The discovery of lysozymes in 1922 by Alexander Fleming marked the genesis of modern day innate immunity. Following this finding, there has been the discovery or development of a multitude of antibiotics and antimicrobial peptides. Presently, there are over 3000 antimicrobial peptides that have been discovered and duly recorded in the antimicrobial peptide database. Different types of antimicrobial peptides tend to have some commonalities: most of them have between 10 and 60 amino acid residues, and they are mostly cationic. But there are also several antimicrobial peptides that are anionic in nature, and they also have several acidic amino acids in their structure, including glutamic acid and aspartic acid.
The resistance of organisms to antimicrobial is becoming a concerning problem especially when you consider the rampant abuse of antibiotics in animal husbandry, agriculture, and medicine. A research study conducted in Kenya observed that there were a substantial amount of antibiotic residues in most of the edible meats that were being sold in most cities in the country.
But from the perspective of most of the pharmaceutical companies in the region, the development and subsequent use of new antibiotic therapies are likely to lead to a dip in profit, and this is not something these companies are willing to accept. Because of this, the process of replacing antibiotics is still a concern within the industry and there is consultation between the concerned parties, including the experts in animal husbandry, the food industries, and the relevant pharmaceuticals.
There are many ongoing studies about antimicrobial peptides, and with each passing year it is believed that more information about these peptides will surface. In this piece, we will explore the action mechanism of the antimicrobial peptides as well as some of the design methods that are currently being used in several studies and research.
Membrane Targeting Mechanisms
There are various models that can be used to describe the membrane targeting mechanisms of antimicrobial peptides. The major ones, however, include the following-:
The Toroidal Pore Model
Also known as the wormhole model, the toroidal pore model is when the antimicrobial peptides vertically embed themselves in the cell membrane where after accumulation, they bend leading to the formation of a ring hole of about 2nm in diameter. Some of the typical examples of this model include lacticin Q, arenicin, and magainin. Also, cationic peptides have also been observed to compromise the cell membranes by creating various types of fluid domains.
With this model of actions, the antimicrobial peptides will aggregate with each other before penetrating the bilayer of the cell membrane in the form of multimers before forming channels that lead to cytoplasmic outflow. In very rare and extreme cases, it is possible for the antimicrobial peptides to induce the collapse of cell membranes which ultimately leads to the death of the cells. For example, alamethicin does its pore-forming activities with the use of this model.
With this model, the antimicrobial peptides start by getting arranged parallel to the cell membranes. With this arrangement, their hydrophilic ends will be organized in a manner such that they end up facing the solution while their hydrophobic ends face the phospholipid bilayer. Antimicrobial peptides will then cover the surface, much like a carpet covers the floor, and as such will destroy the cell membrane in a detergent-like manner.
However, for this pore-forming mechanism, there has to be a certain concentration threshold, and the amount of concentration required of the antimicrobial peptides is normally too high. A good example of a peptide that clearly exhibits this activity is the human cathelicidin.
It is possible to further refine most of the membrane targeting mechanisms as a means of addressing the large differences in the lipid composition of the cell membranes of the fungi, bacteria, and mammals. Some of the main lipids found in cell membranes include lysolipids, sterols, and sphingolipids. Phosphatidylethanolamine (PE) and cardiolipin, (CL) happen to be some of the most common anionic lipids present in bacteria while phosphatidic acid (PA) and phosphatidylcholine (PC) are some of the main glycerophospholipids (GPLs) found in the fungal cell membranes.
Additionally, fungal cell membranes happen to be more anionic compared to mammalian cell membranes and they also tend to have a higher concentration of PC content. On the other hand, ergosterol is the sterol that you will find in the plasma membrane of lower eukaryotes like fungi while that of the animals tend to contain cholesterol. The majority of the antimicrobial peptides pride themselves in the differences in their membrane composition as the unique edge they have over one another when it comes to exerting their various effects.
Antimicrobial peptides have emerged as promising candidates for anti-biofilm agents. However, it should be noted that they are very different from cell-penetrating peptides which usually contain between 5 and 30 amino acids and they have the unique ability to be able to translocate across the cell membranes.
Cell-penetrating peptides are normally classified according to their physicochemical properties and this leads to three main classes: hydrophobic, cationic, and amphipathic. Anti-biofilm peptides, however, have very strict requirements when it comes to these physicochemical properties. The targeting of anti-biofilm peptides is usually through a variety of mechanisms, including the degradation of the signals within the biofilm, permeabilizing within the cytoplasmic membrane, and modulating the production of the EPS.
Non-membrane Targeting Mechanisms
With most of the non-membrane targeting mechanisms, the antimicrobial peptides mainly enter the cells through direct penetration or through endocytosis. After getting through to the cytoplasm, the antimicrobial peptides will then identify and then act appropriately on the target. Depending on what the target is, here is an overview of some of the action mechanisms under that the antimicrobial peptides can use-:
The inhibition of protein biosynthesis
Through their actions, antimicrobial peptides have the ability to affect or influence processes such as translation, transcription, and the assembly of proteins into functional peptides through biological processes such as chaperone folding which normally happen through the interference with the relevant effector molecules and related enzymes. A good example of such a peptide is Bac 7 1-35 which is designed to target ribosomes with the goal of inhibiting protein translation.
Tur1A, on the other hand, can inhibit the synthesis of protein in E.coli by inhibiting the translation starting from the initial phase through to the extension phase. However, the difference between the two – Tur1A and Bac 7 1-35 – can lead to the binding to ribosomes as well as the interaction with the ribosomal peptide exit tunnel. However, some antimicrobial peptides have very different targets. For example, through genome-wide transcription, it has been observed that DM3 may have an effect on a variety of vital intracellular pathways of protein biosynthesis.
Chaperons are vital proteins when it comes to correctly folding and assembling newly synthesized proteins needed to make them stereoisomerism, which in turn will make all the antimicrobial peptides have cell selectivity and the ability to prevent cytotoxicity. According to review and findings from past works, it was observed that both drosocin and pyrhocoricin have the ability to prevent Dnak from refolding misfolded proteins through the induction of a permanent closure of DnaK peptide-binding cavity.
The inhibition of nucleic acid biosynthesis
Some of the antimicrobial peptides have the ability to affect certain key enzymes or simply induce the degradation of nucleic acid molecules to stop the biosynthesis of the nucleic acid. For example, indolicin, which is a C-terminal amidated cationic antimicrobial peptide with 13 amino acids, has demonstrated the ability to specifically target the abasic site of DNA to cross-link single or double-stranded DNA. The peptide has also shown the ability to inhibit the actions of DNA topoisomerase 1. Tissue Factor Pathway Inhibitor is another antimicrobial peptide from the tongues which can easily enter the cytoplasm of the target cells after the rupture of the cell membrane and then cause the degradation of the DNA and RNA.
Inhibition of the activities of Proteases
There are quite a few antimicrobial peptides with the ability to inhibit various metabolic activities by simply inhibiting the actions of protease. For example, histatin 5 is an antimicrobial peptide known to have very strong inhibitory effects on the proteases created by a wide range of bacteria and other forms of hosts. AMPs eNAP-2 and indolicidin have the ability to inhibit the actions of microbial protease, elastase, as well as chymotrypsin. Cathelicidin-BF is another peptide with powerful inhibitory effects with the ability to inhibit thrombin-induced platelet aggregation and subsequently prevent protease-activated receptor 4.
Inhibition of cell division
Many antimicrobial peptides also have the ability to inhibit the process of cell division by simply inhibiting DNA damage response and DNA replication processes, causing the failure of the separation of chromosomes, or blocking the cell cycles. For example, APP which is an antimicrobial peptide with a total of 20 amino acids residues is has been found to be very effective in killing C.albicans because it has very high efficiency when it comes to cell penetration as well the ability to cause S-phase arrest when exposed to intracellular environments. MciZ, another antimicrobial peptide with about 40 amino acid residues, is also a very powerful inhibitor for bacterial cell division, localization, and Z-ring formation.
Additionally, some studies have demonstrated that several AFPs with the ability to damage the organelles of fungi exist. A good example is histamine 5, which has the ability to interact with mitochondria, leading to the production of ROS which ultimately results in the death of the cells.
Apart from the intracellular targets, other potential targets for antimicrobial peptides can be defined by the differences that exist between the cell compositions, mannoproteins, and lipid A. Specifically, Gram-negative and Gram-positive bacteria are usually classified according to their bacterial cell wall structure. With Gram-positive bacteria, they usually have a layer of cross-linked peptidoglycan while Gram-negative bacteria usually feature an extra outer membrane with an inner leaflet that has phosphatidic acid and an out leaf that features LPS.
LPS comes with a negatively charged phosphate group, which then combines with a salt bridge with the help of a divalent cation, leading to the formation of an electrostatic network. The electrostatic zone is normally viewed as the main barrier that stops the hydrophobic antibiotics and is also responsible for creating a low permeability of Gram-negative bacteria. Most of the cell walls are mainly comprised of mannoproteins and chitin.
Any mutations happening in the relevant genes of the LPS pathways as well as in phospholipid trafficking are sufficient to provide resistance to the antimicrobial peptide. Some of the mannoproteins typically found in the cell walls include various proteins including structural proteins, enzymes, and cell adhesion proteins which are normally very much involved in the synthesis and remodeling. These proteins are slightly different from the proteins found in the membranes of human cells and they are very viable targets for AFPs. Additionally, lipoteichoic acid and teichoic acid present in the cell walls can also be potential targets for AMPs. With these theories, there is sufficient support for the design of antimicrobial peptides with very low toxicity.
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