Biochemical characteristics and fatty acid methyl ester (FAME) analysis identified the strain as E. feacalis, whereas 16 S rDNA sequencing identified the strain as E. faecium[19]. Potassium tellurite reduction, however, distinguished the strain as E. faecalis rather than E. faecium. The concentrate made from the CFS of the test strain inhibited 7 multidrug resistant strains of C. albicans.

There are several bacteriocins from E. faecalis and other species origin [15, 24], but antimycotic peptides or proteins are rare. Pseudomonas syringie and some Bacillus species produce antifungal peptides, but no such reports about E. faecalis[25] were found. The genus Enterococcus belongs to a group of important lactic acid bacteria (LAB) that participate and contribute towards different fermentation processes. Their functionality in dairy and meat products has been reported in detail [26, 27]. Several bacteriocins produced by Enterococcus species [24] or other enterococci of different origins [15], have been reported and characterized at the biochemical and genetic levels. Several antifungal peptides (iturins, bacillomycins) were discovered from Bacillus and Pseudomonas. Nikkomycins, produced by Streptomyces tendae and S. ansochromogenes, and polyoxins, produced by S. cacaoi, are the most widely studied antifungal peptides, whereas antifungal peptides from Enterococcus species [25, 28] are rare. Various strains of Bacillus subtilis produce iturin A and bacillomycin L peptide. Iturins inhibited the growth of fungi including Aspergillus niger, C. albicans, and F. oxysporum[29, 30]. Initial clinical trials involving humans and animals showed that iturin A was effective against dermatomycoses and had a wide spectrum of antifungal properties and low allergenic effects [31]. Unfortunately, bacillomycin L and iturin A are haemolytic, which may reduce their potential use as antifungal drugs [32].

In an era of increased incidence of fungal infections in immunocompromised patients [33, 34] and greater resistance to ‘frontline’ antifungal therapies [35], there is a growing need to discover new antifungal therapies. Although newer azole derivatives such as voriconazole are more effective and have cidal activity against filamentous fungi such Aspergillus fumigatus[36], these derivatives are fungistatic and not fungicidal against pathogenic yeasts. The inability to kill yeasts leads to resistance to azole in prolonged infections and increases the likelihood that these agents will lack efficacy in severe Candida infections in immunosuppressed patients. Amphotericin B has also been commonly used to treat serious fungal infections, but in contrast to azoles, amphotericin B is fungicidal against yeasts. Nevertheless, resistance to amphotericin B is slowly developing in selected Candida species [37] and there are significant side effects associated with its use, including nephrotoxicity. Although recently developed antifungal agents, including the peptide-based agents’ micafungin and caspofungin, are very promising, resistance to these therapies has already been reported [38–40] and will no doubt become more widespread. The development of resistance to current antifungal agents, the limited efficacy, and the side effects associated with several of these agents increase the importance of continued development of new alternative approaches.

The identified Enterococcus faecalis strain produces the antimycotic substance, ACP, extracellularly. The activity of the ACP was stable upon treatment at different temperatures, for up to 90°C for 20 min but the activity was lost after boiling and autoclaving. While similar results have been reported for bacillomycin D from B. subtilis[41] and durancin L28-1A from E. durans[42], bacteriocin ST15 from E. faecium was inactivated when subjected to 121°C for 20 min [43]. The antimycotic property of the ACP also remained unaffected in the pH range of 6.0–8.0. At pH values of 5.0 and 9.0, however, the activity was reduced by 50% whereas at values of pH 2.0, 4.0, and 10.0 activity was lost completely. These results are similar to those reported for the bacteriocin produced by E. mundtii[44]. Several bacteriocins produced by enterococci are known to exhibit a wide range of pH stability [45]. The ACP was stable in different organic solvents and surfactants; such stability has been a common feature of many bacteriocins produced by Enterococcus, AMP produced by Bacillus species, and other LAB [43, 46, 47].

The ACP was fully sensitive to proteinase K and partially sensitive to pronase E, confirming its proteinaceous nature. Its resistance to pepsin, lysozyme and trypsin indicated that the anti-Candida active principle may be a cyclic peptide containing unusual amino acids and therefore more resistant to protease hydrolysis [48]. These results suggested that this antimycotic peptide could survive in the intestinal environment and might therefore be administered with food [49]. On the other hand, the ineffectiveness of α-amylase and lipase on antimycotic activity suggested that the ACP might not be glycosylated and might not contain a lipid moiety. When the ACP was heated with 1 mmol and 2 mmol β-mercaptoethaol at 80°C for 10 min to ensure thiol residues existed in the reduced state, no particular change in antimycotic activity was observed. This indicates that the oxidation state of the cysteine residues may not be important for the antimycotic activity [50]. When the dialysed ACP was treated with the reducing agent DTT, no decrease in inhibitory activity was observed, indicating that disulphide bonds are not responsible for biological activity. It was also observed that storage of ACP at −80°C for 1 year did not significantly affect biological activity. Ammonium sulfate salt as well as sodium phosphate buffer did not inhibit ACP activity at the concentration used and did not modify the result of the assay. The dialysed concentrate of ACP, dissolved in 20 mmol sodium phosphate buffer, weakly bound with the DEAE Sepharose matrix, indicating that the ACP bears negative charges. Being weakly negative, it was separated easily in native polyacrylamide gel electrophoresis. After purification by ammonium sulfate fractionation, dialysis, anion exchange chromatography and gel filtration, the final amount of recovered protein (0.45%) was found very low. This could be increased by using protein engineering and optimization methods.

Comparing the partial amino acid sequence of the purified antimycotic protein to other antimicrobial peptides and bacteriocins by using protein-protein BLAST in NCBI revealed no complete homology with other known bacteriocins or AMPs. The combined N-terminal and de novo sequence GPGGPG…WLPPAGLLGRCGRWFRPWLLWLQSGAQYKWLGNLFGLGPK had high amounts of glycine, proline, leucine and tryptophan. This has been observed in many antimicrobial peptides including bacteriocins like enterocin and acidocin.

It was reported earlier that the glycine-rich antifungal peptide tenacin-3 enters the C. albicans cytoplasm [51], although tenacin-3 seems not to induce membrane permeabilisation. Linear peptides with an extended structure were characterised by an unusual proportion of one or more amino acids (most often proline, tryptophan, or glycine) [52, 53]. Penaedins characterised from shrimps and prawns had a high content of Pro/Arg/Gly residues in the extended N-terminal domain [54]. Oxypinin 2 has a GVG motif, and ponericin G has glycine residues flanking the central proline, resulting in a GPG motif with calculated grand average of hydropathicity (GRAVY) of −0.683.20. The presence of Gly-Pro hinges in antimicrobial peptides like oxypinins, ponericins, and cecropins supports the antimicrobial potential of ACP, wherein a similar sequence was observed. The regional flexibility provided by proline was sometimes enhanced by the presence of glycine residues [55]. In another recent report, a penaedin homologue, hyastatin from spider crab [56], was shown to possess a Pro/Gly domain similar to the N-terminal domain of penaedins that bind chitin tightly. This information strengthens the idea that the N-terminal minor sequence GPGGPG of the anti-Candida protein in the present study could interact with the cell wall of Candida as a primer for antimicrobial action [56]. In such a proline-rich sequence, a proline kink has all the potential to create pores [57]. It was cogently argued that in cationic hydrophobic peptides the presence of polar residues confers a hydrophilic property to the proline-rich peptides. In an earlier study conducted on curvaticin FS47, the neutral (Gly [24%]) and hydrophobic (Ala, Ile, Leu, Val, Pro, and Phe [47%]) residues at the N-terminal constitute a significant proportion which helps to explain the hydrophobic interactions that curvaticin FS47 displays. It was reasoned that the high proportion of Gly residues (23.9% in ACP) would likely provide a significant amount of flexibility to the antimicrobial molecule [58]. In fact, the increase of hydrophobicity of the peptides also correlated with fungicidal activity [59]. In accordance with many other bacteriocins of LAB e.g., lactococcin A [60], lactacin F [61], and curvaticin FS47 [58], a high proportion of glycine was likely to provide a significant amount of flexibility to the molecule. A recent study on lactococcin G, enterocin 1071B, and EntC2 suggested that the N-terminal sequence of the peptide of each bacteriocin (LcnGβ, Ent1071B and EntC2) is important for determining target cell specificity [23, 62].

Previously, the N- terminal sequence of the antimicrobial dermaseptin B was reported to be highly hydrophobic which could enable its binding to zwitterionic outer and negatively charged surfaces [63]. In addition, the part of the N-terminal sequence which contains Gly-Pro residues and the combined de novo sequence detected in the anti-Candida protein ACP 43 under current investigation, were supported by the inference that proline-rich peptides (often associated with arginine) enter cells without membrane lysis and after entering the cytoplasm bind to and inhibit the activity of specific molecular targets causing cell death [64]. Other studies with model amphipathic all L- amino acid peptides with the sequence KX3KWX2KX2K, where X = Gly, Ala, Val, or Leu showed that the leucine-rich peptide, rather than the Ile- or Val-containing peptide, was particularly antimicrobial [63]. Our result is in agreement with this observation: leucine amounted to 19.6%, and proline (13.0%) was in association with arginine.

The combined sequence derived from the de novo sequencing, WLPPAGLLGRCGRWFRPWLLWLQ SGAQY KWLGNLFGLGPK, showed high content of glycine (17.5%), proline, leucine and tryptophan. The amino acid content also revealed that the peptide was quite hydrophobic due to the presence of high amounts of leucine (22.5%), and this is believed to play a role in the interactions with the cell membrane [61]. The hydrophobicities (GRAVY) of individual peptides having m/z 718, 1039 and 601 were 0.108, -0.388 and 0.282 respectively, which indicates that these peptides are relatively hydrophobic and characteristic of many bacteriocins isolated from Enterococcus species [65]. High levels of glycine (31%) and glutamine (18%) residues in another cationic antifungal peptide constitutively produced by S. peregrine larva were also reported to bind C. albicans through electrostatic interaction and disturb the osmotic integrity of treated cells [56]. In contrast, a novel glycine/leucine-rich antimicrobial peptide, leptoglycine (glycine 59.1% and leucine 36.4%) derived from Leptodactylus pentadactylus failed to inhibit C. albicans. We have used the combined de novo sequence to predict the structure using the PSIPRED (Protein Structure Prediction) server. The sequence WFRPWLLWLQSGAQYK showed alpha helical structure, which is characteristic of many antimicrobial peptides [63].

The MIC of the ACP against wild-type C. albicans DI was 1067 μg ml^-1, whereas the lowest MIC, 133 μg mL^-1, recorded was against MTCC 183 and MTCC 7315.The MIC of the ACP against MTCC 3958 was 267 μg mL^-1 which was slightly higher than the MICs of iturin and bafilomycin F [25]. In this study, the results of toxicity experiments were of great interest. ACP was non-toxic to human erythrocytes up to a tested concentration of 6.4 mg mL^-1. At this concentration, the percent haemolytic activity was 3.76 which is comparatively much less than the haemolytic activities of baciamin [66] and bafilomycin F [25].

It was also concluded that ACP was not able to hemagglutinate human red blood cells up to the concentration of 1.6 mg ml^-1 (Figure 8), however the concentration higher than this were able to hemagglutinate the human RBC, whereas this concentration is much more than the MIC of the ACP. These properties taken together might render this antimycotic protein ACP, a potent candidate for treating candidiasis, and its related pharmaceutical application can be established in synergy with other relevant antifungal antibiotics of low dosage.

In this study an antimycotic protein, ACP from the bacterial strain E. faecalis was purified to near homogeneity. This antimycotic peptide has negligible haemagglutination and haemolytic activity and hence potentially warrants use in synergy with low dosages of available antifungal drugs to inhibit multidrug resistant C. albicans.