Fungi have been used for centuries for food and beverage processing. In recent years, a better understanding of science and advancement in medical technology has shone a spotlight on their potential of being used as antibiotics to overcome the well-known deficits of the current antibiotics in use. However, it should not be forgotten that fungi are also responsible for causing a myriad of human infections. There is well-documented evidence of an increase in the number of cases for community-acquired fungal infections over the last decade.
Also, a rise in the number of immunodeficiency-related cases, antibiotic resistance development, and limited therapeutic options have made the search for alternatives a serious concern, and one which the research world is currently heavily involved with. There is a need for the new antifungals being formulated currently to be less toxic for the host, and it is also vital for them to have broader or targeted antimicrobial spectra, and have very little chances for triggering resistance in the long run.
With such criteria in mind, a lot of focus has been turned towards antimicrobial peptides that exhibit antifungal properties. These peptides are currently being viewed as powerful and potential candidates because of their high efficacy as well as high selectivity. In this piece, we will be giving an overview of the classification as well as the bioactivity of both natural and synthetic antifungal peptides, including their modes of actions as well as the specific advantages they have over the current drugs being used as antifungal therapies.
Types of antifungal peptides and bioactivity
Currently, it is estimated that there are about 1,133 peptides that possess antifungal properties – this is according to the information given by Antimicrobial Peptide Database (APD). Below is a classification of antifungal peptides based on their structure or mode of operation. However, the most acceptable classification is usually based on whether they are natural, synthetic, or semisynthetic.
Natural Antifungal Peptides
Natural antifungal peptides originate from a variety of species of bacteria, eukarya, and archae obtained from various natural sources. A good number of the natural antifungal bacteria thought antagonist activity testing in vitro on known pathogenic fungi. But with the increased use of sequencing technologies, new strategies have been formulated for predicting and discovering new natural antifungal peptides. Some of the notable new methods that are currently being used for discovery and prediction of these peptides include the hidden Markov model, docking simulations, and various template-based and sequence-based methods that make it possible for the novel in silico prediction of the antifungal peptides.
In most cases, natural antifungal peptides usually have alpha-helix structure, beta-hairpin, or sheet or a blend between alpha/beta-sheet structures whenever they interact with the membranes. Some of the natural antifungal peptides are rich in certain amino acids, making them be classified as glycine-rich arginine-rich, tryptophan-rich, histidine-rich and proline-rich amino acids. However, the actual structure of most of the antifungal peptides is yet to be fully determined.
Synthetic and semisynthetic peptides and structure-activity relationship
Antifungal synthetic and semisynthetic peptides are designed with the aim of improving the pharmacological properties, lowering immunogenicity, and reducing some of the undesirable side effects of the natural peptides. The pharmacological alterations also help in enhancing the bioavailability as well as the stability of the peptides. A very good example of such pharmacological transformation is the reduction of the hemolytic activity of echinocandin B antifungal peptide, which was achieved by replacing the linoleoyl side-chain with a pentyloxyterphenyl or octyloxybenzoyl side chains.
Structure-activity relationship (SAR) are vital factors of considerations in the design and development of synthetic peptides. There are lots of biophysical properties that can be used in the determination of antifungal activities , with the common ones being the peptide length , the secondary structure, amphipathicity, hydrophobicity, net charge, and stereospecificity among others. Some of these characteristics are interdependent on each other, while others are completely independent.
It should be noted, however, that most of the antifungal peptides are non-stereospecific and in a similar manner, we don’t have any dominant conformations among the antifungal peptides. Consequently, the main differentiating factors among the peptides originate from the variations of the sequences in both primary and secondary structures.
Amphipathicity and hydrophobicity are vital factors when it comes to the interactions at the peptide membranes as well as the processes of membrane permeabilization, not forgetting vital variables in designing synthetic peptides. A rise in hydrophobicity and amphipathicity correlates positively to a rise in antifungal activities as well as an increase in hemolytic activities. Studies also suggest that tryptophan can lead to an increase in hemolytic activities because of its ability to alter lipid polymorphism in the membranes.
The length of the antifungal peptides is also vital for the secondary structure as well as the mode of action. Most of the already-discovered antifungal peptides have between 11 and 40 residues. Studies show that about 7 – 8 amino acids are necessary for the formation of amphipathic structures in antimicrobial peptides. Studies also postulate that longer lengths of the amino acids may have potential impacts on the stability, cytotoxicity, and manufacturing costs.
To go around these hurdles, short microbial peptides with no more than amino ten amino acids are coming out as potential alternatives because they are more stable and they have low toxicity. In general, short antimicrobial peptides comprises of simple amino acids and it is easy to synthesize and modify them chemically which brings a lot of control over toxicity, half-life, specificity, and stability. They have also been found to be less immunogenic.
Models of mechanisms of actions of antifungal peptides
Peptides with antifungal properties have a very broad range of antimicrobial spectrum including viruses, fungi, and bacteria. Below is a brief look at the hypothetical mechanisms of how antifungal peptides operate when fighting fungal pathogens.
Inhibiting the actions of 1,3-β-Glucan synthesis
β-glucan synthesis is important for the integrity of the cell walls. However, cyclic lipoproteins may succeed to non-competitively inhibit it causing the destabilization of the cell wall, making it prone to cell lysis and osmotic stress. 1,3-β-Glucans are heavily involved with processes such as septum division and assembly of the walls – an action that makes it possible for β-Glucan synthase inhibitors to affect the structures of the cell walls. When β-Glucan synthase is inhibited, it leads to negative feedback which makes the cell cycles to rest.
Inhibitions of Chitin Biosynthesis in the cell walls
Chitin is present in the walls of the fungal cells. It is a vital amino acid in marinating cell integrity. Aureobasidins belong to this category and they are cyclic lipophilic 8-mer depsipeptides with two major modes of actions: interrupting sphingolipid synthesis and interrupting cell wall membranes by changing the accumulation of actin. Various members of aureobasidin family display anti-candida activity. These antifungal peptides are also known to interfere with the synthesis of chitin found in C. albicans as it was observed in various in vivo and in vitro studies.
Selective activities on membranes
One of the natural antifungal peptides that have shown selective activity on the cell membranes is Rs-ARF2. This is a 50 amino acid residue obtained from plant defensin and exhibits a three-stranded alpha-helix and beta-sheets structures. The amino acid residue has shown super effective properties in targeting fungus specific membrane glucosylceramide which is known to induce membrane permeability that further leads to increased uptake of Ca ions and medium alkalinization. The defensing is also known to cause an increase in the formation of toxic and reactive oxygen species intracellularly. Rs-ARF2 do not have this particular fungus-specific ceramide. They have very little cytotoxicity on mammalian cells when administered in dosages that might cause fungal pathogen inhibition.
Iturins are also natural antifungal peptides with very interesting selective activities on the cell membranes. They are cyclic peptides with a lipophilic amino acid attached to the L and D acids. They are mostly produced by Bacilus subtilis and they are very well-known to cause pore formation in the membranes, leading to leakage of certain key ions.
They have very limited antimicrobial activities with very little effects on bacteria. Sadly, these peptides have been discovered to be toxic to mammalian cell membranes. Also interestingly, the bulk of antimicrobial peptides are usually cationic, but iturins are also cationics but may sometime be anionic or neutral. Bacillomycin F is a member of this family and it is known to effectively inhibit the actions of C. tropicalis, C. albicans, and A. niger.
Histatin 5 is another natural antimicrobial peptide whose activities on the cell membranes are worth pointing out. It is a mammalian peptide with four arginines, seven histidines, and three lysines. Such a structure makes it adopt an alpha-helical structure when present in non-aqueous environments. The peptide works by binding itself to Ssa2p –a cell wall protein that is necessary for internalizing histatin5 into the cells. For the smooth uptake of this peptide, the polyamine transporters of Dur31 and Dur3 are always necessary. These have to be translocated into the cells.
The broad range of antimicrobial peptides
Most of the antimicrobial peptides have various ways of affecting a number of microorganisms. Such organisms include fungi, envelope-containing viruses, and bacteria. Most of the antimicrobial peptides can be collectively grouped as cyclic peptides or linear peptides. Examples of cyclic peptides include poultry gallinacins, syringomycins, macrocyclic peptides, and mammalian defensin among others. Examples of linear peptides include magainins, cathelicidins, lactoferrin-derived peptides, and bombinins.
Most of these peptides are known to cause membrane disruption by forming a toroidal pore that causes essential molecules to leak through the membranes, hence, greatly compromising the integrity and functionality of the membrane. Other mechanisms of the antimicrobial peptide actions include demixing and clustering, alteration of membrane potential, lipid flip-flop, the formation of anionic lipid-peptide domains, and membrane thinning among others. Additionally, some of these antimicrobial peptides may influence cell processes such as inhabitation of DNA replication, cell apoptosis induction, damage of the DNA and protein, and RNA synthesis among others.
Advantages of antifungal peptides
The current antifungal agents have a wide range of shortcomings that hamper their efficacy as therapeutic tools in the fight against fungal infections. There is just a handful of approved antifungal drugs but their future as antifungal therapies is shrouded with a lot of uncertainties because of the persistent resistance to those therapies. Apart from the antifungal resistance, the current therapies have a low diversity of mechanisms of action. Some of them also exhibit adverse reactions on the hosts while others also affect common eukaryotic targets present in both human cells and pathogenic fungi cells. These challenges have caused serious problems in the design and development of novel efficient and non-toxic antifungal therapies.
Antifungal peptides are currently being viewed as the best alternatives for overcoming the shortcomings of the current antifungal drugs. This is because antifungal peptides have multiple microbial targets and this helps to reduce the possibility of the development of resistance as it is the case with most antifungal drugs. Some of the notable antimicrobial targets include molecules for physiological processes such as DNA and RNA, cell cycle, protein synthesis, and different cell wall components.
Due to the lack of side effects, a good number of antifungal peptides are able to target very specific fungal molecules. This ability leads to high pathogen selectivity and also helps to reduce the chances of cytotoxicity on mammalian cells. Sadly, this is not an assurance that there will be no cytotoxicity. However, most of the antifungal peptides have highly reduced cytotoxicity. This is believed to be the case because of two reasons. One: It is believed that there exists a stronger interaction between the cationic charges of the peptide and the negatively charged fungal membrane. Two, some of the antifungal peptides target membrane lipids which are unique to the fungi and are not found in mammalian cells. This phenomenon greatly helps to reduce toxicity.
More and more evidence is being gathered to show that antimicrobial peptides and antifungal peptides are multifactorial. Host defense peptides such as the cathelicidins and defensins are known to be anti-inflammatory, immunomodulatory, and angiogenic. They also display the ability to initiate adaptive immune responses. With further insights in the development of antifungal therapies using antifungal peptides, resistance to the current antifungal drugs will be a thing of the past, and there will be a whole new regime for dealing with fungal and microbial infections.
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