Anionic Antimicrobial Peptides: Definition, Structure, Mechanism and Examples
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What are the anionic antimicrobial peptides?
Anionic antimicrobial peptides (AAMPs) have been known to play an important role in the innate immune systems of many different kinds of organisms since their discovery in the 1980s. The intended recipients of these peptides include bacteria, fungi, insects, and viruses. AAMPs are a rare kind of antimicrobial peptides that, under physiological pH, have a net negative charge. AAMPs can be created or constitutively produced; in some cases, their antibacterial effect seems to be a byproduct, whereas other biological processes constitute their primary purpose.
Structure of Anionic antimicrobial peptides
Structural study indicates that AAMPs often exhibit a net charge between -1 and -7 and vary in length from 5 to around 70 residues; for some of these peptides, post-translational changes are crucial for antibacterial efficacy. The contact with membranes is crucial to the antimicrobial efficacy of AAMPs, and to promote these interactions, these peptides typically assume amphiphilic conformations. These topologies range from the α-helical peptides seen in some amphibians to the cyclic cystine knot structures present in specific plant proteins. Certain AAMPs seem to utilize metal ions to establish cationic salt bridges with negatively charged elements of microbial membranes, thus promoting interaction with their target organisms. Like cationic antimicrobial peptides (CAMPs), AAMPs include both hydrophobic and hydrophilic domains, enabling interaction with lipid bilayers, but by distinct mechanisms with microbial cells.
The ovine pulmonary surfactant associated anion peptide (SAAP), the first identified anionic antimicrobial peptide containing 5-7 aspartate residues, exhibited antibacterial action against the ovine pathogen Mannheimia haemolytica in the presence of zinc ions. The addition of 0.14 mol/L NaCl and EDTA to the surfactant solution significantly decreased its bactericidal action, which was subsequently recovered upon the reintroduction of ZnCl2. The amidated C-terminal segment of the α-helical anionic antimicrobial peptide maximin H5 establishes an intra-peptide hydrogen bond with the N-terminal region, which is crucial for sustaining the tilted α-helix conformation.
Structure and characteristics of AAMPs. (Zhang Q Y., et al., 2021)
Mechanism of Anionic antimicrobial peptides
AAMPs function distinctively from CAMPs because of their negative charge. Rather than employing electrostatic interactions to directly attach to microbial cell membranes. AAMPs sometimes need metal ions (such as zinc, calcium or magnesium) to connect their negative charges with the negatively charged microbial membrane. The presence of these ions enables them to efficiently bind to and break microbial membranes or cell walls. Upon attachment to the microbial membrane, typically aided by metal ions, AAMPs can penetrate the membrane, resulting in physical disturbances. This compromises membrane integrity, resulting in the release of cellular contents and subsequent cell death. Certain AAMPs may disrupt intracellular targets, including vital enzymes or metabolic processes, upon traversing the microbial membrane. This mechanism of action diminishes the probability of resistance emergence. AAMPs frequently function synergistically with other antimicrobial agents or enzymes, enhancing their efficacy. This is particularly significant in settings where AAMPs exist at minimal amounts.
Anionic antimicrobial peptide interacts with Zinc(II). (Almarwani B., et al., 2020)
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Examples of Anionic antimicrobial peptides
AAMPs from ruminants
Recent years have seen the discovery of several bovine AAMPs encoded within the main protein structures of chromaffin cells, a type of neuroendocrine cell found in the adrenal gland's medulla and other sympathetic nervous system ganglia, and their secretory granules. It was discovered that a naturally cleaved segment prochromacin, which matched to residues 79-431 of Chromogranin A (CGA)—a prominent glycoprotein (Swiss-Prot accession code: P05059) present in these granules—was very active against both Gram-positive and Gram-negative bacteria. These bovine AAMPs, which belong to the peptide B / enkelytin family and have been extensively studied, were first found in the secretory granules of bovine chromaffin cells but have subsequently been found in their plasma and infectious fluids as well (Swiss-Prot entry code: P01211). Several investigations demonstrated that these pep tides had strong antibacterial properties, effectively inhibiting the growth of several Gram-positive bacteria, such as Micrococcus luteus, Staphylococcus aureus, and Bacillus megaterium, with minimum inhibitory concentrations (MICs) often below 3 μM.
One of the first AAMPs to be identified from bovine milk was kappacins, which have just lately been found in cheeses derived from this milk. These AAMPs are phosphorylated, non-glycosylated variants of caseinomacropeptide (CMP). They are made by enzymatically cleaving -casein, a common milk protein (Swiss-Prot accession code: P02668), to yield a fragment consisting of residues 106 to 169. Although other kappacins have been identified, the most well-studied are kappacins A and B. With MICs in the low μM range, both peptides have demonstrated efficacy against several Gram-positive and Gram-negative oral bacteria.
AAMPs from Ruminants. (Harris F., et al., 2009)
AAMPs from plants
AAMPs were identified from the processing of a 7S globulin protein in the kernels of Macadamia integrifolia, commonly known as the Macadamia nut. The initial AAMP, MiAMP2a (Swiss-Prot accession code: Q9SPL3/4/5), remains uncharacterized; nonetheless, it notably possesses a net charge of -6, making it one of the most negatively charged plant AAMPs documented. The second of these proposed AAMPs, MiAMP2b (Swiss-Prot accession code: Q9SPL3/4/5), has a net charge of -2 and was extracted from seed exudate. The peptide shown efficacy against fungal plant diseases, including Leptosphaeria maculans and Verticillium dahliae, at lethal concentrations in the low μM range. The antifungal mechanism of MiAMP2b remains unexamined; nevertheless, it has recently been suggested that the poisonous effect of vicilins derives from their capacity to attach to the cell wall and plasma membrane of fungi and yeasts. The aforementioned authors suggested an analogous mode of action for hevein and hevein-like peptides, which constitute another recognized category of plant antifungal peptides. Research on hevein (Swiss-Prot accession code: P02877), initially extracted from the rubber latex of Hevea brasiliensis, the para rubber tree, indicated that the peptide had a net charge of -2 and is post-translationally cleaved from its precursor protein, pro hevein. The investigation into the antibacterial properties of the peptide revealed its inhibition of chitin-containing fungi and a significant association with chitin, suggesting that hevein may contribute to the protection of plant wound areas against fungal invasion. Recently, WjAMP-1, a hevein-like peptide with a net charge of -1 (Swiss-Prot accession code: Q8H950), was isolated from the leaves of Wasabia japonica. The inoculation of W. japonica with fungal pathogens elicited the expression of WjAMP-1 across the plant's tissues. The peptide exhibited action against fungi and bacteria, leading to the suggestion that WjAMP-1 serves a defensive function in W. japonica.
AAMPs from Plants. (Harris F., et al., 2009)
AAMPs from insects
The recent application of this approach to analyze antimicrobial peptides within the haemolymph of immune-challenged G. mellonella larvae resulted in the identification of two potential AAMPs. The peptides were named Gm anionic peptide 1 (Swiss-Prot accession code: P85211) and Gm anionic peptide 2 (Swiss-Prot accession code: P85216). The AAMPs exhibited net charges of -4 and -3, respectively, and shown efficacy against Gram-positive bacteria, including M. luteus and L. monocytogenes, with MICs below 90 μM; however, they were ineffective against Gram-negative bacteria. Furthermore, each peptide exhibited a restricted range of antifungal action, with MICs consistently below 90 μM. Sequence research revealed that Gm anionic peptide 1 had considerable similarities with segments from precursors of lebocins, which are weak antibacterial chemicals derived from the silk moth, Bombyx mori; nonetheless, it remains uncertain if the AAMP is a G. mellonella lebocin-like peptide. Conversely, Gm anionic peptide 2 exhibited no similarity with any known proteins and has not been further described to date.
AAMPs from insects. (Harris F., et al., 2009)
Applications of anionic antimicrobial peptides
Antibacterial activity
AAMPs were initially extracted from epithelial cells and pulmonary secretions. These compounds have been shown to inhibit bacterial proliferation in the presence of divalent metal ions. Brogden's team has demonstrated that these tiny peptides (under 1 kD) impede the proliferation of both Gram-negative and Gram-positive bacteria in 0.14 M NaCl with 2.5 μM ZnCl2. The antimicrobial action is located in the central Asp hexapeptide homopolymeric region, and growth inhibition escalates with the quantity of Asp residues in the peptide. They need zinc for optimal action and establish a complex with it. Zinc is hypothesized to provide a cationic salt bridge, enabling the peptide to surmount the net negative charge on the microbial surface.
Implant coatings
AAMPs can be employed to coat medical equipment, including catheters, implants, and surgical instruments, to inhibit bacterial colonization and biofilm development, which are major contributors to infections in healthcare environments. Coating these devices with AAMPs inhibits bacterial colonization and biofilm formation, which exhibit significant resistance to antibiotics.
Agricultural and veterinary medicine
To improve livestock health and decrease spoilage and microbiological contamination, AAMPs can be utilized as natural preservatives in animal feed. In contrast to conventional antibiotics, which can amplify resistance, AAMPs provide a safer and more specific method. For agricultural purposes, AAMPs have shown useful in preventing plant illnesses caused by bacteria and fungi. To safeguard crops from microbes, increase output, and decrease reliance on chemical pesticides, plants can synthesize anionic peptides with inherent antifungal characteristics, such as peptides high in aspartic acid.
Reducing product spoilage
The presence of moisture makes many cosmetics more susceptible to contamination by microbes. By inhibiting the growth of bacteria and fungus, AAMPs can be added to personal care products, increasing their shelf life and preserving their integrity. The skin is often less affected by AAMPs than by other synthetic preservatives. Skincare formulations targeted at sensitive skin or products used regularly might benefit from their moderate action and low toxicity.
References
- Zhang Q Y., et al., Antimicrobial peptides: mechanism of action, activity and clinical potential, Military Medical Research, 2021, 8: 1-25.
- Dennison S R., et al., An atlas of anionic antimicrobial peptides from amphibians, Current Protein and Peptide Science, 2018, 19(8): 823-838.
- Harris F., et al., Anionic antimicrobial peptides from eukaryotic organisms, Current Protein and Peptide Science, 2009, 10(6): 585-606.
- Almarwani B., et al., Interactions of an anionic antimicrobial peptide with Zinc (II): application to bacterial mimetic membranes, Langmuir, 2020, 36(48): 14554-14562.
