Antimicrobial peptides are degraded by the cytosolic proteases of human erythrocytes

Abstract

Well-studied and promising antimicrobial peptides (AMPs), with potent bactericidal activity, in vitro, have yet to have a significant impact in human medicine beyond topical applications. We previously showed that interactions of AMPs with concentrated human erythrocytes inhibit many of them, and suggested that screens and assays should be done in their presence to mimic host cell inhibition. Here, we use AMPs to characterize the activity of proteases that are associated with human erythrocytes. The representative AMPs, ARVA and indolicidin, are degraded significantly during incubation with dilute, washed erythrocytes and yield a variety of degradation products, suggesting significant exopeptidase activity. Comparison of these fragments with those obtained from incubation with serum shows that the proteolytic activity associated with cells yields unique products that are not explained by residual serum proteases. By separately testing the membrane and cytosolic fractions, we show that erythrocyte proteolytic activity is found only in the cytosol. Finally, we incubated a diverse cross-section of natural and synthetic linear AMPs with human erythrocyte cytosolic extracts and observed degradation of all of them. These results show that, in addition to cell binding, proteolysis can also contribute significantly to host cell inhibition of AMPs in vitro and possibly also in vivo.

Synthetic molecular evolution of host cell-compatible, antimicrobial peptides effective against drug-resistant, biofilm-forming bacteria

“…These two sequences, which belong to distinct families (35), both have potent, sterilizing antimicrobial activity in standard laboratory assays (38) against a range of gram-negative and gram-positive bacteria, including drug-resistant strains (35). However, like many known AMPs, ARVA and NATT are strongly inhibited by host cells (23), are susceptible to proteolysis (22), have poor solubility, and are somewhat cytotoxic (35).…”

“…With a few exceptions, discovery of new AMPs has been achieved by the trial-and-error modification of known sequences. Even then, the major impediments to therapeutic applications discussed here, one or more of which likely apply to most known AMPs (22,23), are not usually assessed until after the peptide design/discovery phase. This approach reduces opportunities for feedback and down-selection of AMPs that have impediments, ultimately limiting the flow of promising new AMP candidates into the drug development pipeline.…”

“…Despite their promise, most known AMPs have impediments to therapeutic utility. For example, AMPs can have limited solubility, residual host cell toxicity, susceptibility to proteolytic degradation, and binding to host cells, tissue, and serum proteins, all of which are expected to reduce or eliminate the activity of an AMP in vivo (22,23).…”

Sensitivity of Antibacterial Activity to Backbone Sequence in Constitutionally Isomeric OligoTEAs

“…Selectivity for bacteria over mammalian cells is attributed to the reduced anionic character of the mammalian cell membrane and its rigidity due to the presence of cholesterol . While some AMPs display potent cell lysis, they generally suffer from reduced activity in serum containing physiological fluids due to protease degradation and protein binding . To combat these drawbacks, recent efforts have been made toward synthesizing AMP mimics comprised of proteolytic‐resistant backbones.…”

Investigating Antibacterial Effects of Latrodectus Dahli Crude Venom on Escherichia coli, Staphylococcus aureus and Bacillus subtilis

“…AMPs have amphipathic features that mirror phospholipids, thus allowing them to interact with and exploit vulnerabilities inherent in essential microbial structures such as cell membranes (3). Until now, the antibacterial activity of more than 1000 peptides from different eukaryotic and prokaryotic sources have been investigated to find a suitable antimicrobial alternative for antibiotics (4,5). It has been demonstrated that venom of snakes, scorpions and spiders has strong antibacterial effects (6).…”