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

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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.

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Abbreviations

AMP:

Antimicrobial peptide

BAC:

Benzalkonium chloride

CFU:

Colony-forming units

Esc-1a(1-21)NH2 :

Esculentin-1a(1-21)NH2

hTCEpi:

Telomerase-immortalized human cornel epithelial cells

LB:

Luria-Bertani

MH:

Mueller–Hinton

MIC:

Minimal inhibitory concentration

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MPO:

Myeloperoxidase

PB:

Phosphate buffer

PBS:

Phosphate buffered saline

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Acknowledgments

We thank Prof. Santi Maria Recupero, Head of DESMOS Department, Faculty of Medicine and Psychology, Sapienza University of Rome, for allowing access to the ambulatory centre of S. Andrea Hospital to collect tears from consenting volunteers. We thank Dr. Anna Rita Blanco, at SIFI, Catania, Italy, for providing the clinical isolates from human ocular surface infections. This work was supported by grants NIH EY13175 (AMM), NIH EY07551 (UHCO Core grant), Sapienza University of Rome (prot. C26A12NPXZ).

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Authors and Affiliations

  1. College of Optometry, The Ocular Surface Institute, University of Houston, Houston, TX, USA

    Satya Sree N. Kolar, Hasna Baidouri & Alison M. McDermott

  2. Department of Biochemical Sciences “A. Rossi Fanelli”, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Via degli Apuli 9, 00185, Rome, Italy

    Vincenzo Luca & Maria Luisa Mangoni

  3. Ophthalmology Unit, NESMOS Department, S. Andrea Hospital, Faculty of Medicine and Psychology, Sapienza University of Rome, Rome, Italy

    Giuseppe Mannino

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  1. Satya Sree N. Kolar
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  2. Vincenzo Luca
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  6. Maria Luisa Mangoni
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Correspondence to Maria Luisa Mangoni.

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Kolar, S.S.N., Luca, V., Baidouri, H. et al. Esculentin-1a(1-21)NH2: a frog skin-derived peptide for microbial keratitis. Cell. Mol. Life Sci. 72, 617–627 (2015). https://doi.org/10.1007/s00018-014-1694-0

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