Histone H1: An Antimicrobial Protein of Atlantic Salmon ( Salmo salar) Robert C. Richards,* David B. O’Neil,* Pierre Thibault,† and K. Vanya Ewart* ,1 *NRC Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia B3H 3Z1, Canada; and †NRC Institute for Biological Sciences, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada Received May 3, 2001 Antimicrobial activity was detected in acid extracts of liver, intestine, and stomach of healthy Atlantic salmon (Salmo salar). An antimicrobial protein was isolated from salmon liver using acid extraction fol- lowed by ammonium sulfate precipitation, large-scale gel filtration chromatography, reverse-phase HPLC, and size exclusion HPLC. The salmon antimicrobial (SAM) protein was found to have a molecular mass of 20,734 Da by MALDI TOF mass spectrometry. Peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry identified the protein as histone H1. The protein had a minimal in- hibitory concentration of 31 g/mL against E. coli D31 in a plate clearing assay. The effect of the SAM protein on bacterial morphology was indistinguishable from that of (Ala- 8,13,18 )-magainin II, as shown by scanning electron microscopy, which suggests that the protein disrupts E. coli membranes in a manner similar to that of most antimicrobial peptides. This protein may act as an antimicrobial in vivo through active secretion or by release from cells during infection-related apop- tosis. © 2001 Academic Press Key Words: histone H1; antimicrobial; Atlantic salmon; Salmo salar; mass spectrometry; scanning electron microscopy; E. coli D31. Antimicrobial peptides play key roles in innate im- munity (1, 2). These peptides and proteins interact directly with bacteria and kill them. First identified in frogs and insects, antimicrobial peptides are now known to be widespread throughout the animal king- dom. Although many are cationic amphiphilic alpha helices, such as the frog magainins, they show remark- able diversity in structure and size (1). A small number of these agents have been identified in teleost fish. A peptide named pardaxin obtained from skin mucus of Moses sole (Pardachirus marmoratus) was found to have antimicrobial and cytolytic activity (3). Another flatfish, the winter flounder (Pleuronectes americanus) produces a distinct antimicrobial peptide named pleu- rocidin (4, 5) which exists in several forms encoded by a multigene family (5). Antimicrobial agents have also been isolated from two catfish species. Parasin, a pep- tide derived from the N-terminus of histone H2A, was found in Parasilurus asotus (6) and a much larger protein related or identical to histone H2B was found in the skin mucus of another catfish (Ictalus punctatus) (7). Antimicrobial activity in the skin of carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss), and other species suggests the possibility of other large antimicrobial proteins (8, 9). The endogenous antimicrobial peptides and proteins of salmonids have not been widely explored. The diver- sity of antimicrobial peptides (1) and their variety, even within a single species (5), suggests that salmo- nids might have one or more novel antimicrobial agents. The present study was therefore undertaken to identify the source of antimicrobial activity in Atlantic salmon (Salmo salar). MATERIALS AND METHODS Materials. Healthy adult Atlantic salmon (S. salar) were ob- tained from net pens in the Bay of Fundy at the Atlantic Salmon Demonstration and Development Farm, St. George, New Brunswick, Canada, in November 1997. E. coli strain D31 (a lipopolysaccharide mutant) was obtained from the CGSC E. coli Genetic Stock Centre, Yale University (Princeton, NJ). The synthetic magainin analogue, (ala- 8,13,18 )-magainin II (referred to here as Ala-magainin), was ob- tained from Sigma (St. Louis, MO). All laboratory chemicals used were of reagent grade. Peptide purification. Skin, stomachs, intestines and livers were dissected from freshly caught Atlantic salmon, frozen quickly on dry ice and stored at -80°C until use. An initial screen for antimicrobial activity was performed on the four tissues to determine the best source for isolation of an antimicrobial peptide. For this screen, 1 g of each tissue was homogenized in 6 ml 60% acetonitrile, 0.1% TFA (trifluoroacetic acid), centrifuged at 21,500g, and the supernatant lyophilized and finally dissolved in 1 ml of water. Samples (20 l) were combined with 10 l of 2.5 mg/mL pronase in 100 mM Tris-HCl, pH 7.0 or 10 l of 2.5 mg/mL proteinase K in 100 mM Tris-HCl, pH 1 To whom correspondence should be addressed. Fax: (902) 426- 9413. E-mail: vanya.ewart@nrc.ca. Biochemical and Biophysical Research Communications 284, 549 –555 (2001) doi:10.1006/bbrc.2001.5020, available online at http://www.idealibrary.com on 549 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.