Two distinct amphipathic peptide antibiotics with systemic efficacy.

Antimicrobial peptides are important candidates for developing new classes of antibiotics because of their potency against antibiotic-resistant pathogens. Current research focuses on topical applications and it is unclear how to design peptides with systemic efficacy. To address this problem, we designed two potent peptides by combining database-guided discovery with structure-based design. When bound to membranes, these two short peptides with an identical amino acid composition can adopt two distinct amphipathic structures: A classic horizontal helix (horine) and a novel vertical spiral structure (verine). Their horizontal and vertical orientations on membranes were determined by solid-state 15N NMR data. While horine was potent primarily against gram-positive pathogens, verine showed broad-spectrum antimicrobial activity. Both peptides protected greater than 80% mice from infection-caused deaths. Moreover, horine and verine also displayed significant systemic efficacy in different murine models comparable to conventional antibiotics. In addition, they could eliminate resistant pathogens and preformed biofilms. Significantly, the peptides showed no nephrotoxicity to mice after intraperitoneal or intravenous administration for 1 wk. Our study underscores the significance of horine and verine in fighting drug-resistant pathogens.

Low cationicity is important for systemic in vivo efficacy of database-derived peptides against drug-resistant Gram-positive pathogens.

As bacterial resistance to traditional antibiotics continues to emerge, new alternatives are urgently needed. Antimicrobial peptides (AMPs) are important candidates. However, how AMPs are designed with in vivo efficacy is poorly understood. Our study was designed to understand structural moieties of cationic peptides that would lead to their successful use as antibacterial agents. In contrast to the common perception, serum binding and peptide stability were not the major reasons for in vivo failure in our studies. Rather, our systematic study of a series of peptides with varying lysines revealed the significance of low cationicity for systemic in vivo efficacy against Gram-positive pathogens. We propose that peptides with biased amino acid compositions are not favored to associate with multiple host factors and are more likely to show in vivo efficacy. Thus, our results uncover a useful design strategy for developing potent peptides against multidrug-resistant pathogens.

Nanofiber Dressings Topically Delivering Molecularly Engineered Human Cathelicidin Peptides for the Treatment of Biofilms in Chronic Wounds.

Biofilms of multidrug-resistant bacteria in chronic wounds pose a great challenge in wound care. Herein, we report the topical delivery of molecularly engineered antimicrobial peptides using electrospun nanofiber dressings as a carrier for the treatment of biofilms of multidrug-resistant bacteria in diabetic wounds. Molecularly engineered human cathelicidin peptide 17BIPHE2 was successfully encapsulated in the core of pluronic F127/17BIPHE2-PCL core-shell nanofibers. The in vitro release profiles of 17BIPHE2 showed an in initial burst followed by a sustained release over 4 weeks. The peptide nanofiber formulations effectively killed methicillin-resistant Staphylococcus aureus (MRSA) USA300. Similarly, the 17BIPHE2 peptide containing nanofibers could also effectively kill other bacteria including Klebsiella pneumoniae (104 to 106 CFU) and Acinetobacter baumannii (104 to 107 CFU) clinical strains in vitro without showing evident cytotoxicity to skin cells and monocytes. Importantly, 17BIPHE2-containing nanofiber dressings without debridement caused five-magnitude decreases of the MRSA USA300 CFU in a biofilm-containing chronic wound model based on type II diabetic mice. In combination with debridement, 17BIPHE2-containing nanofiber dressings could completely eliminate the biofilms, providing one possible solution to chronic wound treatment. Taken together, the biodegradable nanofiber-based wound dressings developed in this study can be utilized to effectively deliver molecularly engineered peptides to treat biofilm-containing chronic wounds.

Structure and Activity of a Selective Antibiofilm Peptide SK-24 Derived from the NMR Structure of Human Cathelicidin LL-37.

The deployment of the innate immune system in humans is essential to protect us from infection. Human cathelicidin LL-37 is a linear host defense peptide with both antimicrobial and immune modulatory properties. Despite years of studies of numerous peptides, SK-24, corresponding to the long hydrophobic domain (residues 9-32) in the anionic lipid-bound NMR structure of LL-37, has not been investigated. This study reports the structure and activity of SK-24. Interestingly, SK-24 is entirely helical (~100%) in phosphate buffer (PBS), more than LL-37 (84%), GI-20 (75%), and GF-17 (33%), while RI-10 and 17BIPHE2 are essentially randomly coiled (helix%: 7-10%). These results imply an important role for the additional N-terminal amino acids (likely E16) of SK-24 in stabilizing the helical conformation in PBS. It is proposed herein that SK-24 contains the minimal sequence for effective oligomerization of LL-37. Superior to LL-37 and RI-10, SK-24 shows an antimicrobial activity spectrum comparable to the major antimicrobial peptides GF-17 and GI-20 by targeting bacterial membranes and forming a helical conformation. Like the engineered peptide 17BIPHE2, SK-24 has a stronger antibiofilm activity than LL-37, GI-20, and GF-17. Nevertheless, SK-24 is least hemolytic at 200 µM compared with LL-37 and its other peptides investigated herein. Combined, these results enabled us to appreciate the elegance of the long amphipathic helix SK-24 nature deploys within LL-37 for human antimicrobial defense. SK-24 may be a useful template of therapeutic potential.

Short and Robust Anti-Infective Lipopeptides Engineered Based on the Minimal Antimicrobial Peptide KR12 of Human LL-37.

This study aims to push the frontier of the engineering of human cathelicidin LL-37, a critical antimicrobial innate immune peptide that wards off invading pathogens. By sequential truncation of the smallest antibacterial peptide (KR12) of LL-37 and conjugation with fatty acids, with varying chain lengths, a library of lipopeptides is generated. These peptides are subjected to antibacterial activity and hemolytic assays. Candidates (including both forms made of l- and d-amino acids) with the optimal cell selectivity are subsequently fed to the second layer of in vitro filters, including salts, pH, serum, and media. These practices lead to the identification of a miniature LL-37 like peptide (d-form) with selectivity, stability, and robust antimicrobial activity in vitro against both Gram-positive and negative bacteria. Proteomic studies reveal far fewer serum proteins that bind to the d-form than the l-form peptide. C10-KR8d targets bacterial membranes to become helical, making it difficult for bacteria to develop resistance in a multiple passage experiment. In vivo, C10-KR8d is able to reduce bacterial burden of methicillin-resistant Staphylococcus aureus (MRSA) USA300 LAC in neutropenic mice. In addition, this designer peptide prevents bacterial biofilm formation in a catheter-associated mouse model. Meanwhile, C10-KR8d also recruits cytokines to the vicinity of catheters to clear infection. Thus, based on the antimicrobial region of LL-37, this study succeeds in identifying the smallest anti-infective peptide C10-KR8d with both robust antimicrobial, antibiofilm, and immune modulation activities.

Sequence Permutation Generates Peptides with Different Antimicrobial and Antibiofilm Activities.

Antibiotic resistance poses a threat to our society, and 10 million people could die by 2050. To design potent antimicrobials, we made use of the antimicrobial peptide database (APD). Using the database filtering technology, we identified a useful template and converted it into an effective peptide WW291 against methicillin-resistant Staphylococcus aureus (MRSA). Here, we compared the antibacterial activity and cytotoxicity of a family of peptides obtained from sequence permutation of WW291. The resulting eight WW peptides (WW291-WW298) gained different activities against a panel of bacteria. While WW295 inhibited the growth of Escherichia coli, WW298 was highly active against S. aureus USA300 LAC. Consistently with this, WW298 was more effective in permeating or depolarizing the S. aureus membranes, whereas WW295 potently permeated the E. coli membranes. In addition, WW298, but not WW295, inhibited the MRSA attachment and could disrupt its preformed biofilms more effectively than daptomycin. WW298 also protected wax moths Galleria mellonella from MRSA infection causing death. Thus, sequence permutation provides one useful avenue to generating antimicrobial peptides with varying activity spectra. Taken together with amino acid composition modulation, these methods may lead to narrow-spectrum peptides that are more promising to selectively eliminate invading pathogens without damaging commensal microbiota.

Resistome of Staphylococcus aureus in Response to Human Cathelicidin LL-37 and Its Engineered Antimicrobial Peptides.

Staphylococcus aureus is notoriously known for its rapid development of resistance to conventional antibiotics. S. aureus can alter its membrane composition to reduce the killing effect of antibiotics and antimicrobial peptides (AMPs). To obtain a more complete picture, this study identified the resistance genes of S. aureus in response to human cathelicidin LL-37 peptides by screening the Nebraska Transposon Mutant Library. In total, 24 resistant genes were identified. Among them, six mutants, including the one with the known membrane-modifying gene (mprF) disabled, became more membrane permeable to the LL-37 engineered peptide 17BIPHE2 than the wild type. Mass spectrometry analysis detected minimal lysyl-phosphatidylglycerol (lysylPG) from the mprF mutant of S. aureus JE2, confirming loss-of-function of this gene. Moreover, multiple mutants showed reduced surface adhesion and biofilm formation. In addition, four S. aureus mutants were unable to infect wax moth Galleria mellonella. There appears to be a connection between the ability of bacterial attachment/biofilm formation and infection. These results underscore the multiple functional roles of the identified peptide-response genes in bacterial growth, infection, and biofilm formation. Therefore, S. aureus utilizes a set of resistant genes to weave a complex molecular network to handle the danger posed by cationic LL-37. It appears that different genes are involved depending on the nature of antimicrobials. These resistant genes may offer a novel avenue to designing more potent antibiotics that target the Achilles heels of S. aureus USA300, a community-associated pathogen of great threat.

Antimicrobial peptides: Possible anti-infective agents.

Multidrug-resistant bacterial, fungal, viral, and parasitic infections are major health threats. The Infectious Diseases Society of America has expressed concern on the decrease of pharmaceutical companies working on antibiotic research and development. However, small companies, along with academic research institutes, are stepping forward to develop novel therapeutic methods to overcome the present healthcare situation. Among the leading alternatives to current drugs are antimicrobial peptides (AMPs), which are abundantly distributed in nature. AMPs exhibit broad-spectrum activity against a wide variety of bacteria, fungi, viruses, and parasites, and even cancerous cells. They also show potential immunomodulatory properties, and are highly responsive to infectious agents and innate immuno-stimulatory molecules. In recent years, many AMPs have undergone or are undergoing clinical development, and a few are commercially available for topical and other applications. In this review, we outline selected anion and cationic AMPs which are at various stages of development, from preliminary analysis to clinical drug development. Moreover, we also consider current production methods and delivery tools for AMPs, which must be improved for the effective use of these agents.