Rapid systemic and local treatments with the antibacterial peptide dimer A3-APO and its monomeric metabolite eliminate bacteria and reduce inflammation in intradermal lesions infected with Propionibacterium acnes and meticillin-resistant Staphylococcus aureus
Introduction
In the past few years, classical antimicrobial peptides (AMPs) have received much attention due to their efficacy in mouse models of systemic infections [1]. However, their path to the clinic remains tortuous due to the generally low therapeutic indices that originate from the relatively large amounts of peptides needed to fully kill bacteria in vitro and in vivo and the inherent bleeding of animals at not much higher doses [2]. Toxicity concerns are not unexpected as the major mode of action of AMPs is the disintegration or weakening of bacterial membranes and at elevated doses selectivity to prokaryotic membranes is seldom achievable [3]. The outlook is more optimistic for proline–arginine-rich peptides that exhibit improved therapeutic indices owing to their multimodal action involving deactivation of internal target biopolymers [4].
In addition to intracellular targets, many AMPs modulate the immune responses of the host [5]. Improved protection in infection models can be achieved through either activation of macrophage performance [6] or upregulation of anti-inflammatory cytokine production [7]. The immunostimulatory and immunoprotective effects may explain the remarkable efficacies of the proline–arginine-rich antibacterial peptide dimer A3-APO and its monomeric metabolite in mouse models of systemic and local infections. A3-APO and the Chex1-Arg20 monomer moderately destroy bacterial membranes and inhibit chaperone-assisted bacterial protein folding [8], but these activities cannot explain the in vivo efficacy against pathogens to which the minimum inhibitory concentration (MIC) is >32 mg/L and that do not express proteinaceous products needed for survival or replication. Despite the modest in vitro MICs, A3-APO and the single-chain monomer exhibit superior efficacy in mouse models of Escherichia coli and Acinetobacter baumannii systemic infections [9], [10]. In a burn infection model, A3-APO administered three times intramuscularly at 5 mg/kg sterilises mouse wounds following multidrug-resistant A. baumannii inoculation and is more efficacious than any current therapy regimen [11]. In parallel, we documented that A3-APO heals uninfected wounds. Perhaps more surprisingly, A3-APO cures mice with meticillin-resistant Staphylococcus aureus (MRSA) burn infections with a single 5 mg/kg intramuscular (i.m.) dose when it cannot kill the bacterium at all in vitro [12]. One of the likely reasons for this activity is anti-inflammatory cytokine production [12].
In the current report, we intended to harness the immune stimulatory and anti-inflammatory properties of A3-APO and its Chex1-Arg20 metabolite in intradermal infections and using administration modes with the highest need for new antimicrobials and patient compliance. The major pathogens in skin and soft-tissue infections are Gram-positive bacteria, predominantly S. aureus and Propionibacterium acnes, which also develop resistance to current antibiotics at an alarming rate [13], [14]. Ideally, these infections are treated topically to reduce further mutations in bacterial genes as well as to reduce systemic side effects [15]. Here we report that peptide dimer A3-APO and the monomer, administered intramuscularly or as topical cream/spray, can almost completely sterilise S. aureus-infected wounds and that A3-APO fights P. acnes-induced inflammation in intradermal ear infections.
Section snippets
Antibiotics and minimum inhibitory concentration determination
Peptide A3-APO and the Chex1-Arg20 metabolite were synthesised on solid-phase and were purified by reversed-phase high-performance liquid chromatography as previously described [16]. The sequences of the peptides are as follows: A3-APO, (H-Chex-Arg-Pro-Asp-Lys-Pro-Arg-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-Arg-Pro-Val-Arg)2-Dab; and Chex1-Arg20 monomer, H-Chex-Arg-Pro-Asp-Lys-Pro-Arg-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-Arg-Pro-Val-Arg-NH2, where non-natural amino acid residues are Chex
In vitro activity against a panel of resistant strains
First, the in vitro activities of the A3-APO dimer and its Chex1-Arg20 monomeric metabolite were compared against a panel of eight resistant reference strains (Table 1). Overall, the two peptides exhibited very similar MICs, with no more than a two-fold difference against any of the isolates studied. Both peptides killed extended-spectrum β-lactamase-producing E. coli and Klebsiella pneumoniae strains as well as the K. pneumoniae carbapenemase (KPC)-expressing K. pneumoniae isolate at 16–32
Discussion
Many AMPs exhibit better in vivo efficacies than their MICs against the pathogens would indicate [23]. For example, peptide LL-37 with no antimicrobial activity protects experimental animals from S. aureus and Salmonella infection. Antibacterial peptides are especially useful in wound repair [24], suggesting that they genuinely exert anti-inflammatory properties [25]. Elimination of bacteria from wounds, however, also requires upregulation of macrophage (or other monocyte) production and
Acknowledgment
The authors thank Feng Lin (Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Australia) for peptide synthesis.
Funding: Part of this work was supported by Australian Research Council Discovery grant DP120101963 to JDW, who is also a National Health and Medical Research Council (Australia) Principal Research Fellow. Research at the Florey Institute of Neuroscience and Mental Health (University of Melbourne, Melbourne, Australia) is supported by the Victorian
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