Mechanisms to Combat Drug Resistance: Overview of Antimicrobial Peptides

What are Antimicrobial Peptides?

Antimicrobial peptides (AMPs) are naturally occurring peptide molecules found in various organisms, including humans, animals, plants, and microorganisms. These peptides play a crucial role in the innate immune system's defense against microbial infections, exhibiting antimicrobial properties that can inhibit the growth or kill bacteria, fungi, viruses, and even some parasites.

AMPs possess broad-spectrum activity, targeting various pathogens. AMPs typically rapidly disrupt the integrity of microbial cell membranes, leading to cell lysis and death. This rapid mode of action contrasts with many traditional antibiotics that target specific cellular processes. In addition to their direct antimicrobial effects, some AMPs can modulate immune responses, enhancing phagocytosis and even exhibiting anti-inflammatory properties for localized treatment of skin infections, respiratory infections, and systemic infections. Due to their targeting of the physical structure of microbial cell membranes rather than specific cellular processes, AMPs have a lower likelihood of inducing microbial resistance, making them potential candidates against drug-resistant strains. Researchers are actively studying AMPs to better understand their mechanisms of action, optimize their use in clinical settings, and develop new therapeutic strategies to combat infections and antibiotic-resistant pathogens.

The First Medicinal Antimicrobial Peptides

In 1947, the first antimicrobial peptide, polymyxins, was discovered. This family was produced by Paenibacillus polymyxa and includes members such as polymyxin B1, B2, and E (colistin).

The first clinical application of polymyxins was in 1959 with colistin (also known as polymyxin E). Colistin is an antibiotic used to treat multidrug-resistant (MDR) Gram-negative bacterial infections, including pneumonia. Isolated in 1949 in Japan from a fermented culture of Paenibacillus polymyxa, colistin was first used clinically in 1959 under the trade name Cortisporin®.

Colistin sodium, a less toxic prodrug, became available for injection in 1959. In the 1980s, due to renal and neurotoxicity concerns, the use of polymyxins was widely discontinued. However, with the increasing prevalence of multidrug-resistant bacteria in the 1990s, colistin was reconsidered as an emergency solution, despite its toxicity.

Colistin is a surfactant that penetrates and disrupts bacterial cell membranes. It is a cationic peptide with multiple 2,4-diaminobutyric acid (Dab) residues, possessing both hydrophobic and lipophilic portions. It interacts with bacterial cytoplasmic membranes, altering their permeability.

Antimicrobial Peptides (AMPs)

Common antibiotics, based on pharmacology and chemical structure, can be broadly categorized into penicillins, cephalosporins, tetracyclines, aminoglycosides, macrolides, clindamycin, sulfonamides, trimethoprim, metronidazole, tinidazole, quinolones, nitrofurantoin, and peptides.

AMPs include actinomycin D, bacitracin, colistin, and polymyxin B. Actinomycin D has been used in cancer chemotherapy. Most other AMPs are too toxic for systemic administration but can be safely used topically on the skin for superficial cuts and abrasions.

Mechanisms of Action of Antimicrobial Peptides

The mechanisms of antibiotic action can be broadly classified into three categories: inhibition of cell wall synthesis, increased cell membrane permeability, and interference with protein synthesis, nucleic acid metabolism, and other metabolic processes (such as folate synthesis).

AMPs primarily exert their antimicrobial effects through two different mechanisms: 1) membrane-targeting AMPs disrupt the integrity of bacterial cell membrane structures, and 2) non-membrane-targeting AMPs mainly inhibit the synthesis of nucleic acids, enzymes, and other functional proteins. In both mechanisms, the former destabilizes bacterial membranes, while the latter can translocate across membranes without disrupting cell membranes but may impair normal cell functions.

Membrane Disruption Mechanism

Membrane-active AMPs can interact with the surface of microbial cells through receptor-mediated or non-receptor-mediated interactions. Some highly efficient AMPs can even initiate interactions with general targets on cell surfaces without the need for specific receptors. The physicochemical properties of AMPs, such as net charge, hydrophobicity, amphipathicity, membrane curvature, and self-aggregation tendency, play a crucial role in governing the peptide-membrane interactions that lead to membrane integrity disruption. This inhibition results in increased permeability of the cell envelope, leakage of cellular contents, and ultimately cell death.

The mechanism of action of membrane-active AMPs primarily relies on cationic and hydrophobic interactions. Particularly, electrostatic interactions between positively charged residues (such as arginine and lysine) of AMPs and the negatively charged surface of bacterial cell membranes provide a significant driving force for AMP-membrane binding. Bacterial cell membranes are characterized by abundant negative charges, such as high anionic lipid content, including phosphatidylglycerol (PG), cardiolipin, and phosphatidylserine. These negative charges interact strongly with the positive charges (mainly ammonium ions) on AMPs, creating powerful electrostatic attractions, while animal cell membranes have zwitterionic phospholipids, such as phosphatidylcholine (PC) and sphingomyelin.

Additionally, components like lipoteichoic acid, lipophosphoglycan, and lipopolysaccharides (LPS) with negative charges on the surface of bacterial cells are considered potential targets for AMPs. Therefore, compared to interactions between AMPs and bacterial membranes, the electrostatic interactions between AMPs and mammalian cell membranes are relatively weak. Furthermore, mammalian cell membranes contain cholesterol, enhancing membrane stability and preventing the insertion of AMPs, providing safety for the use of AMPs.

Besides abundant negative charges, hydrophobicity is a primary characteristic of peptides (both in the main chain and hydrophobic side chains), controlling the interaction of hydrophobic residues with the acyl chains of membrane lipids. This control influences the insertion of peptide segments across the membrane and distribution into the hydrophobic core of the bilayer, constituting an essential mechanism for AMPs to interfere and disrupt bacterial cell membranes. Generally, peptides with moderate hydrophobicity exhibit optimal activity, while highly hydrophobic peptides show strong hemolytic activity and reduced antimicrobial activity. Additionally, the amphipathicity of AMPs contributes to the affinity of their α-helical secondary structure with the membrane, where hydrophobic residues interact with the lipid bilayer, and hydrophilic residues interact with phospholipid groups. As the concentration of membrane-binding AMPs increases, peptide-peptide or lipid-peptide complexes are formed. When the accumulation of AMPs in the membrane reaches a critical aggregation concentration, AMPs penetrate the hydrophobic core of the bilayer and form transmembrane pores in the cytoplasmic membrane.

Non-membrane-active Mechanisms

Although the initial antimicrobial action of AMPs was predominantly membrane-active, subsequent studies revealed that many AMPs target essential cellular components and functions, leading to bacterial death. These AMPs initially translocate into the cytoplasm without disrupting the cell membrane and then hinder critical cellular processes by interacting with intracellular targets, including inhibiting protein and nucleic acid synthesis, as well as the degradation of enzymes and proteins. Specifically, AMPs can prevent the transport of peptidoglycan precursors synthesized in the cytoplasm to the bacterial cell wall without interfering with the cell membrane, a crucial factor in bacterial cell wall formation.

Multidrug-Resistant of Antimicrobial Peptides

Many infectious pathogens, especially Gram-negative bacteria, have developed resistance to conventional antibiotics. This resistance phenomenon is not limited to bacteria but extends to pathogenic fungi, viruses, and parasites. AMPs face the challenge of resistance. Resistance is the result of bacteria mutating in response to the use of drugs, such as developing resistance by blocking binding sites. This method of resistance may explain why AMPs cannot act on Gram-negative bacteria, which have a thin peptidoglycan layer that changes due to variations in growth media, rendering AMPs ineffective.

Relatively speaking, resistance is less common in peptide antibiotics, such as bacitracin. In most cases, the ability of peptides to overcome resistance stems from their mechanism of inhibiting cell wall synthesis, thereby preventing bacterial cell proliferation before resistance can form. These novel antibiotics can serve as alternatives to traditional antibiotics. AMPs isolated from Bacillus species, in particular, have the most promising potential to overcome current antibiotic drawbacks. Bacillus species produce a variety of peptides with different basic chemical structures, including bacteriocins, glycopeptides, lipopeptides, and cyclic peptides.

Listed Antimicrobial Peptides

Currently, AMPs such as nisin, gramicidin, polymyxins, daptomycin, and melittin are used clinically for their antimicrobial efficacy.

Nisin

Nisin is an FDA-approved Generally Regarded as Safe (GRAS) peptide with recognized clinical potential. In recent decades, the application of nisin has expanded to the biomedical field. Studies report that nisin can inhibit the growth of resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, Enterococcus faecalis, and Clostridium difficile. Nisin has demonstrated antibacterial activity against Gram-positive and Gram-negative pathogenic organisms. Additionally, similar to host defense peptides, nisin can activate adaptive immune responses and has immunomodulatory effects. Increasing evidence suggests that nisin can influence tumor growth and exhibit selective cytotoxicity against cancer cells.

Gramicidin D

Gramicidin D is used to treat skin diseases and ophthalmic infections. It is a heterogeneous mixture of three antibiotics—Gramicidin A, B, and C—derived from the soil bacterium Bacillus brevis, collectively known as Gramicidin D. Gramicidin is a 15-residue peptide with alternating D and L amino acids, assembling into β-helices within the hydrophobic interior of the cell membrane lipid bilayer. Gramicidin D is active against most Gram-positive bacteria and some Gram-negative organisms, mainly used as a topical antibiotic due to its high hemolytic activity, making it unsuitable for oral administration. It is commonly used in eye drops in combination with two other antibiotics, neomycin and polymyxin B.

Gramicidin D binds to and inserts into the bacterial membrane, leading to membrane rupture and permeabilization. This results in (i) the loss of intracellular solutes such as K+ and amino acids; (ii) dissipation of transmembrane potential; (iii) respiratory inhibition; (iv) reduction of ATP pools; (v) inhibition of DNA, RNA, and protein synthesis, ultimately causing cell death.

Daptomycin

Daptomycin is a cyclic lipopeptide antibiotic used to treat complex skin and skin structure infections caused by susceptible Gram-positive bacteria, as well as bacteremia caused by Staphylococcus aureus.

Vancomycin

Vancomycin is a glycopeptide antibiotic used to treat severe and susceptible bacterial infections, such as infections caused by MRSA. The antimicrobial action of vancomycin primarily results from inhibiting cell wall biosynthesis. Vancomycin prevents the incorporation of N-acetyl-muramic acid (NAM) and N-acetyl-glucosamine (NAG) peptide subunits into the peptidoglycan matrix, which is a major structural component of the cell wall in Gram-positive bacteria. Vancomycin forms hydrogen bonds with the D-Ala-D-Ala portion at the end of NAM/NAG peptides. Additionally, vancomycin alters the permeability of bacterial cell membranes and inhibits RNA synthesis. There is no cross-resistance observed between vancomycin and other antibiotics.

Oritavancin

Oritavancin is used to treat acute bacterial skin and skin structure infections (ABSSSI) caused by susceptible Gram-positive bacteria. Oritavancin combats susceptible Gram-positive organisms through three distinct mechanisms. Firstly, it inhibits the transglycosylase activity by binding to peptidoglycan precursors, a process that typically occurs during bacterial cell wall synthesis. Secondly, oritavancin inhibits cross-linking in the bacterial cell wall biosynthesis process by binding to the peptide bridge segment of the cell wall pentaglycan. Lastly, the drug exerts its action by disrupting the integrity of bacterial cell membranes.

Dalbavancin

Dalbavancin is used to treat ABSSSI caused by susceptible Gram-positive strains. It shares a similar spectrum of activity and mechanism of action with vancomycin. Dalbavancin inhibits cell wall biosynthesis and alters the permeability of bacterial cell membranes and RNA synthesis.

Telavancin

Telavancin is used to treat complex skin and skin structure infections, as well as various types of acquired bacterial pneumonia. Telavancin is a lipoglycopeptide with activity against various Gram-positive bacteria. Telavancin prevents the aggregation of NAM and NAG by binding to D-Ala-D-Ala, and it inhibits the cross-linking of peptidoglycans. The synthesis of bacterial cell walls is consequently inhibited.

References:

1. Vaara, M., Polymyxin Derivatives that Sensitize Gram-Negative Bacteria to Other Antibiotics, Molecules, 2019, 24(2), 249.
2. Bahar, A. A., and Ren, D., Antimicrobial peptides. Pharmaceuticals, 2013, 6, 1543-1575.