The treatment of infectious diseases poses major challenges for healthcare systems worldwide due to rapid increase in bacterial strains resistant to conventional antibiotics. AMPs have emerged as novel promising agents for both topical and systemic treatment of infections. The AMPs are considered less prone to resistance development than conventional antibiotics due to the rapid microbicidal effect combined with broad mode of action (Fjell et al. 2012).
Drug development based on AMPs is complicated by the fact that the antimicrobial activities of numerous naturally occurring AMPs are antagonised by physiological salt concentrations, which might reflect the increasing ionic strength under high salt conditions weakening the electrostatic charge interactions of AMPs with bacterial membranes (Tam et al. 2002). Thus, attempts are directed towards developing therapeutic AMPs where salt-resistant properties are achieved through chemical modifications of the peptide structure (Fjell et al. 2012; Park et al. 2004; Tam et al. 2002). Peptides isolated from marine organisms, that are adapted to salt-rich environments, are likely to be less sensitive to ionic strength, and are therefore highly interesting from a drug development perspective.
In this study, the peptide previously isolated from coelomocyte (blood cell) extracts of the green sea urchin, S. droebachiensis, centrocin 1 heavy chain (CEN1 HC-Br) (Li et al. 2010) and the variants thereof, were evaluated for the potential as novel anti-infectious agents by measuring their antibacterial and anti-inflammatory effect in vitro. CEN1 HC-Br contains a brominated tryptophan in position two in the amino acid sequence (Li et al. 2010) with unknown function. AMPs containing brominated tryptophan have previously been characterized from several marine organisms such as hagfish intestinal antimicrobial peptides (HFIAPs), isolated from Atlantic hagfish or styelin D, isolated from tunicate Styela clava (Taylor et al. 2000; Uzzell et al. 2003). It has been speculated that the unusual amino acid bromotryptophan makes these AMPs less susceptible to proteolytic degradation and may increase the biological activity of the peptide (Li et al. 2010). To elucidate the functional significance of the bromination of the tryptophan residue, the debrominated variant of the centrocin 1 heavy chain, i.e. CEN1 HC, was included in the assessments. To map the peptide region necessary of the biological activity, the C-terminal truncated variants of both peptides, CEN1 HC-Br (1–20) and CEN1 HC (1–20), were also tested. A free cysteine residue in the peptide sequence may potentially complicate the product development process due to the possible heterogeneity of the product caused by formation of disulphide bonds between molecules. Therefore, we investigated if the cysteine residue in CEN1 HC (position 25) could be replaced by a similar amino acid. The most commonly used replacement for cysteine is serine, which in terms of geometry and volume occupancy is a highly isosteric analogue of cysteine. A cysteine to serine modified variant of CEN1 HC, CEN1 HC (Ser), was compared for microbicidal and anti-inflammatory properties to its non-substituted equivalent.
When the CEN1 HC-Br derived peptides were evaluated for anti-bacterial activity against S. aureus and P. aeruginosa, all peptides showed pronounced microbicidal effect in assay medium containing low salt concentrations and no serum (0.037% BHI). The peptides CEN1 HC-Br and CEN1 HC were generally salt and wound fluid tolerant and significant microbicidal activity was observed in the presence of 150 mM NaCl and in 50% h.i. SWF. Notably, physiological sodium chloride concentrations as well as h.i SWF severely decreased or fully eliminated the bactericidal effect of CEN1 HC-Br (1–20), CEN1 HC (1–20) and CEN1 HC (Ser). The microbicidal effect of the peptides and their sensitivity to physiological-like conditions varied dependently on the bacterial strain tested, possibly due to differences between the cell wall/membrane properties of the Gram-positive (S. aureus) and Gram-negative (P. aeruginosa) bacteria. Interestingly, debromination of tryptophan in CEN1 HC-Br did not impair the bactericidal effect in neither condition of low salt nor under physiological-like conditions.
The anti-inflammatory properties of CEN1 HC-Br, CEN1 HC, CEN1 HC (1–20) and CEN1 HC (Ser) were evaluated by measuring secretion of the most commonly used inflammation markers TNF-α and IL-6 in human monocyte derived macrophages stimulated with LPS. In this assay, CEN1 HC-Br, CEN1 HC and the Ser-substituted peptide CEN1 HC (Ser) exerted a more potent anti-inflammatory effect compared to the C-terminal truncated variant CEN1 HC (1–20). No obvious difference in efficacy was seen comparing CEN1 HC-Br and CEN1 HC, while the first peptide had higher cytotoxicity compared to the debrominated equivalent.
Based on these experiments, we conclude that debromination of the tryptophan residue of CEN1 HC-Br did not result in any reduced effect under the experimental conditions tested. Importantly, CEN1 HC had a more favourable safety profile compared to the original peptide, with no lytic activity observed against mammalian cells at the concentrations tested, thus showing a clear dissociation of antibacterial and anti-eukaryotic cell activities. C-terminal truncation of the full-length peptide variants resulted in reduction in salt-tolerability of the microbicidal effect as well as in reduced anti-inflammatory properties. Also, the peptide with the free cysteine residue, CEN1 HC had improved salt and serum tolerability profile in the MMC99 assay compared to the serine-modified equivalent. Based on these observations, it was concluded that from the panel of peptide variants tested, CEN1 HC showed the best efficacy and safety profile. Thus, CEN1 HC was selected to be further evaluated for its in vitro and in vivo antimicrobial properties.
CEN1 HC was shown to have broad microbicidal effect against microorganisms appearing in topical and parenteral infections including Gram-positive bacteria (S. aureus, S. pyogenes, S. epidermidis, and P. acnes), Gram-negative bacteria (E. coli, P. aeruginosa, K. pneumonia, and A. baumannii) and the yeast C. albicans. CEN1 HC also showed pronounced effect against the pathogen MRSA. Importantly, during the cultivation of 14 days, the bacterial strains tested (S. aureus and MRSA) failed to develop significant resistance towards CEN1 HC, indicating that these strains could not circumvent the action of this AMP, while significant resistance profile was observed toward the conventional antibiotic mupirocin used as comparator.
CEN1 HC demonstrated marked effect on reducing bacterial counts in infected wound models in two different animal species. In a rat model of infected full thickness excision wounds, treatment by CEN1 HC significantly reduced the bacterial counts of MRSA as well as P. aeruginosa, the most common pathogens in topical infections in man. The antibacterial effect against S. aureus was confirmed in an infected wound model in pig skin. In line with the in vitro tests, CEN1 HC (Ser) had markedly lower efficacy in these two animal models, compared to the cysteine-containing equivalent.
In summary, we demonstrate that CEN1 HC, a chemically synthesised 30 amino acid peptide sequentially derived from the previously described AMP centrocin 1, has a broad spectrum microbicidal effect, resistant to physiological salt concentration and serum containing wound fluid, combined with anti-inflammatory action. Importantly, S. aureus and MRSA failed to develop resistance against this peptide. CEN1 HC significantly reduced bacterial counts of S. aureus, MRSA and P. aeruginosa in animal models of infected wounds. Based on this study, CEN1 HC appears to be a promising agent in the topical treatment of infections, and further studies are warranted to evaluate the applicability of this AMP in clinical settings.