Esculentin-1a(1-21)NH2: a frog skin-derived peptide for microbial keratitis

Abstract

Pseudomonas aeruginosa is the primary bacterial pathogen causing contact lens related keratitis. Available ophthalmic agents have reduced efficacy and antimicrobial peptides (AMPs) hold promise as future antibiotics. Here we investigated the in vitro and in vivo anti-Pseudomonal activity of esculentin-1a(1-21)-NH2, derived from a frog skin AMP. The data revealed a minimum inhibitory concentration between 2 and 16 μM against reference strains or drug-resistant clinical isolates of P. aeruginosa without showing toxicity to human corneal epithelial cells up to 50 μM. At 1 μM the peptide rapidly killed bacterial cells and this activity was fully retained in 150 mM sodium chloride and 70% (v/v) human basal tears, particularly against the virulent ATCC 19660 strain. Furthermore, its dropwise administration at 40 μM to the ocular surface in a murine model of P. aeruginosa keratitis (three times daily, for 5 days post-infection) resulted in a significant reduction of infection. The mean clinical score was 2.89 ± 0.26 compared to 3.92 ± 0.08 for the vehicle control. In addition, the corneal level of viable bacteria in the peptide treated animals was significantly lower with a difference of 4 log10 colony counts, compared to 7.7 log10 cells recovered in the control. In parallel, recruitment of inflammatory cells was reduced by half compared to that found in the untreated eyes. Similar results were obtained when esculentin-1a(1-21)NH2 was applied prior to induction of keratitis. Overall, our findings highlight esculentin-1a(1-21)NH2 as an attractive candidate for the development of novel topical pharmaceuticals against Pseudomonas keratitis.

The Frog Skin-Derived Antimicrobial Peptide Esculentin-1a(1-21)NH2 Promotes the Migration of Human HaCaT Keratinocytes in an EGF Receptor-Dependent Manner: A Novel Promoter of Human Skin Wound Healing?

“…The established ability of Esc(1-21) to kill microbes without harming mammalian cells, namely its high anti- Pseudomonal activity also in physiologically relevant conditions [46,51] makes this short-sized AMP a particularly attractive candidate wound healing promoter, especially in the management of chronic, often Pseudomonas -super-infected, human skin ulcers [52,100]. …”

d-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions

“…In addition, in vivo experiments revealed that Esc(1-21) prolongs survival of murine models of P. aeruginosa-induced sepsis or lung infection, upon intravenous or intra-tracheal administration, respectively . Despite this and other studies showing an in vivo antimicrobial activity of AMPs (Giacometti et al 2006;Pini et al 2010;Uccelletti et al 2010;Xiong et al 2011;Luca et al 2014;Jung Kim et al 2014;Kolar et al 2015), inherent limitations for the development of these molecules as new anti-infective therapeutics include: (1) cytotoxicity outside their natural environment, which can strongly dampen the in vivo antimicrobial efficacy of AMPs, and (2) problems with stability, bioavailability and delivery systems. With reference to the latter, studies on the effect of the incorporation of d-amino acids into AMPs were mainly focused on the antimicrobial activity of the peptides, their reduced cytotoxicity and their increased stability to circulating enzymes (Hamamoto et al 2002;Papo et al 2002).…”

Fighting microbial infections: A lesson from amphibian skin-derived esculentin-1 peptides

“…Note that the treatment approach used is in sharp contrast with standard of care antibiotic regimen for bacterial keratitis in human patients in which drops are administered every 15 min up to 1 h, during the first two days of treatment. In parallel, a 2-4 log reduction in the number of viable Pseudomonas cells was observed in the cornea of peptide-treated eyes compared to the vehicle control, as well as a lower number of recruited inflammatory cells like neutrophils, as determined by the relative myeloperoxidase activity in the harvested corneas [59]. In another set of experiments, prophylactic treatment was done by applying the peptide 3 times in 24 h before inducing keratitis, then the treatment was continued post-infection.…”

“…The two selected esculentin derivatives are endowed with a large spectrum of antibacterial activity encompassing Grampositive and Gram-negative species, including clinical isolates with a different antibiotic susceptibility profile, with MIC values in the range of 1-16 M, with the exception of Staphylococcus aureus strains toward which they are practically inactive [59]. In contrast with the majority of mammalian AMPs [51,52], both esculentin derivatives retain their bactericidal activity at high ionic strength and in the presence of biological fluids e.g.…”

“…In contrast with the majority of mammalian AMPs [51,52], both esculentin derivatives retain their bactericidal activity at high ionic strength and in the presence of biological fluids e.g. serum and tears [54,59,71]. Moreover, they are not toxic toward mammalian cells e.g.…”