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Antimicrobial Agents and Chemotherapy, August 2000, p. 2039-2045, Vol. 44, No. 8
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of a Fish Antimicrobial Peptide: Gene Expression, Subcellular Localization, and Spectrum of Activity†

Alexander M. Cole,1 Rabih O. Darouiche,2 Diana Legarda,1 Nancy Connell,3 and Gill Diamond1,*

Departments of Anatomy, Cell Biology and Injury Sciences1 and Microbiology and Molecular Genetics,3 University of Medicine and Dentistry of New Jersey-New Jersey Medical School and Graduate School of Biomedical Sciences, Newark, New Jersey, and Infectious Disease Section, Veterans Affairs Medical Center, Houston, Texas2

Received 25 February 2000/Returned for modification 21 April 2000/Accepted 11 May 2000

Antimicrobial peptides are proposed to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against pathogenic microbes. Pleurocidin, a new 25-residue linear antimicrobial peptide, was recently isolated from the skin secretions of winter flounder (Pleuronectes americanus). The present study identifies the cDNA and gene encoding pleurocidin. The pleurocidin gene comprises four exons. Its upstream region demonstrates consensus binding sequences for transcription factors found in host defense genes in mammals, including sequences identical to the NF-IL6 and alpha and gamma interferon response elements. Pleurocidin is predicted to exist as a 68-residue prepropeptide that undergoes proteolytic cleavage of its amino-terminal signal and carboxy-terminal anionic propiece to form the active, mature peptide. Transmission electron microscopy localized pleurocidin to the mucin granules of skin and intestinal goblet cells. Significant synergy was shown to occur between pleurocidin and D-cycloserine targeting Mycobacterium smegmatis. Pleurocidin was functionally active at physiologic concentrations of magnesium and calcium; however, high concentrations of these divalent cations ablated pleurocidin's activity against a standard test strain, Escherichia coli D31. Pleurocidin was tested against bacterial and fungal clinical isolates and showed broad-spectrum antimicrobial activity. Together, these data support the hypothesis that pleurocidin participates in innate mucosal immunity, and it may prove to be a beneficial therapeutic agent.

* Corresponding author. Mailing address: Department of Anatomy, Cell Biology and Injury Sciences, University of Medicine and Dentistry of New Jersey, MSB-G604, 185 South Orange Ave., Newark, NJ 07103. Phone: (973) 972-3324. Fax: (973) 972-7489. E-mail: gdiamond{at}umdnj.edu.

dagger New Jersey Sea Grant Publication NJSG-00-441.

Antimicrobial Agents and Chemotherapy, August 2000, p. 2039-2045, Vol. 44, No. 8
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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