ANTIMICROBIAL COMPONENTS OF THE MUCUS AND EXTRUDED SLIME

OF HAGFISH {Myxine glutinosά)

FIELD OF THE INVENTION The present invention relates to the preparation and characterization of antimicrobial extracts from hagfish {Myxine glutinosά) mucus and extruded slime and the bioassay guided fractionation and characterization of the antimicrobial peptide (AMP), myxinidin, from the acidic epidermal mucus extract and extruded slime of hagfish.

BACKGROUND OF THE INVENTION

All living organisms coexist with microorganisms including bacteria, fungi and viruses. Many of these microbes are commensals or saprophytes but some are pathogenic and capable of causing severe pathological disorders (Gabay, 1994; Nicolas and Mor, 1995). Under normal conditions, hosts protect themselves from infectious microbes through unique defense mechanisms such as innate defense systems (Kimbrell and Beutler, 2001). Antimicrobial peptides (AMPs) are one of the essential components of innate immunity and have been found to play a defensive role in almost all living species (Boman, 1995). The search and study of novel host defense AMPs gained the interest of investigators following the discovery of insect cecropins, mammalian defensins and amphibian magainins (Steiner et al., 1981; Selsted et al., 1984; Zasloff, 1987). To date more than 880 AMPs have been characterized from various organisms including plants, invertebrates and animal species (http://www.bbcm. univ.trieste.it/~tossi.).

Antimicrobial peptides vary in biochemical properties such as amino acid sequences, length and structure, but share several common features. Most AMPs are

constitutively produced or produced in response to the presence of microbes, have a net positive charge of +2 to +8 (cationic) (Ganz et al., 1985; Boman, 1995; Hancock, 1997; Park et al., 1998) and display a broad-spectrum of activity against numerous pathogenic organisms including Gram positive and Gram negative bacteria, yeast, fungi, enveloped viruses and parasites with little or no toxicity to host cells (Boman, 1995; Vizioli and Salzet, 2002). Antimicrobial peptides function in synergy with conventional antibiotics and other host defense components such as lysozyme to kill pathogens (Hancock and Scott, 2000; Patrzykat et al., 2001). In addition to microbicidal capabilities, some AMPs also perform diverse immunomodulatory functions such as the stimulation of monocytes, chemo attraction of neutrophil and the promotion of tissue or wound repair (Territo et al., 1989; Murphy et al., 1993; Verbanac et al., 1993; Huang et al., 1997; Befus et al., 1999; Scott et al., 2000).

Fish have adapted to survive in a pathogen rich aquatic environment. Their primary protection against invading pathogens is the epidermal mucus which contains a variety of antimicrobial components such as antimicrobial peptides, lysozyme, proteases, and lectins (Ellis, 2001a). The antimicrobial property of epidermal mucus against infectious pathogens has been demonstrated previously in rainbow trout (O. mykiss) (Austin and Mclntosh, 1988), ayu {Plecoglossu altivelis), turbot (Scophthalmus maximus) and carp (Cyprinus carpio) (Kanno et al., 1989; Fouz et al., 1990; Lemaϊtre et al., 1996). Increased expression of one or more of the above mentioned antimicrobial components in fish epidermal mucus has been observed following microbial stress (Park et al., 1998; Aranishi and Mano, 2000; Patrzykat, 2001) thus supporting the role of epidermal mucus in protecting fish from infectious pathogens.

Antimicrobial peptides are most often produced in sites that come in contact with pathogens such as mucus layers on skin, gill and intestine and in circulating immune cells (Barra and Simmaco, 1995; Silphaduang and Noga, 2001; Namjoshi et al, 2007). While a number of novel AMPs have been identified from the mucus of numerous amphibian and mammalian species (Krisanaprakornkit et al., 1998; Smet and Contreras, 2005; Pukala et al., 2006; Ming et al., 2007; Kim et al., 2007), only a few families of fish have been investigated for the presence of epidermal mucus-derived AMPs.

Antimicrobial peptides have been shown to be a key innate defense component in the mucosal immunity of fish species including Moses sole fish (P. marmoratus) winter flounder (P. americanus), catfish (P. asotus), Atlantic halibut (H. hippoglossus), rainbow trout (O. mykiss) and Atlantic cod (G. morhuά).

Pardaxin (a 33-residue peptide from Moses sole fish, Pardachirus marmoratus; Oren and Shai, 1996), pleurocidin (a 25-residue peptide from winter flounder, Pleuronectes americanus; Cole et al., 1997), parasin 1 (a 39-redidue peptide from catfish, P. asotus; Park et al., 1998), hipposin (a 51 -residue peptide from Atlantic halibut, H. hippoglossus; Birkemo et al., 2003), oncorhycin III ( a 6671 Da peptide from rainbow trout, Oncorhynchus mykiss; Fernandes et al., 2003), oncorhycin II (a histone Hl-derived peptide from rainbow trout O. mykiss; Fernandes et al., 2004) and a histone H2B derived peptide from Atlantic cod (Gadus morhua; Bergsson et al., 2005) are examples of AMPs that have been identified in fish epidermal mucus. Antimicrobial peptides derived from fish epidermal mucus have shown higher activity against a broad spectrum of fish and human pathogens than AMPs derived from amphibian mucus (Park et al., 1998). A distinguishing feature of fish epidermal mucus AMPs relative to mammalian AMPs is that fish AMPs exhibit potent activity at relatively high sodium chloride concentrations

(Cole et al, 1997; Noga and Silphaduang, 2003). These features make AMPs derived from fish epidermal mucus potential therapeutic agents for the maintenance of fish and human health.

The antimicrobial properties of the epidermal mucus extracts of Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp (Cyprinus carpio sub sp. koi), striped bass (Morone saxatilis), haddock (Melanogrammus aeglefinus) and hagfish (Myxine glutinosa) have been previously studied in our laboratory (Subramanian et al., 2008). The acidic epidermal mucus extracts of brook trout, haddock and hagfish showed significant antimicrobial activity against a wide range of pathogens (Subramanian et al., 2008). The hagfish acidic epidermal mucus extract was found to be the most potent of the fish species studied (Subramanian et al., 2008).

The mechanism by which fish derived AMPs kill microbes is still unclear but, previous studies have proposed that interactions of lytic peptides with the cytoplasmic membrane of the target microbe may lead to membrane permeabilization, channel formation, leakage of cellular contents and cell death (Oren and Shai, 1996; Saint et al., 2002; Syvitski et al., 2005). Other mechanisms such as inhibition of DNA and integral membrane proteins syntheses (Boman et al., 1993; Carlsson et al., 1998; Park et al., 1998; Patrzykat et al., 2002) have also shown to be involved.

A number of fish-derived AMPs have been shown to be active at high salt (NaCl) concentrations as compared to their mammalian counterparts, which have activity that is sensitive to high salt concentrations (Smith et al., 1996; Goldman et al., 1997). This is an especially important feature as fish-derived AMPs have potential applications for the control of bacterial infections in the lungs of cystic fibrosis patients (Cole et al., 1997). Pleurocidin, piscidin and histone H2B-derived peptide of cod epidermal mucus have all

been shown to exhibit antimicrobial activity at varying salt concentrations (Cole et al, 1997; Noga and Silphaduang, 2003; Bergsson et al., 2005). Oncorhyncin III from the epidermal mucus of an anadromous rainbow trout showed poor activity at higher salt concentrations (Fernandes et al., 2003). The habitat of the fish species therefore appears to predict the activity of fish AMPs in saline environments.

Antimicrobial peptides such as melittin and pardaxin have been shown to be cytotoxic to mammalian cell membranes (Habermann, 1972; Lazarovici et al., 1986).

Hagfish (M glutinosa) are the most primitive vertebrates (Bardack, 1998). Immune tissues or genes for immunoglobulins, major histocompatibility complex (MHC), and T cell receptor proteins, which are the crucial components of adaptive immunity in cartilaginous and teleost fish, are absent in the hagfish (Raison and dos Remedios, 1998; Flanjnik, 2004; Rolff, 2007). Therefore, hagfish depend highly on their innate immune mechanisms for protection against pathogens (Raison and dos Remedios, 1998). Innate immune parameters including lysozyme, cathepsin B and other proteases have been previously reported in hagfish epidermal mucus (Subramanian et al., 2007). The mucosal epidermal layer is thought to play a crucial role in the defense of hagfish against pathogenic infection.

The hagfish, in addition to the typical epidermal mucus secretion, have been observed to produce copious amounts of "slime" in the traps used by hagfish fishermen. The slime is extruded upon stimulation from the numerous slime glands that line on the either side of the ventrolateral body walls (Leppi, 1968; Downing et al., 1981; Helfman et al., 1997; Spitzer and Koch, 1998). It is thought to defend hagfish from predators. The extruded slime is a significant by-product of the hagfish fishing and processing industry.

To date no value-added use of hagfish slime waste has been identified except for its use in a low value fishmeal product.

Fish waste generated by the fishing and fish processing industries, such as the skin, viscera, fins, skeleton, head, liver and by-catch, had largely been used in the past to produce fish oil, fishmeal, fertilizer, pet food and fish silage (F aid et al., 1997; Kotzamanis et al., 2001; Aidos et al., 2003; Bechtel, 2003; Kim and Mendis, 2006). However, recent studies have reported the presence of a number of bioactive compounds in fish waste that could be used in human and animal dietary or health supplements (Cancre et al., 1999; Cho et al., 2005; Kim and Mendis, 2006).

SUMMARY OF THE INVENTION

The instant invention focuses on the antimicrobial activity and structure characterization of a novel antimicrobial component from the acidic epidermal mucus of hagfish using bioassay-guided fractionation. The isolation of an antimicrobial component from the epidermal mucus of hagfish supports the immune defensive role of the mucus. This invention also focuses on accessing the antimicrobial activity and presence of myxinidin in the extruded slime of hagfish to identify potential value-added uses of this waste product.

The present invention discloses the bioassay-guided fractionation and characterization of the AMP, myxinidin, from the acidic epidermal mucus extract of hagfish. Myxinidin was isolated from the crude extract using Cl 8 SPE, FPLC size- exclusion and HPLC reversed-phase chromatographies. The structure elucidation of H6, the final active fraction, was determined using tricine SDS-PAGE, mass spectrometry (MS), automated amino acid sequencing and solid-phase synthesis. The molecular mass

of myxinidin, [M+ H] ⁺ 1327.68 m/z, agreed with the theoretical mass of the myxinidin amino acid sequence obtained from an Edman degradation and tricine SDS-PAGE analysis. The molecular mass of myxinidin was lower than that observed for peptides previously isolated from the epidermal mucus of fish species such as winter flounder (2711.0 Da), catfish (2000.4 Da) and Atlantic halibut (5459.0 Da) (Cole et al, 1997; Park et al., 1998; Birkemo et al., 2003). Myxinidin's mass was different from the AMPs that have been previously isolated from the skin of Pacific hagfish, Eptatretus burgeri (1279.5 Da) and the intestines of Atlantic hagfish, M. glutinosa (3551.9, 4564.0 and 4643.3 Da) (Shinnar et al., 1996; Hwang et al., 1999). The amino acid sequence of myxinidin was determined by Edman degradation and ESI/MS/MS analysis. No homology to the protein/peptides in the Swissprot, NCBI and antimicrobial peptide databases was observed for the amino acid sequence of myxinidin. Definitive confirmation of the proposed structure was achieved through solid- phase synthesis of myxinidin and comparison of MS/MS fragmentation profiles. The AMPs from hagfish intestinal tissue (HFIAP 1, 2 and 3) contained bromo-tryptophan residues (Shinnar et al., 1996) but this chemical feature was not observed in myxinidin.

Synthetic myxinidin showed the same antimicrobial activity as compared to the myxinidin that was isolated from hagfish epidermal mucus (Table 1). Myxinidin exhibited antimicrobial activity towards Gram-positive and Gram-negative bacteria and a yeast strain (Table 1). The minimum bactericidal concentrations (MBC) of myxinidin were found to be in the range of 1-10 μg protein/mL. Fish pathogens including A. salmonicida A449, L. anguillarum 02-11 and Y. ruckeri 96-4 were highly sensitive to myxinidin. When compared to the activity of other fish epidermal mucus derived- AMPs tested against fish pathogens, myxinidin was found to be 1-100 times more active

(Shinnar et al., 1996; Kjuul et al, 1999; Hwang et al, 1999; Birkemo et al, 2003). This suggests that myxinidin has a significant role in hagfish defense against pathogenic infection in the aqueous environment. The MBC of myxinidin versus pleurocidin (NRC- 17) (Table 1) showed myxinidin was more potent than pleurocidin against all of the organisms tested except S. enterica C610, E. coli D31 and S. epidermis C621. The antimicrobial activity of other reported hagfish AMPs including HFS-I (hagfish skin peptide) and HFIAP 1, 2 and 3 (Shinnar et al., 1996; Hwang et al., 1999) indicated that myxinidin is highly potent against most of the human and fish pathogens screened in the study which leads to the present invention.

Table 1

Microbial strains MBC (μg / mL)

Epidermal Mucus Pleurocidin Synthetic Myxinidin (NRC-17) Myxinidin

Human Pathogens

Escherichia coli D31 2 0 <0.3 2 0

Salmonella enterica Serovar Typhimurium CβlO 2 5 1.0 2.5

Staphylococcus epidermis C621 8.5 6.0 8.5

Pseudomonas aeruginosa Z61 7.0 >19.2 7.0

P aeruginosa K799 10 0 24.0 10 0

Candida albicans C627 10 0 16 0 10 0

Fish Pathogens

Aeromonas salmonicida sub sp. salmonicida 2.0 8.0 2.0 A449 Listonella anguillarum 02-11 1.0 16.0 1 0

Yersinia ruckeri 96-4 2 0 12.8 2 0

Clean-up of the acidic extruded slime extract, using a 5 kDa cut-off centrifugal filter and semi-preparative RP-HPLC, was conducted to prepare the sample for LC/DAD and/or LC/MS analysis of the myxinidin content. The presence of myxinidin in the extruded slime extract sub fraction SL-I was confirmed using LC/ESI/MS. The concentration of myxinidin in the dried crude extruded slime was determined to be 0.37 mg/g. The total protein concentration of the extruded slime indicated that myxinidin contributed 0.13% to the total extruded slime protein. Extruded slime acid extract showed activity at a MBC of 8 μg protein/mL against S. enterica C610, while the synthetic myxinidin MBC was 2.5 μg/mL against the same pathogen. Hence myxinidin in the extruded slime acid extract would not be expected to contribute significantly to the overall antimicrobial activity. This suggests that the acidic extract of the extruded slime contains other antimicrobial components. However, myxinidin may function in synergy with these other agents. The HPLC chromatograms of the extruded slime showed a number of unidentified components (Figure 10) indicating the presence of other components that may have promising antimicrobial activity. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the isolation of myxinidin from the acidic extract of the epidermal mucus of hagfish (Myxine glutinosa L.).

Figure 2 shows a FPLC chromatogram of the Sep-Pak treated acidic hagfish epidermal mucus extract. The active fraction was loaded on a Superdex™ peptide 10/300 GL column and eluted isocratically with 0.1 M ammonium acetate buffer (pH 6.8) at a flow rate of 0.5 mL/min. The elution was monitored at 280 nm. Fractions Fl to F6 were collected (as marked by perpendicular lines on X-axis) and evaluated for bioactivity. The black bar shows the fractions that exhibited antimicrobial activity against E. coli D31.

Figure 3 shows the reversed-phase HPLC chromatogram of fraction F3. The fraction was loaded on a semi-preparative Cl 8 RP-HPLC which was eluted with a linear gradient of solvent A [0.1 % (v/v) trifiuoroacetic (TFA) in Milli Q water) and solvent B (0.1 % (v/v) TFA in acetonitrile (ACN)] from 10 to 90 % for 45 min at a flow rate of 1.0 niL/min. The elution was monitored at 214 nm. The diagonal line shows the concentration of acetonitrile in the eluting solvent. The black bar indicates the fraction, FA, which exhibited antimicrobial activity against E. coli D31.

Figure 4 shows a Tricine SDS-PAGE electrophoretic gel of active epidermal mucus fractions during each stage of bioassay guided fractionation. 1. Epidermal mucus acid extract, 2. Fraction F3 from size-exclusion chromatography, 3. Fraction FA from Cl 8 reversed- phase semi-preparative HPLC fractionation, and 4. Fraction H6 from analytical RP-HPLC fractionation. The arrow indicates the molecular mass of the active component of H6. The samples were loaded onto a separating gel of 16.5 % acrylamide with a 10 % acrylamide spacer gel and 4 % stacking gel. The electrophoretic gel was run for 3.5 to 4 h at 90 V and the bands were visualised using silver staining. The molecular mass markers are displayed along the left side of the gel.

Figure 5 shows a reversed-phase HPLC chromatogram of fraction FA. Fractionation of FA was accomplished by loading it onto an analytical C18 RP-HPLC which was eluted with a two step shallow gradient of solvent A (0.1 % (v/v) trifiuoroacetic acid (TFA) in Milli Q water) and solvent B (0.1 % (v/v) TFA in acetonitrile (ACN)) from 10 to 20 % and 20 to 29 % over a period of 12 and 23 min, respectively, at a flow rate of 1.0 mL/min. The column elution was monitored at 214 nm. The diagonal line shows the concentration of acetonitrile in the eluting solvent. The black

bar indicates the fraction, H6 that showed potent antimicrobial activity against both fish and human pathogens.

Figure 6 shows a mass spectra of H6 determined by Q TRAP ^® ESI/MS. The MS determined the +1 and +2 ions at mass-charge ratio (m/z) of 1327.68 Da and 664.84 Da, respectively.

Figure 7 shows a fragment ion pattern resulting from the collision of the [M+H] ⁺ m/z 1,327.68 of myxinidin using Q TRAP® LC-ESI/MS/MS (m/z 100-1,120). The mass difference of the b3 and b4 and the y4 and y5 fragment ions, 115.08 and 115.2 Da, respectively, confirms that the amino acid at position 4 is ASP. The observed ions are indicated in bold.

Figure 8 shows a Tricine SDS-PAGE of the hagfish extruded slime. The acidic extract of hagfish extruded slime and a 5 kDa molecular weight cut-off filtrate of the acidic extract of the extruded slime are shown. Molecular mass (kDa) markers are on the left side of the gel. Extruded slime samples were loaded onto a separating gel of 16.5 % acrylamide with a 10 % acrylamide spacer gel and 4 % stacking gel. After subjecting to electrophoresis for 3.5 to 4 h at 90 V the gels were silver stained.

Figure 9 shows the antimicrobial activity of the acidic extract of hagfish extruded slime against Salmonella enterica C610 using the broth dilution method in duplicate (A and B)(128 μg protein/ mL). Lanes C and D are the positive controls (acidic extract of hagfish epidermal mucus, 132.8 μg protein/ mL ), E and F are the negative control (wells containing only bacteria ), and G and H are blank lanes that do not contain bacteria. Numbers on the top of the plate indicates the serially two-fold dilution of sample and controls.

Figure 10 shows HPLC chromatograms of the low molecular weight fraction of the extruded slime extract of hagfish: a reversed-phase HPLC chromatogram of the low molecular weight fraction of the extruded slime extract and a reversed-phase HPLC chromatogram of the low molecular weight fraction of the extruded slime extract co- injected with synthetic myxinidin. The arrow indicates the peak (27-28 min) that was enhanced in absorbance.

Figure 11 shows the absorbance of formazan (mean OD) of HUVEC cells treated with and without synthetic myxinidin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The antimicrobial peptide, myxinidin, of the present invention can be prepared by either recombinant methods or by chemical synthesis.

(I) Isolation of AMP myxinidin from the epidermal mucus and extruded slime of hagfish (M. glutinosa) and antimicrobial activity and salt resistance of same

Materials and Methods Fish and Their Maintenance

Hagfish (M glutinosa) weighing 150 ± 35 g were obtained from a fisherman from Cape Sable Island, NS, Canada. The fish were maintained at the National Research Council-Institute for Marine Biosciences (NRC-IMB), Marine Research Station, Ketch harbour, NS. Hagfish were placed in a 1500 L capacity flow-through tank at a stocking density of 6-7 Kg/m ³ (~ 45 fish per tank) and a water temperature of 8 ± 2 ⁰ C. The guidelines set by the Canadian Council on Animal Care (CCAC) were strictly adhered to

while maintaining and handling the fish. Only healthy fish were sampled for epidermal mucus collection. Dead fish or fish with lesions were removed from the tanks. The fish were fed twice a month with oven dried herring.

Epidermal Mucus Collection and Extraction

Epidermal mucus was collected from 70 to 80 fish as per the method of Ross et al. (2000) with slight modifications. The fish were starved for 24 h and the epidermal mucus was obtained non-lethally by anaesthetizing the fish with a sub-lethal dose (100 mg/L) of tricaine methanesulphonate (MS-222, 100 mg/L; Syndel Laboratories, BC, Canada). Each anaesthetized fish was transferred into a polyethylene bag (27 x 28 cm, Ziploc ^® , Johnson and Sons, Ltd., ON, Canada) containing 10 mL of 100 mM NaCl. The fish was gently moved back and forth inside the bag for 1-2 min to slough-off the epidermal mucus. The hagfish was then returned to the recovery tank. Individual fish epidermal mucus samples (-10 mL) were extracted by adding 10 mL of 10% (v/v) acetic acid and heated for 5 min in a boiling water bath. The acid mucus mixtures from individual fish were then pooled and homogenized for 1 min using a polytron homogenizer (PTA 10TS, Kinematica®, Brinkmann Instruments, Inc., NY, USA). The homogenate was centrifuged at 18,000 x g for 35 min at 4 ⁰ C. The supernatant was collected and applied onto four reversed-phase Sep-Pak Vac 1 g C18 cartridges (125 A, 55-105 μm; Waters Corporation, MA, USA) for solid phase extraction (SPE). Prior to the addition of sample, each of the cartridges was equilibrated first with 30 mL of methanol and then 10 mL of 10% (v/v) acetic acid. After the supernatant was loaded, the cartridge was washed with 10 mL of 0.1% (v/v) trifluoroacetic acid (TFA) and then eluted with 40 mL of acetonitrile (ACN) / water / TFA (80.0: 19.9:0.1, v/v/v). The eluates from each Sep-Pak cartridges were pooled,

evaporated under a stream of nitrogen, freeze dried, reconstituted in water and then assayed for antimicrobial activity against Escherichia coli D31 or Salmonella enterica Serovar Typhimurium C610.

Extruded Slime Collection and Extract Preparation

Hagfish were induced to produce "extruded slime" in a bucket of seawater. Individual hagfish were transferred carefully into a bucket containing 3 L of seawater. The water inside the bucket was agitated gently by hand; this action induced the hagfish to release larger masses of slime into the water, which was immediately scooped out of the bucket and squeezed to remove excess water. Each hagfish was then transferred back into a recovery tank. The extruded slime was collected from 40 fish using this method was pooled and freeze dried.

Freeze dried extruded slime (10 g) was extracted with 100 mL of 10% (v/v) acetic acid for 5 min in a boiling water bath. The acidic slime mixture was then homogenized for 1 min using a polytron homogenizer (PTA 10TS, Kinematica®, Brinkmann Instruments, Inc., NY, USA). The resulting homogenate was centrifuged at 18,000 x g for 35 min at 4 ⁰ C to yield a supernatant that was loaded onto two equilibrated reversed-phase Sep-Pak Vac 1 g C18 cartridges (125 A, 55-105 μm; Waters Corporation, Milford, MA, USA). The elution of the cartridges was carried out using the method previously described by Subramanian et al. (2008). The 80% acetonitrile (ACN)/ 19.9% water/ 0.1% trifluoroacetic acid (TFA) eluates from each Sep-Pak cartridges were pooled, evaporated under a stream of nitrogen, freeze dried and assayed for antimicrobial activity.

Bioassay-Guided Fractionation of Acidic Epidermal Mucus Extract

The dried eluate generated from the Sep-Pak cartridges was suspended in distilled water and subjected to size fractionation on an AKTA™ Fast Protein Liquid Chromatography (FPLC, Amersham Pharmacia Biotech, NJ, USA) that was equipped with a Superdex™ peptide 10/300 GL column (GE Healthcare Bio-Sciences Corporation, NY, USA). The column was equilibrated with 0.1 M ammonium acetate buffer, pH 6.0, and the sample fractions were eluted isocratically with 0.1 M ammonium acetate buffer, pH 6.0, at a flow rate of 0.5 mL/min. The elution profile was monitored at 280 nm. The resulting fractions were freeze dried, re-dissolved in distilled water and tested for antimicrobial activity against E. coli D 31. The fraction showing the most potent antibacterial activity was further fractionated on an Agilent 1100 HPLC system (Agilent Technologies, Inc., CA, USA) equipped with a semi-preparative Jupiter C18 (RP-HPLC; 5 μm, 300 A, 10 x 250 mm) column. The separation was carried out using a linear gradient of solvent A (0.1% (v/v) TFA in Milli Q water) and solvent B (0.1% (v/v) TFA in ACN) from 10 to 90% for 45 min, at a flow rate of 1.0 mL/min. The column effluent was monitored at 214 nm. The eluted fractions were evaporated under a stream of nitrogen, freeze dried, reconstituted in distilled water and analyzed for antimicrobial activity against E. coli D31.

The most bacteriolytic fraction was further purified using an analytical Jupiter Cl 8 RP-HPLC (5 μm, 300 A, 4.6 x 250 mm) column following a two step shallow gradient of solvent A and B from 10 to 20% and 20 to 29% over a period of 12 and 23 min, respectively. The elution was achieved at a flow rate of 1.0 mL/min and the profile was monitored at 214 nm. The collected fractions were evaporated under a stream of nitrogen, freeze dried, reconstituted in distilled water and tested for activity against E. coli D31.

Fractionation of the Extruded Slime Extract

The freeze dried acidic extract of the extruded slime was resuspended in water and size fractionated using a 5 kDa MWCO (molecular weight cut-off) centrifugal filter device (Amicon® Ultra-15, Millipore Corporation, Boston, MA, USA). The filtrate was subsequently injected onto a semi-preparative Jupiter Cl 8 Reversed-phase-High Performance Liquid Chromatography (RP-HPLC; 5 μm, 300 A, 10 x 250 mm) column installed in an Agilent 1100 HPLC DAD system (Agilent Technologies, Inc., CA, USA). The column was eluted with a linear gradient of 0.1% (v/v) TFA in Milli Q water and 0.1% (v/v) TFA in ACN going from 10 to 90% for 45 min, at a flow rate of 1 mL/min and monitored at 214 nm.

Co-injection of the extruded slime extract with synthetic myxinidin allowed for the collection of fraction SL-I at 27-28 min which was identified using this method to potentially contain myxinidin.

LC/MS Analysis of the Myxinidin Content of the Extruded Slime

Analysis of the myxinidin content of fraction SL-I was accomplished using liquid chromatography coupled with electrospray ionisation mass spectrometry (LC/ESI/MS). An Agilent 1100 HPLC MSD (Agilent Technologies, Inc., CA, USA) instrument equipped with a Jupiter Cl 8 RP-HPLC (5 μm, 300 A, 4.5 x 250 mm) column and was operated at a flow rate of 1 mL/min. Analysis of SL-I was performed using a shallow gradient of 10 to 20% of solvent B for 12 minutes and 20 to 29% for a further 11 minutes (solvent A was 0.5% (v/v) formic acid (FA) in Milli Q water and solvent B was 0.5% (v/v) TFA in ACN). The column eluate was monitored on the MS using electrospray

ionization which was carried out in a positive mode under the following conditions: capillary voltage, 5000 V; capillary gas temperature, 350 ⁰ C; nebulizer pressure, 30 psi and fragmentation voltage, 75 V. The ionization and the mass spectroscopic parameters were optimized to obtain maximum sensitivity at unit resolution. The selected ion monitoring (SIM) experiment was conducted by examining the mass to charge (m/z) ratio of precursor ion 1327.68 (+1) and the fragment ions 443.56 (+3) and 664.84 (+2) for the standard (synthetic myxinidin) and SL-I.

The concentration of myxinidin in the extruded slime was determined by constructing a calibration curve. A stock solution of synthetic myxinidin (2.75 mg/mL) was diluted with Milli Q water to give a series of working standards with a linear concentration of 0.08, 0.14, 0.55, 0.82 and 1.40 mg/mL. A five point standard curve was prepared by injecting 2 μL of each standard solution in triplicate. Blank runs were carried out between each triplicate run of a standard or sample using solvent A to avoid sample carry over. Peak areas were integrated automatically using Agilent LC3D chemstation software. The regression equation of the standard curve was used to calculate the myxinidin concentration in SL-I.

Microbial Cultures

Antimicrobial activity of hagfish epidermal mucus was studied using a range of pathogenic Gram positive and Gram negative bacteria and yeast (Table 2) including human pathogens (E. coli D31, S. enterica C610, Staphylococcus epidermis C621,

Pseudomonas aeruginosa Z61, P. aeruginosa K799 and Candida albicans C627) and fish pathogens (Aeromonas salmonicida sub sp. salmonicida A449, Listonella anguillarum

02-11 and Yersinia ruckeri 96-4). All strains were obtained from individual lab collections at NRC- 1MB. The strains were stored at - 8O ⁰ C until use.

Table 2

Microbial strains Gram (+/-) Description References

Human Pathogens

Escherichia coll D31 Lipopolysacchaπde mutant Boman et al (1974)

S enterica serovar Typhimuπum C610 Defensm-supersusceptible Fields et al (1989)

Staphylococcus epidermis C621 Human clinical isolate Patrzykat et al (2003)

Pseudomonas aeruginosa Z61 Antibiotic-supersusceptible Patrzykat et al (2003)

P aeruginosa K" '99 The parent strain of Z61 Angus et al (1982)

Candida albicans C627 Yeast Human clinical isolates 1MB strain collection

Fish Pathogens

A salmonicida sub sp salmonicida A449 Isolated from brown trout Umelo & Trust ( 1997)

L anguillarum 02- 1 1 Isolated from chum salmon 1MB strain collection

Yersinia ruckeri 96-4 Field isolates 1MB strain collection

Most human bacterial and yeast pathogens were grown at 37 ⁰ C in Mueller-Hinton (MH) broth (Difco Laboratories, MI, USA) except for E. coli D31 which was grown at 37 ⁰ C in Luria-Bertani (LB) broth (EM Science, NJ, USA). The fish pathogens were cultured at 16 ⁰ C in tryptic soy broth (Difco) containing 0.85% NaCl.

Antimicrobial Assay

The antimicrobial activity of hagfish epidermal mucus acid extract was screened for activity against S. enterica C610 and E. coli D31 using the broth dilution method as described previously (Subramanian et al., 2008). The S. enterica C610 used was a mutant bacterial strain that has shown a high susceptibility to various antibiotics and antimicrobial peptides (Fields et al., 1989). The E. coli D31 strain used was a lipopolysaccharide mutant that had been found to be resistant to streptomycin and ampicillin (Bomen et al., 1974). During each stage of fractionation, the activity was examined against E. coli D31 by the broth dilution method. Briefly, 50 μL of sample (known concentration) was diluted serially two-fold with 50 μL of LB broth in a 96-well polypropylene microtitre plate (Costar; Corning, Inc., NY, USA). Assays were carried out in triplicate. The bacteria were grown overnight to mid-logarithmic phase and diluted to give a final density of 2 x 10 ⁴ CFU/mL. Fifty μL of diluted bacterial culture was added to the wells containing epidermal mucus fractions. The plate was then incubated overnight at the appropriate growth temperatures. The controls, which consisted of 50 μL of distilled water or the buffers being used in each fractionation step, were assayed in parallel with the epidermal mucus fractions in the same microtitre plate. The controls were two-fold diluted with 50 μL of LB broth and incubated with 50 μL of bacterial cells. The bactericidal or antimicrobial activity was determined by visual inspection (clear well contents) and then confirmed by streaking an aliquot of the well contents on LB agar plates.

The antimicrobial activity of the acidic extract of the extruded slime was assessed using S enterica (C610). The antimicrobial activity was determined using a broth microdilution method that was carried out in duplicate. The acidic epidermal mucus extract was used as a positive control in the antimicrobial assay. The epidermal mucus

control was prepared as described previously (Subramanian et al., 2008). The bactericidal activity was determined by visual inspection (clear well contents) and then confirmed by streaking an aliquot of the well contents on Mueller-Hinton (MH) agar plates. The minimum concentration of extruded slime protein (μg/mL) that exhibited antimicrobial activity was presented as the minimal bactericidal concentrations (MBC).

Determination of Minimal Bactericidal Concentrations (MBC)

The MBC of the RP-HPLC-purifϊed active fraction was determined for the nine different bacterial and yeast strains mentioned in Table 2 using the broth dilution method. To determine the significance of the antimicrobial activity of the active component, the antimicrobial assays were carried out in parallel using a synthetic broad-spectrum antimicrobial peptide, pleurocidin NRC- 17 (Patrzykat et al., 2003). The MBC was defined as the minimal concentration of antimicrobial component that resulted in complete (100%) inhibition of bacterial growth.

Protein Quantification and Electrophoresis

The protein concentration of the epidermal mucus extract and resulting fractions was determined using a protein-dye binding assay kit (Bradford, Bio-Rad laboratories, Inc., CA, USA) assay, which was based on the Bradford protein analysis method (Bradford, 1976) using bovine gamma globulin as a standard. The approximate molecular mass (Da) and purity of active fractions generated during each fractionation step were analyzed using tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis (tricine SDS-PAGE) as described by Schagger et al. (1987). A separating gel of 16.5% acrylamide with a 10% acrylamide spacer gel and 4% stacking gel was used. The gel was

run in a Bio-Rad electrophoresis apparatus for 3.5 to 4 h at 90 V. Sodium dodecyl sulphate-PAGE standard markers (polypeptide, Bio-Rad) were included to estimate the molecular mass of active components. The bands were visualized using silver staining (Blum et al, 1987). The protein profile of hagfish extruded slime extracts were analyzed using tricine sodium dodecyl sulphate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE) as described by Schagger et al. (1987). Protein samples (6 μg total protein) were diluted 1 :1 with sample buffer [4% (w/v) SDS, 50 mM Tris-HCl, 2% mercaptoethanol (v/v), 12% (v/v) glycerol and 0.5% (w/v) bromophenol blue adjusted with HCl to pH 6.8] and loaded onto a separating gel of 15% acrylamide with a 10% acrylamide spacer gel and 4% stacking gel. The gel was run in a Bio-Rad electrophoresis apparatus for 3.5 to 4 h at 90 V. Sodium dodecyl sulphate-PAGE standard markers (polypeptide, Bio-Rad laboratories, Inc., CA, USA) were included to estimate the molecular mass of proteins. Proteins were visualized using silver staining (Blum et al., 1987).

Electroblotting and Edman Degradation

The active fraction obtained from analytical RP-HPLC was diluted 1:1 in the tricine SDS-PAGE loading buffer [3% (w/v) SDS, 125 mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 5% (v/v) and 2-mercaptoethanol] and subjected to electrophoresis on a 16.5% gel. Prior to sample loading, the separating gel was aged for 48 h in 200 mL of 0.02 M Tris and 0.4% (w/v) SDS. After electrophoresis, the sample from the gel was electroblotted on to a polyvinylidene difluroide (PVDF) membrane (0.22 μm, Sequi- Blot™, Bio-Rad) at 50 V for 60 min in the presence of CAPS buffer (10 mM (w/v) 3- (cyclohexylamino)- 1 -propane sulphonic acid, pH 11.0; Sigma- Aldric, Inc., MO, USA).

The blotted membrane was stained with 0.1% Coomassie Brilliant Blue R-250 (Sigma) in MeOH / H ₂ O / AcOH (40:50:10) for 5 min and destained with MeOH / H ₂ O / AcOH (40:50:10) for 10 min.

The band of interest was submitted for N-terminal amino acid Edman sequencing by the ABI 492 Procise cLC sequencer at the Advanced Protein Technology Centre of the Hospital for Sick Children, ON, Canada.

Mass Spectrometry

The molecular mass and the amino acid sequence of the RP -HPLC -purified active fraction were determined using the nanoelectrospray mass spectrometry (nESI/MS) (Q Trap ^® LC-ESI7MS/MS, Applied Biosystems MDS-SCIEX, CA, USA) at the Atlantic Research Centre, Dalhousie University, NS, Canada. The instrument was calibrated with peptides of known molecular mass in the range from 100 to 2000 Da and had an accuracy of ± 0.01% for mass determinations. The freeze dried RP -HPLC -purified active fraction was dissolved in 0.1% formic acid (FA) / 50% ACN and infused at a flow rate of 5 μl/min. Ions were detected and analyzed in the positive mode on the basis of their m/z ratio. The precursor ions selected by the first quadrapole were trapped and fragmented in a collision cell set at 35 eV. The amino acids in the sequence were identified by calculating the mass differences between adjacent b ions or adjacent y ions. Homology searches of the amino acid sequence of the isolated antimicrobial component were performed on the SwissProt database using the basic local alignment search tool (BLAST) (Altschul et al., 1997) provided by the NCBI Server (http://www.ncbi.nlm.nih.gov/BLAST) and peptide databases (http://aps.unmc.edu/AP/ main.html., Wang and Wang, 2004). The charge was predicted using Expert Protein

Analysis System (ExPASy) proteomics tool at the Swiss Institute of Bioinformatics (http://www. expasy.ch/).

Peptide Synthesis Myxinidin was synthesized at Dalton Pharma Services, ON, Canada and desalted using Sep-Pak Vac 1 g C18 cartridge (Waters). Prior to the addition of myxinidin, the cartridge was equilibrated first with 30 ml of MeOH and then 10 ml of 10% (v/v) acetic acid. After myxinidin was loaded, the cartridge was washed with 10 mL of 0.1% (v/v) TFA and then eluted with 40 ml of ACN / water/ TFA (80.0: 19.9:0.1, v/v/v). The eluate obtained from the Sep-Pak cartridge was evaporated under a stream of nitrogen, freeze dried, reconstituted in water and then evaluated for antimicrobial activity.

Antimicrobial Activity and Salt Resistance of Synthetic Myxinidin

The antimicrobial assay previously outlined in this text was used to determine the microbial killing activity of synthetic myxinidin against bacterial and yeast strains (Table

2). The activity of myxinidin at different salt concentrations was determined with a modified broth dilution protocol using E. coli D31 in LB broth and by adding NaCl at increasing concentrations. Fifty μl of bacterial cells diluted to 2 x 10 ⁴ CFU/ml were added to the wells containing two-fold diluted myxinidin and NaCl (0.1-0.6 M) and the plates were incubated overnight at 37°C. The activity was assessed by visual inspection (clear well contents) and then confirmed by streaking an aliquot of the well contents on LB agar plates.

Hemolysis Assay

The hemolytic activity of synthetic myxinidin was determined using rabbit red blood cells (Lamp ire Biological Laboratories, Inc., PA, USA) as described previously

(Bignami, 1993). Briefly, a 1:9 dilution of the red blood cell pellet (v/v) in PBS (50 μl) was added to 50 μL of synthetic myxinidin solutions. The myxinidin solutions had been previously serially two fold-diluted with PBS to give a concentration range of 500 to 1 μg myxinidin/ml. The red blood cell suspensions were centrifuged at 1300 x g for 10 min following a 1 h incubated at 37 ⁰ C. The level of hemolytic activity in the resulting supernatants was measured at 540 run using a microplate reader (Spectramax Plus ³⁸⁴ Molecular Devices, CA, USA).

(II) Cytotoxicity of chemically synthesized myxinidin using MTT (3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay

Materials and Method

The human umbilical vein endothelial cells (HUVEC) cells were purchased from ATCC (Manassas, VA). All chemicals used were sterile and cell culture standard ( Dulbecco's modified Eagle's medium (DMEM) (Mediatech Inc., Herndon, VA), fetal bovine serum (FBS) (Gibco Invitrogen Co., Carlsbad, CA), penicillin/ streptomycin (MP Biomedicals, Solon, Ohio), Dulbecco's phosphate buffered saline (PBS) (Sigma), trypsin/ethylenediaminetetra-acetic acid (Sigma) and L-glutamine (Sigma Chemicals, St.Louis, MO)). Dimethyl sulfoxide (DMSO) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) (Sigma) were used for the assay.

HUVEC Cell Culture Conditions

Human HUVEC cells were grown in 75 cm ² culture flasks (Corning Inc., Corning, NY) containing 20 mL of complete growth medium consisting of DMEM supplemented with 10% (v/v) FBS, 1% (v/v) IOOX non-essential amino acids, penicilln/streptomycin (100 U/mL) and L- glutamine (2 mmol/L). The cells were incubated at atmospheric pressure with 5% CO ₂ in air at 37° C. When the cells were 90-95% confluence, the medium was removed and the cell monolayer was washed twice with 10 mL of PBS. Cells were then incubated in 2 mL of trypsin/ ethylene diamine tetra-acetic acid for 5 min at 37° C in 5% CO ₂ . Five mL of complete medium was added to the culture flasks. The flasks were then agitated to ensure complete cell layer elevation. The cell suspension was transferred to a 15 mL falcon centrifuge tube (Corning) and centrifuged at 500 x g for 5 min. The resultant pellet was suspended in 1 mL of complete growth medium and the cell concentration was determined manually using a haemocytometer.

MTT Tetrazolium Assay

The cytotoxicity of synthetic myxinidin was determined using a modified MTT tetrazolium assay (Wang et al, 2006). Briefly, 100 μL of medium containing 20,000 HUVEC cells were seeded to each well of a 96 well tissue culture plate (Costar, Corning) and incubated at 37° C in 5% CO ₂ overnight. In the morning the medium was replaced by 100 μL of DMEM medium supplemented with 5% (v/v) FBS containing either no myxinidin (control) or myxinidin at 100 μM/well. The assay was carried out in triplicate. The cells were incubated for 22 h at 37° C in 5% CO _2. Twenty μL of MTT (5 mg/ml in PBS, pH 7.4) was then added to each well and incubated at 37° C in 5% CO ₂ for 2 h. The plate was centrifuged at 1400 x g for 10 min. The supernatant was discarded, and 100 μL

of DMSO was added to each well and incubated at room temperature for 1 h. The absorbance was measured at 490 nm using a plate reader.

(Ill) Antimicrobial activity of synthetic myxinidin against methicillin-resistant Staphylococcus aureus (MRSA; strain C623) by broth dilution method

Microbial Culture

The MRSA (C623) culture was obtained from a culture collection at NRC- 1MB. The pathogen was grown at 37° C in Mueller-Hinton (MH) broth (Difco Laboratories Inc., Detroit, MI).

Antimicrobial Assay

The antimicrobial activity was studied using the broth microdilution method as described previously (Subramanian et al., 2008). Briefly, 50 μL of synthetic myxinidin (known concentration) was diluted serially two-fold with 50 μL of MH broth in a 96-well polypropylene microtitre plate (Costar, Corning). The assay was carried out in triplicate. The MRSA was grown overnight to mid-logarithmic phase and diluted to give a final cell density of 2 x 10 ⁴ CFU/mL. Fifty micro litres of diluted bacterial culture was added to the wells containing synthetic myxinidin. The plate was then incubated overnight at 37° C. The controls were assayed in parallel with the sample in the same microtitre plate. For a control, 50 μL of bacterial cells in MH broth was incubated. The bactericidal or antimicrobial activity was determined by visual inspection (clear well contents) and then confirmed by streaking an aliquot of the well contents on MH agar plates.

RESULTS

In previous experiments, the inventors of the present invention observed that the acidic epidermal mucus extract of hagfish had antimicrobial activity against several fish and human pathogens and the crude acidic epidermal mucus extract showed proteins predominantly of molecular mass below 20 kDa (Subramanian et al., 2008). To investigate the active component(s) responsible for the observed activity, a larger volume of hagfish epidermal mucus was collected, acid extracted, partially purified by Sep-Pak

Cl 8 cartridges and the activity was tested against E. coli D31 and S. enterica C610. The

Sep-Pak C18 eluates exhibited antimicrobial activity at 6.1 and 8.3 μg protein/mL against E. coli D31 and S. enterica C610, respectively.

Bioassay-Guided Fractionation of Hagfish Epidermal Mucus

The acidic hagfish epidermal mucus extract that resulted from the Sep-Pak cleanup was initially fractionated using an FPLC equipped with a size-exclusion Superdex™ peptide column. Six major fractions (Fl to F6; Figure 2) were assayed for antimicrobial activity. The highest level of activity against E. coli D31 was exhibited by fraction F3 (eluted between 16.5 and 21.5 min) which was further fractionated using semipreparative Cl 8 RP-HPLC. Tricine SDS-PAGE analysis showed that fraction F3 had strong bands below 6.0 kDa and a few less intense bands between 6.0 and 14.0 kDa (Figure 4, lane 2). Antimicrobial activity of the 14 fractions collected from the fractionation of F3 showed that fraction FA (eluted between 27 and 31 min corresponding to 58—63% ACN; Figure 3) exhibited the highest level of antimicrobial activity against E. coli D31. This fraction contained low molecular mass bands of approximately 6.0 and 1.4 kDa, respectively, when examined by tricine SDS-PAGE (Figure 4, lane 3). Fractionation of FA using

analytical Cl 8 RP-HPLC with a shallow gradient (Figure 5) resulted in the collection of fraction H6 [eluted at 21.5 min (24.2% ACN); 600 μg protein/mL] that exhibited a high level of microbicidal activity. Reinjection of H6 on the same column (analytical Cl 8 RP- HPLC) resulted in a chromatogram containing a single peak. Electrophoretic analysis of H6 revealed that it contained a single band corresponding to a molecular mass of approximately 1.4 kDa (Figure 4, lane 4).

Primary Structure and Sequence Determination

The molecular mass analysis of H6 using a Q Trap® LC-ESI/ MS showed molecular ion at a m/z of 1,327.68 (M+H) ⁺ (Figure 6). The molecular mass and the chromatographic properties suggested that this compound was a peptide. The molecular mass of H6 agreed with the molecular mass that was observed on a tricine SDS-PAGE gel.

Edman degradation of H6 revealed that it was a peptide of 12 amino acids with the following sequence from the N terminus, GLY-ILE-HIS-X-ILE-LEU-LYS-TYR- GLY-LYS-PRO-SER, where X was either aspartic acid (ASP) or histidine (HIS). The average mass of the Edman sequence with X replaced by HIS or ASP gave theoretical masses of 1,349 and 1,328 Da, respectively. The theoretical mass of the ASP containing peptide was in best agreement with the observed mass spectrometry (MS) mass. To confirm the assignment of ASP at position 4, H6 was subjected to de novo sequence analysis using Q Trap® LC-ESI/MS/MS. The molecular mass differences between the resulting fragment ions revealed that the molecular mass differences between ions 423.24 (b4) and 308.16 (b3) correlates with the molecular mass of ASP (115.08 Da; Figure 7). No b4 peak was observed at 445.21 which would have indicated the presence of a HIS

(137.05 Da) at position 4. Similarly, the molecular mass differences of y4 and y5 (115.2 Da) ions indicated the presence of ASP (Figure 7). The identity of the amino acid at position 4 of the sequence of H6 was, therefore, confirmed to be an ASP. The ESI/MS/MS de novo sequence analysis confirmed the identity of other amino acids in H6. Searches for amino acid sequence homology using the basic local alignment search tool (BLAST) against Swissprot, NCBInr and AMP databases showed no significant sequence identity with the existing data. The highest sequence similarity of (41.2%) was observed for the insect-derived AMPs bombolitin II and III (Argiolas and Pisano, 1985). The antimicrobial peptide represented by H6 was thus determined to be novel and was assigned the name myxinidin. Further sequence analysis showed the myxinidin was cationic (+2), composed of three positively charged (1 HIS and 2 LYS) and one negatively charged amino acids (ASP) and had about 50% hydrophobic amino acid content.

Antimicrobial Activity of Myxinidin

The antimicrobial activity of isolated and synthetic myxinidin were examined against Gram-positive and Gram-negative bacteria and yeast that were either human or fish pathogens. The antimicrobial activity of myxinidin was compared with pleurocidin (NRC- 17), a synthetic AMP previously reported from winter flounder. The minimal concentration of the peptide that resulted in no viable growth was taken as the minimal bactericidal concentrations (MBC). Results showed that isolated and synthetic myxinidin samples were equally active against all the screened pathogens (Table 1). Most notably, S. enterica C610, E. coli D31 and fish pathogens (A. salmonicida A449, Y. ruckeri 96-4 and L. anguillarum 02-11), were found to be highly sensitive to myxinidin at MBCs of 1-2.5

μg/mL. Myxinidin was found to be less effective against S. epidermis C621 and P. aeruginosa K799 with an MBC of 10 μg/mL. The MBC of myxinidin was found to be 4- 10 fold lower than pleurocidin against fish pathogens and 1.6-2.5 times lower against P. aeruginosa Z61 and K799 and C. albicans C627. The effect of NaCl on the antimicrobial property of myxinidin was determined by incubating E. coli D31 with synthetic myxinidin at different concentrations of NaCl (0.1— 0.6 M). Myxinidin actively killed bacteria up to 0.3 M NaCl. Microbial growth was inhibited by NaCl at concentration above 0.4 M.

Hemolytic Activity of Synthetic Myxinidin

The ability of synthetic myxinidin to lyse mammalian cells was determined using rabbit red blood cells. When myxinidin was incubated with red blood cells at a concentration of 1-500 μg/mL, no change in absorbance (540 nm) was observed when compared to the negative hemolysis control (PBS). The assay results showed no detectable levels of hemolytic activity for myxinidin at concentrations 50-fold higher than its antimicrobial activity.

Cytotoxicity of Synthetic Myxinidin

From the cytotoxicity of synthetic myxinidin study, the absorbance of formazan derived from MTT indicates the viability of HUVEC cells. The mean ± standard deviation (n = 3) of myxinidin treated and control cells are shown in Figure 11. In comparison to control, the myxinidin exhibited no cytotoxicity to the HUVEC cells.

Activity of Synthetic Myxinidin against Methicillin-Resistant Staphylococcus aureus (MRSA C623)

The synthetic myxinidin caused complete inhibition of MRSA growth at a MBC of 32 μg/mL The activity of myxinidin against MRSA was found to fall within the bactericidal range of a potent fish-derived antimicrobial peptide, pleurocidin (Patrzykat et al., 2008).

Antimicrobial Activity of Extruded Slime of Hagfish

The collection of crude extruded hagfish slime was successfully afforded using a mild physical disturbance of the water in the hagfish holding bucket. The crude freeze dried extruded slime was extracted with an acidic solvent, centrifuged and desalted using solid phase extraction (SPE) Sep-Pak C18 cartridges. Elution of the SPE cartridges with

80% ACN yielded an extract that predominantly contained proteins below 20 kDa as determined by Tricine SDS-PAGE (Figure 8). When assayed against S. enterica C610, the acidic extruded slime extract exhibited antimicrobial activity at a minimum bactericidal concentration (MBC) of 8 μg protein/mL (Figure 9). This level of antimicrobial activity was equivalent to that observed for the acidic extract of hagfish epidermal mucus (8.3 μg protein/mL). The acid extracted extruded slime MBC was similar or higher than that which have been observed for other fish by-products such as skate skin (5 mg/niL) (Cho et al., 2005) and the acidic epidermal mucus extracts of haddock (14 μg protein/mL), brook trout (10 μg protein/mL) and rockfish (180 μg protein/mL) (Nagashima et al., 2003; Subramanian et al., 2008). The extruded slime acidic extracts showed the presence of low molecular mass proteins/peptides and

therefore had a comparable protein profile to that of the acidic extracts of hagfish epidermal mucus (Subramanian et al., 2008).

Myxinidin Content of the Extruded Slime of Hagfish Clean-up of the acidic extruded slime extract was conducted to prepare the sample for LC/DAD and/or LC/MS analysis of the myxinidin content. The acidic extruded slime extract was first subjected to size separation using a 5 kDa cut-off centrifugal filter to remove the larger molecular weight proteins. This yielded a fraction containing the low molecular mass components visualized in Figure 8 (lane 2). Analysis of this fraction using RP-HPLC revealed a complex chromatogram with numerous peaks indicating the need for further fractionation (Figure 10). Co-injection of synthetic myxinidin with the low molecular weight subfraction of the extruded slime extract, showed a noticeable increase in the absorbance (mAU) intensity of a peak eluting between 27-28 min in comparison to the chromatogram of the low molecular weight subfraction (Figure 10 A and B). Semi -preparative RP-HPLC was therefore used to obtain fraction SL-I by collecting the column eluent between 27-28 min.

The presence and concentration of myxinidin in SL-I was determined using a synthetic myxinidin standard and LC/ESI/MS in the selected ion monitoring (SIM) mode. The precursor ion (1327.68) and the doubly (664.84) and triply (443.56) charged fragment ions of myxinidin were identified in the MS data for SL-I for a peak with the same retention time as the myxinidin standard (20.1 min). The presence of myxinidin in the extruded slime extract subfraction SL-I was therefore confirmed. The MS spectrum of SL-I also showed the presence of another peak at 22.3 min that potentially has structural similarities to myxinidin.

A Standard curve, prepared using synthetic myxinidin, was used to determine the level of myxinidin present in SL-I. Both the standard and SL-I were injected in triplicate and linear regression analysis was performed using the average peak areas of each standard versus its concentration. The concentration of myxinidin in the dried crude extruded slime was determined to be 0.37 mg/g. The total protein concentration of the extruded slime is estimated to be 0.29 g/g of dried crude extruded slime indicating that myxinidin contributes 0.13% of the total extruded slime protein. Extruded slime acid extract showed activity at a MBC of 8 μg protein/niL against S. enterica C610, while the synthetic myxinidin MBC was 2.5 μg/mL against the same pathogen. Hence myxinidin in the extruded slime acid extract would not be expected to contribute significantly to the overall antimicrobial activity. This suggests that the acidic extract of the extruded slime contains other antimicrobial components. However, myxinidin may function in synergy with these other agents. The HPLC chromatograms of the extruded slime showed a number of unidentified components (Figure 10) indicating the presence of other components that may have promising antimicrobial activity.

DISCUSSIONS

Fish have adapted to survive in pathogen-rich aquatic environment. Their primary protection against invading pathogens is the epidermal mucus which contains a variety of antimicrobial components such as AMPs, lysozyme, proteases, and lectins (Ellis 2001). The antimicrobial property of epidermal mucus against infectious pathogens has been demonstrated previously in rainbow trout (O. mykiss) (Austin and Mclntosh 1988), ayu (Plecoglossu altivelis), turbot (Scophthalmus maximus), and carp (Cyprinus carpio) (Kanno et al. 1989; Fouz et al. 1990; Lemaϊtre et al. 1996). Increased expression of one or

more of the above-mentioned antimicrobial components in fish epidermal mucus has been observed following microbial stress (Aranishi and Mano 2000; Patrzykat et al. 2001), thus supporting the role of epidermal mucus in protecting fish from infectious pathogens.

AMPs were found to be important innate defense components in the epidermal mucosal layer of Moses sole fish (P. marmoratus), winter flounder (P. americanus), catfish (P. asotus), Atlantic halibut (H. hippoglossus), rainbow trout (O. mykiss), and Atlantic cod (G. morhuά). The present study describes the bioassay-guided fractionation and characterization of the AMP, myxinidin, extracted from the acidic epidermal mucus of hagfish. Myxinidin was isolated from the crude extract by FPLC size-exclusion and RP-HPLC chromatographies and characterized using tricine SDS-PAGE and MS. The molecular mass of the isolated myxinidin (1,327.68 Da) agreed with the theoretical mass of the myxinidin amino acid sequence obtained from an Edman degradation and tricine SDS-PAGE analysis. The myxinidin molecular mass was lower than that observed for AMPs previously isolated from the intestinal tissues of Atlantic hagfish, M. glutinosa (3,551.9, 4,564.0, and 4643.3 Da) (Shinnar et al. 1996) and from the epidermal mucus of other fish species such as winter flounder (2,711.0 Da), catfish (2,000.4 Da), and Atlantic halibut (5,459.0 Da) (Cole et al. 1997; Park et al. 1998; Birkemo et al. 2003). The AMPs from hagfish intestinal tissue (HFIAP 1, 2, and 3) contained bromotryptophan residues, but this chemical feature was not observed in myxinidin. The amino acid sequence of myxinidin showed no similarity to protein/ peptides in the SwissProt, NCBI, and antimicrobial peptide databases. Definitive confirmation of the proposed structure was achieved through solid-phase synthesis and MS/MS fragmentation.

Synthetically prepared myxinidin showed identical antimicrobial activity to myxinidin isolated from hagfish epidermal mucus. Myxinidin exhibited antimicrobial

activity towards gram-positive and gram-negative bacteria and one yeast strain. The MBCs of myxinidin were found to be in the range of 1-10 μg/mL. Fish pathogens including A. salmonicida A449, L. anguillarum 02-11, and Y. ruckeri 96-4 were highly sensitive to myxinidin. When compared, the myxinidin was found to be two to 16 times more active than pleurocidin and other AMPs derived from hagfish (Shinnar et al. 1996; Kjuul et al. 1999; Hwang et al. 1999). Myxinidin also showed bactericidal activity against a highly resistant human pathogen, MRSA C623. This suggests that myxinidin may have a significant role not only in hagfish defense against pathogenic infection but also in human therapeutic development. The mode of action of myxinidin is yet to be determined but studies have proposed various killing mechanisms for fish-derived AMPs such as cytoplasmic membrane disruption, pore/channel formation (Syvitski et al. 2005), and inhibition of cell wall and nucleic acid synthesis (Patrzykat et al. 2002; Brogden 2005).

Pleurocidin, piscidin, and histone H2B-derived peptide of cod epidermal mucus exhibit antimicrobial activity at varying salt concentrations (Cole et al. 1997; Noga and Silphaduang 2003; Bergsson et al. 2005). Oncorhyncin III from the epidermal mucus of an anadromous rainbow trout showed poor activity at higher salt concentrations (Fernandes et al. 2003). This suggests that the habitat of the fish species may correlate with the ability of the fish-derived AMPs to maintain their antimicrobial activity in saline environments. Epithelial AMPs from mammalian origin such as human /?-defensins (hBD-1) often appear sensitive to high salt (NaCl) conditions associated with diseases such as cystic fibrosis, which thereby allows the colonization of bacterial pathogens in the lungs (Goldman et al. 1997). In contrast to mammalian AMPs, a number of fish-derived AMPs including myxinidin are active at salt (NaCl) concentrations well above the physiological saline concentration.

AMPs such as melittin and pardaxin are cytotoxic to mammalian cell membranes (Habermann 1972; Lazarovici et al. 1986) thus limiting their use as therapeutics. When tested in rabbit red blood cells and HUVEC cells, myxinidin showed no cytotoxic activity even at peptide concentrations up to 50-fold higher than its MBC. The potency, specificity to bacterial cells, and preservation of antimicrobial activity of myxinidin at high salt concentrations suggests it may have potential uses in human health-related applications. Thus, in a preferred embodiment, one or more pharmaceutically acceptable carriers may be added to the AMPs of the present invention for use to treat human and fish pathogens, respiratory infections in cystic fibrosis and for promoting wound healing. In conclusion, the present invention describes the identification of a novel AMP, myxinidin, from the epidermal mucus of hagfish (M. glutinosa). As scavengers, hagfish encounter various pathogenic microorganisms in dead and decaying organisms. The secretion of myxinidin in the epidermal mucus suggests that myxinidin has a role in the mucosal innate immunity of hagfish. Myxinidin exhibited a broad spectrum of antimicrobial activity against a wide range of pathogens with no cytotoxic activity to mammalian red blood and HUVEC cells and the bactericidal activity found was retained at high NaCl concentrations. These properties of myxinidin suggest that it may be beneficial in aquaculture and human health-related applications. Further studies on the mechanism of antimicrobial action and therapeutic applications of myxinidin are in progress. The present invention focused on identifying a constitutively secreted AMP in the epidermal mucus of hagfish but further studies are underway to identify other AMPs present in hagfish epidermal mucus and the investigation of value-added uses of the hagfish by-products of this fishing industry.

The present invention also showed that the extruded slime of hagfish has promising and potentially economically important antimicrobial activity. The presence of the antimicrobial peptide myxinidin in hagfish extruded slime was confirmed and quantitated. The possible presence of other antimicrobial components that need to be further investigated to ascertain if the extruded slime acid extract could be used as a source of antimicrobial peptides for commercial use.

The present invention also demonstrated that a synthetically derived peptide having the amino acid sequence of hagfish myxinidin has identical antimicrobial and toxicological properties as myxinidin derived from hagfish mucus and slime. It is to be understood that the embodiments and variations shown and described herein are merely illustrative of the principles of this invention and that various modifications may be implemented by those skilled in the art without departing from the scope and spirit of the invention.

References:

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389-3402

Aranishi F, Mano N (2000) Response of skin cathepsins to infection of Edwardsiella tarda in Japanese flounder. Fish Sci 66:169—170 Argiolas A, Pisano JJ (1985)

Bombolitins, a new class of mast cell degranulating peptides from the venom of the bumblebee {Megabombus pennsylvanicus). J Biol Chem 260:1437-1444

Austin B, Mclntosh D (1988) Natural antibacterial compounds on the surface of rainbow trout, Salmo gairdneri Richardson. J Fish Dis 11 :275— 277

Bardack D (1998) Relationships of living and fossil hagfishes. In: Jorgensen JM, Lomholt JP, Weber RE, Malte H (eds) The Biology of Hagfishes. Chapman and Hall, London, U.K., pp 3-14

Befus AD, Mowat C, Gilchrist M, Hu J, Solomon S, Bateman A (1999) Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action. J Immunol 163:947-953

Bergsson G, Agerberth B, Jδrnvall H, Gudmundsson GH (2005) Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua). FEBS J 272:4960-4969

Bignami GS (1993) A rapid and sensitive hemolysis neutralization assay for palytoxin. Toxicon 31:817-820

Birkemo GA, Luders T, Anderson O, Nes IF, Nissen-Meyyer J (2003)

Hipposin, a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.). Biochim Biophys Acta 1646:207-215

Blum H, Beier H, Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8:93-99

Boman HG, Nilsson-Faye I, Paul K, Rasmuson T Jr (1974) Characteristics of an inducible cell-free antibacterial reaction in hemolymph of Samia Cynthia pupae. Infect Immun 10:136-145

Boulanger N, Munks RJL, Hamilton JV, Vovelle F, Brun R, Lehane MJ, Bulet P (2002) A novel antimicoribal peptide with antiparasitic activity in the blood-sucking insect Stomoxys calcitrans. J Biol Chem 277:49921-49926

Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of proteins using the principle of protein-dye binding. Anal Biochem 72:248—

254

Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev 3:238-250 Cole AM, Weis P, Diamond G (1997) Isolation and characterization of pleurocidin, an antimicrobial peptides in the skin secretions of winter flounder. J Biol Chem 272:12008-12013

Ellis AE (2001) Innate host defense mechanisms of fish against viruses and bacteria. Dev Comp Immunol 25:827-839

Fernandes JMO, Molle G, Kemp GD, Smith VJ (2004) Isolation and characterization of Oncorhyncin II, a histone Hl -derived antimicrobial peptide from skin secretions of rainbow trout, Oncorhynchus mykiss. Dev Comp Immunol 28:127-138

Fernandes JMO, Saint N, Kemp GD, Smith VJ (2003) Oncorhyncin III: a potent antimicrobial peptide derived from the non-histone chromosomal protein H6 of rainbow trout, Oncorhynchus mykiss. Biochem J 373:621—628

Fields PI, Groisman EA, Heffron F (1989) A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells. Science 243:1059-1062

Fouz B, Devesa S, Gravningen K, Barja JL, Toranzo AE (1990) Antibacterial action of the mucus of turbot. Bull Eur Assoc Fish Pathol 10:56-59

Goldman MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M, Wilson JM (1997) Human /?-defensin-l is a salt-sensitive antibiotic in ling that is inactivated in cystic fibrosis. Cell 88:553-560

Habermann E (1972) Bee and wasp venoms. Science 177:314- 322

Hancock REW (1997) Peptide antibiotics. Lancet 349:418-422

Hancock REW (2001) Cationic peptides: effectors in innate immunity and novel antimicrobials. Lancet Infect Dis 1:156-164

Hwang EY, Seo JK, Kim CH, Go HJ, Kim EJ, Chung JK, Ryu HS, Park NG (1999) Purification and characterization of a novel antimicrobial peptide from the skin of the hagfish, Eptatretus burgeri. J Food Sci 4:28-32

Kanno T, Nakai T, Muroga K (1989) Mode of transmission of vibriosis among ayu, Plecoglossus altivelis. J Aquat Anim Health 1 :2— 6

Kennedy J, Baker P, Piper C, Cotter PD,WalshM,MooijMJ, Bourke MB, Rea MC, O'Connor PM, Ross RP, Hill C, O'Gara F, Marchesi JR, Dobson ADW (2008) Isolation and analysis of bacteria with antimicrobial activitites from marine sponge Haliclona simulans collected from Irish waters. Mar Biotechnol. doi:10.1007/sl0126- 008-9154-1

Kimbrell DA, Beutler B (2001) The evolution and genetics of innate immunity. Nat Rev Genet 2:256-267

Kjuul AK, Bϋllesbach EE, Espelid S,Dunham R, Jørgensen T0,WarrGW, Styrvold OB (1999) Effects of cecropin peptides on bacteria pathogenic to fish. J Fish Dis 22:387-394

Lazarovici P, Primor N, Loew LM (1986) Purification and poreforming activity of two hydrophobic polypeptides from the secretion of the Red Sea Moses sole (Pardachirus marmoratus). J Biol Chem 261:16704-16713

Lemaϊtre C, Orange N, Saglio P, Saint N, Gagnon J, Molle G (1996) Characterization and ion channel activities of novel antibacterial proteins from the skin mucosa of carp (Cyprinus carpiό). Eur J Biochem 240:143-149

Lϋders T, Birkemo GA, Meyer JN, Andersen 0, Nes IF (2005) Proline conformation- dependent antimicrobial activity of a proline-rich histone Hl N-terminal peptide fragment isolated from the skin mucus of Atlantic salmon. Antimicrob Agents Chemother 49:2399-2406

Ming L, Xiaoling P, Yan L, LiIi W, Qi W, Xiyong Y, Boyao W, Ning H (2007) Purification of antimicrobial factors from human cervical mucus. Hum Reprod 22:1810— 1815

Nagashima Y, Kikuchi N, Shimakura K, Shiomi K (2003) Purification and characterization of an antibacterial factor in the skin secretion of rock fish Sebastes schlegeli. Comp Biochem Physiol 136C:63-71

Noga EJ, Silphaduang U (2003) Piscidins: a novel family of peptide antibiotics from fish. Drug News Perspect 16:87-92

Oren Z, Shai Y (1996) A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from Moses sole Pardachirus marmoratus. Eur J Biochem 237:303-310

Palakaha KJ, Shin GW, Kim YR, Jung TS (2008) Evaluation of non-specific immune components from the skin mucus of olive flounder {Paralichthys olivaceus). Fish Shellfish Immunol 24:479-488

Park IY, Park CB, Kim MS, Kim SC (1998) Parasin I, an antimicrobial peptide derived from histone H2A in the catfish, Parasilurus asotus. FEBS Lett 437:258-262

Patrzykat A, Friedrich CL, Zhang L, Mendoza V, Hancock REW (2002) Sublethal concentrations of pleurocidin-derived antimicrobial peptides inhibit macromolecular synthesis in Escherichia coli. Antimicrob Agents Chemother 46:605—614

Patrzykat A, Gallant JW, Seo JK, Pytyck J, Douglas SE (2003) Novel antimicrobial peptides derived from flatfish genes. Antimicrob Agents Chemother 47:2464—2470

Patrzykat A, Zhang L, Mendoza V, Iwama GK, Hancock REW (2001) Synergy of histone-derived peptides of coho salmon with lysozyme and flounder pleurocidin. Antimicrob Agents Chemother 45:1337- 1342

Pukala TL, Bowie JH, Maselli VM, Musgrave IF, Tyler MJ (2006) Hostdefence peptides from the glandular secretions of amphibians: structure and activity. Nat Prod Rep 23:368-393

Raison RL, dos Remedios NJ (1998) The hagfish immune system. In: Jorgensen JM, Lomholt JP,Weber RE, MalteH(eds) The Biology of Hagfishes. U.K.:Chapman and Hall, London, pp 334—344

Robinson WE Jr, McDougall B, Tran D, Selsted ME (1998) Anti-HIV-1 activity of indolicidin, an antimicrobial peptide from neutrophils. J Leukoc Biol 63:94-100

Rolff J (2007) Why did the acquired immune system of vertebrates evolve? Dev Comp Immunol 31 :476-482

Sarma§ik A (2002) Antimicrobial peptides: a potential therapeutic alternative for the treatment of fish diseases. Turk J Biol 26:201—207

Schagger H, Jagow GV (1987) Tricine- sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368-379

Scott MG, Rosenberger CM, Gold MR, Finlay BB, Hancock REW (2000) An α-helical cationic antimicrobial peptide selectively modulates macrophage responses to lipopolysaccharide and directly alters macrophage gene expression. J Immunol 165:3358-3365

Shephard KL (1993) Mucus on the epidermis of fish and its influence on drug delivery. Adv Drug Deliv Rev 11 :403-417

Shinnar AE, Uzzell T, Rao MN, Spooner E, Lane WS, Zasloff M (1996) New family of linear antimicrobial peptides from hagfish intestine contains bromotryptophan as novel amino acid. In: Kaumaya PTP, Hodges RS (eds) Proceedings of the fourteenth American peptide symposium. U.S.A. Mayflower Scientific Ltd., Ohio, pp 189-191

Subramanian S, MacKinnon SL, Ross NW (2007) A comparative study on innate immune parameters in the epidermal mucus of various fish species. Comp Biochem Physiol 148B:256-263

Subramanian S, Ross NW, MacKinnon SL (2008) Comparison of antimicrobial activity in the epidermal mucus extracts of fish. Comp Biochem Physiol 150B: 85-92

Syvitski R, Burton I, Mattatall NR, Douglas SE, Jakeman DL (2005) Structural characterization of the antimicrobial peptide pleurocidin from winter flounder. Biochemistry 44:7282-7293

Verbanac D, Zanetti M, Romeo D (1993) Chemotactic and proteaseinhibiting activities of antibiotic peptide precursors. FEBS Lett 317:255-258

Wang, X., Ge, J., Wang, K., Qian, J. and Zou, Y. (2006). Evaluation of MTT assay for measurement of emodin- induced cytotoxicity. Assay and Drug Develop Technol. 4: 203-

207.

Wang Z, Wang G (2004) APD: the antimicrobial peptide database. Nucleic Acids Res 32:590-592

Yano T (1996) The non-specific immune system: humoral defence. In: Iwama G, Nakanishi T (eds) The fish Immune system: Organism, Pathogen and Environment. Academic press, San Diego, U.S.A, pp 105-157