Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua) Gudmundur Bergsson 1 , Birgitta Agerberth 1 , Hans Jo ¨ rnvall 1 and Gudmundur Hrafn Gudmundsson 2 1 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden 2 Institute of Biology, University of Iceland, Reykjavı ´ k, Iceland The Atlantic cod, Gadus morhua, is widespread in the North Atlantic. It is an ectothermic, cold-water species that generally resides near the sea floor, ranging from inshore regions to deep waters. Cod supports an important commercial fishery industry but in recent times many stocks have collapsed. This has resulted in decreasing catches, leading to protection programs of the resource and higher prices for wild fish. Therefore cod has become a subject for aquaculture. Cod is in intimate contact with its environment, which is rich in both saprophytic and pathogenic microbes. For temperate fish species, such as cod, adaptive immune responses are slow and temperature dependent, e.g. antibody production for salmonids takes at least 4–6 weeks [1]. In contrast, innate immu- nity is fast acting and temperature independent [1]. This innate defence constitutes both a physical and a chemical barrier to infections and is important for fish health in an environment rich in microbes. The low infection rate of fish is remarkable and has inspired further studies of its defence system. One arm of these defences are antimicrobial proteins and peptides, which have previously been found in some fish tissues, e.g. mucus [2–6], liver [7,8] and gills [9]. The integumental secretion of fish, such as the multi- functional skin mucus [10], has been shown to play a significant role in host defence against bacteria and viruses [1,11]. Antimicrobial polypeptides have been identified as parts of the innate immunity and are widespread, both in the plant and the animal kingdom [12], e.g. the mammalian defensins and cathelicidins [13] and magainins from the skin of frogs [14]. There is a limited knowledge about the defence mechanisms of the epidermal mucus of cod. However, both consti- tutive and inducible innate defense mechanism have been suggested to be involved [1]. The aim of this study was to isolate, identify and characterize antimicrobial proteins and peptides in epi- dermal mucus from healthy cod. Increased knowledge of compounds taking part in innate defences can be of Keywords antimicrobial activity; innate immunity; fish; antimicrobial polypeptides; mucus Correspondence G. Bergsson, Department of Medical Biochemistry, Karolinska Institutet, SE-171 77 Stockholm, Sweden Fax: +46 8 337462 Tel: +46 8 524 87699 1 E-mail: bergsson@here.is (Received 22 June 2005, revised 2 August 2005, accepted 5 August 2005) doi:10.1111/j.1742-4658.2005.04906.x The epidermal mucus of fish species has been found to contain antimicro- bial proteins and peptides, which is of interest in regard to fish immunity. An acidic extract from the epidermal mucus of the Atlantic cod (Gadus morhua) was found to exhibit antimicrobial activity against Bacillus mega- terium, Escherichia coli and Candida albicans. This activity varied signifi- cantly when salt was added to the antimicrobial assay, and was eliminated by pepsin digestion. No lysozyme activity was detected in the extract. By using weak cationic exchange chromatography together with reversed-phase chromatography, and monitoring the antimicrobial activity, we have iso- lated four cationic proteins from the mucus extract. Using N-terminal and C-terminal amino acid sequence analysis, together with MS, the antimicro- bial proteins were identified as histone H2B (13 565 Da), ribosomal protein L40 (6397 Da), ribosomal protein L36A (12 340 Da) and ribosomal protein L35 (14 215 Da). The broad spectra of antimicrobial activities in the cod mucus and the characterization of four antimicrobial polypeptides suggest that mucus compounds contribute to the innate host defence of cod. Abbreviations HFBA, heptafluorobutyric acid; RP, reversed phase; WCEX, weak cationic exchange. 4960 FEBS Journal 272 (2005) 4960–4969 ª 2005 FEBS great importance, both as a means for using com- pounds of the innate immunity in aquaculture of cod and for anti-infective agents in animals. Results Antimicrobial activity of the cod mucus extract The mucus extract, comprising approximately 42% protein, was assayed for antimicrobial activity against Bacillus megaterium, Escherichia coli and Candida albi- cans. The 80% acetonitrile OASIS eluates (30 lg) caused a zone inhibition with a diameter of 0–16.4 mm, depending on the salt concentration and the microbe tested (Fig. 1), while the 100% acetonitrile OASIS eluates exhibited no activity. Without addition of medium E to the agarose, the Gram-positive bacter- ium B. megaterium was the most sensitive to the mucus extract (Fig. 1). With medium E added, B. megaterium and the Gram-negative bacterium E. coli were found equally sensitive to the extract, while the activity against the fungus C. albicans was fully eliminated (Fig. 1). In contrast to C. albicans, the extract showed significantly greater (P<0.01) activity against E. coli when medium E was added to the assay (Fig. 1). No difference was noted in activity against B. megaterium with or without medium E. The sensitivity of the microbes was further studied by measuring growth inhibition when incubated in serial dilutions of the 80% OASIS eluates at different concen- trations of NaCl (Table 1). Bacillus megaterium was found to be the most sensitive at all concentrations of NaCl but sensitivity was reduced with increased concen- trations of NaCl. The extract showed intermediate effect, minimum inhibitory concentration of 1.25–0.625 gÆl )1 , against C. albicans at 0 mm NaCl but none at 125 mm or higher concentration of NaCl. In contrast to both B. megaterium and C. albicans, the inhibition of the extract against E. coli was increased with increased concentration of NaCl showing maximum inhibition at 500 mm NaCl. No bacterial growth was observed for B. megaterium at the highest NaCl concentration tested (2000 mm) and for E. coli at 2000 and 1000 mm. Growth of C. albicans was observed at all concentra- tions of NaCl. After incubation of the extracts with pepsin, the antibacterial activity of the extract was greatly reduced when assayed without medium E, and fully eliminated when medium E was added to the agarose. No lyso- zyme activity was detected (data not shown). Identification of antimicrobial proteins from cod mucus extract The extract was fractionated by semipreparative weak cationic exchange (WCEX)-HPLC (Figs 2A and 3A). Fig. 1. Antimicrobial activity of 30 lg protein ⁄ peptide extract from the epidermal mucus of cod against C. albicans, B. megaterium and E. coli, as measured by an inhibition zone assay. The activity was tested with and without medium E in the agarose. Each meas- urement is the average of at least three experiments. The bars indi- cate 99% comparison intervals by the GT2 method for the means of the activity data. Controls were 3 lg of LL-37 for B. megaterium and E. coli, and 3 lg of nystatin for C. albicans. *No activity was recorded against C. albicans when medium E was added to the agarose. Table 1. Inhibitory concentrations (gÆL )1 ) of mucus extract that inhibits the growth of microbes. The results of two independent experiments are shown. NA, Not applicable; >, the highest concentration tested caused no inhibition. NaCl (mM) B. megaterium E. coli C. albicans Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 0 0.0195–0.0098 0.039–0.0195 > 10 > 10 1.25–0.625 1.25–0.625 125 0.039–0.0195 0.039–0.0195 > 10 10–5 > 5 > 10 250 0.039–0.0195 0.039–0.0195 > 10 5–2.5 > 5 > 10 500 > 1.56 0.3125–0.156 1.25–0.625 5–2.5 > 5 > 10 1000 > 1.56 0.3125–0.156 NA NA > 5 > 10 2000 NA NA NA NA > 5 > 10 G. Bergsson et al. Antimicrobial components of cod mucus FEBS Journal 272 (2005) 4960–4969 ª 2005 FEBS 4961 Fractions containing antimicrobial activity were fur- ther purified by two steps of RP-HPLC, using 0.1% trifluoracetic acid (TFA) as a counter ion in the first step (Figs 2B and 3B,D) and 0.1% heptafluorobutyric acid (HFBA) in the second (Figs 2C and 3C,E,F). Fractions collected from the HPLC runs were assayed for antibacterial activity against E. coli and ⁄ or B. megaterium, with medium E included in the assays. A component, active against E. coli, eluted in 1.0 m ammonium acetate upon WCEX-HPLC (fraction 79 in Fig. 2A). This component further eluted at 52% acetonitrile in the first RP-HPLC step (fraction 35 in Fig. 2B), and purified to apparent homogeneity at 52% acetonitrile in the last step (fractions 58 and 59 in Fig. 2C). SDS ⁄ PAGE of fractions 58 and 59 revealed a protein band with a mobility corresponding to a molecular weight close to 13.5 kDa. This component was identified as histone H2B by N- and C-terminal sequence analyses (Table 1). Analysis by MALDI-MS showed the mass to be 13565 Da, which is similar to that of histone H2B from other Actinopterygii species (ray-finned fish) (Table 2) [15–17]. A component eluting at 0.82 m ammonium acetate (fraction 50 in Fig. 3A) was active against both E. coli and B. megaterium. This component was purified by elution at 37% acetonitrile in the initial RP-HPLC step (fraction 25 in Fig. 3B) and at 42% in the final step (fraction 48 in Fig. 3C). Edman degradation and mass determination by MALDI-MS identified this component as 60S ribosomal protein L40 with a molecular mass of 6397 Da. This protein was identified as ribosomal protein L40 by high similarity to ribo- somal protein L40 from Ictaluridae punctatus [18], the ribosomal protein L40 family domain from Oncorhyn- chus mykiss, Pagrus major and Sebastes schlegli, and an unnamed product from Tetraodon nigroviridis (Table 2). Further, in the other Actinopterygii this ribosomal protein is synthesized as carboxyl extensions with ubiquitin (Table 2). This has been observed in other species where ubiquitin and ribosomal protein L40 are frequently produced by genes that encode a fusion protein consisting of ubiquitin at the N termi- nus and ribosomal protein L40 at the C terminus [19]. A fraction eluting at 0.96 m ammonium acetate from WCEX-HPLC (fraction 60 in Fig. 3A) with antimicro- bial activity against both B. megaterium and E. coli was further purified. In the first reversed phase (RP)- HPLC step (fraction 25 in Fig. 3D), this component eluted at 38% acetonitrile, and in the second step at 49% (fraction 47 in Fig. 3E). N-terminal sequence analysis for 13 residues identified this component as the 60S ribosomal protein L36A (Table 2). The mass of the protein was 12 340 Da as measured by MALDI-MS, which is in a good agreement with the same protein in other fish species of Actinopterygii (Table 2) [18]. Fig. 2. Purification of an antimicrobial com- ponent from 12 mg protein ⁄ peptide extract, prepared from skin mucus of cod, by use of HPLC. The antimicrobial activity was monit- ored against E. coli including medium E in the agarose. The height of columns repre- sents the magnitude of antimicrobial activity and can be read on the right Y axis scale in mm. The initial step was performed utilizing WCEX chromatography and the fractions were dissolved in 100 lL 0.1% TFA before the antimicrobial activity was analysed against E. coli (A). The material in fraction number 79 (A) indicated by an arrow was loaded onto an RP column using 0.1% TFA as a counter ion (B). The antimicrobial com- ponent in fraction 35 of panel (B) was puri- fied by loading the fraction onto an RP column using 0.1% HFBA as a counter ion. The active component was identified as his- tone protein H2B in fractions number 58 and 59 (C). Antimicrobial components of cod mucus G. Bergsson et al. 4962 FEBS Journal 272 (2005) 4960–4969 ª 2005 FEBS An additional antibacterial polypeptide was identi- fied in fraction 60 of the WCEX fractionation (Fig. 3A) after two additional steps of RP-HPLC. In the first RP-HPLC step this polypeptide eluted at 46% acetonitrile (Fraction 31 in Fig. 3D) and in the second at 50% (fractions 67 and 68 in Fig. 3F). This polypep- A B CE F D Fig. 3. Purification of antimicrobial compo- nents from 20 mg protein ⁄ peptide extract, prepared from the skin mucus of cod, by use of HPLC and monitoring the antibacte- rial activity. The initial fractionation was per- formed utilizing WCEX chromatography, where each fraction was dissolved in 150 lL of 0.1% TFA, and tested against both E. coli and B. megaterium (A). Fraction 50 (A) indicated by an arrow was loaded onto a RP column using 0.1% TFA as a counter ion and fractions were dissolved in 50 lL (B). The active component was then purified and identified as a 60S ribosomal protein L40 by loading fraction 25 onto a RP chromatography column, utilizing 0.1% HFBA as a counter ion (C). Fraction 60 in the WCEX chromatography (A) was further purified using RP-HPLC utilizing 0.1% TFA as a counter ion (D). Two microbicidal com- ponents were identified in fractions 25 and 31 (D) by one additional RP chromatography using 0.1% HFBA as a counter ion (E and F, respectively). 60S ribosomal protein L36A was identified in fraction 47 (E) and 60S ribosomal protein L35 in fraction 67 and 68 (F). G. Bergsson et al. Antimicrobial components of cod mucus FEBS Journal 272 (2005) 4960–4969 ª 2005 FEBS 4963 tide was active against B. megaterium, and a single molecular band corresponding to 14.4 kDa was detec- ted by SDS ⁄ PAGE. N-terminal sequence analysis for 30 residues and C-terminal analysis for four residues showed it to be identical or highly similar to the 60S ribosomal protein L35 (Table 2) [18,20]. Analysis of the material in this fraction by ESI-MS revealed a mass of 14 215 Da, which is similar to that of ribo- somal proteins L35 from other ray-finned fish species (Table 2). Discussion Fish live in intimate contact with their aqueous envi- ronment, which is densely populated with microorgan- isms. The protective role of the epidermal mucus of fish has been known for many years [1,10], indicating a source for isolation of antimicrobial components. The aim of the present study was to identify antimicro- bial components from the skin mucus of healthy Atlantic cod (Gadus morhua). The mucus extract collected from the skin exhibited high antimicrobial activity against Gram-positive and Gram-negative bacteria, as well as against the yeast C. albicans. As seen in Fig. 1 and Table 1, the anti- Candida activity was fully inhibited when the salt concentration was increased in the assay by addition of medium E or NaCl. In contrast, the activity against E. coli increased significantly in both antimi- crobial assays with elevated salt concentration. This suggests that the antimicrobial components are salt dependent, and might be affected by the levels of salt in seawater. Medium E and NaCl are known to enhance the antimicrobial activity of a-helical pep- tides [21]. This suggests that salt-dependent a-helical peptides, participating in the activity against E. coli are active at the salt levels present in seawater, which is close to 3.5% (w ⁄ v). However, the activity against C. albicans is salt sensitive as the antifungal activity was abolished when both medium E and NaCl were added. The increased concentrations needed to inhibit growth of B. megaterium at increased NaCl concen- trations (Table 1) can be explained by the cations interfering with the electrostatic interaction of the positively charged components found in the mucus and the negatively charged microbial surface. The fact that mucus components are found to be active against both Gram-positive and Gram-negative bac- teria in a condition that is likely to mimic the natural environment of cod further supports the role of the mucus as a defence barrier. Because we observed an abolition of the extract activities by pepsin treatment we concluded that the activities are of protein ⁄ peptide origin. We identified four evolutionarily conserved [22,23], cationic, bactericidal polypeptides from the skin mucus of cod, i.e. histone H2B and three 60S ribosomal pro- teins, L40, L36A and L35. As seen (Figs 2A and 3A) by the number of antimicrobial fractions there are numerous unidentified antimicrobial components in cod mucus. Predictably, due to the appearance of low molecular weight peptides ⁄ polypeptides in SDS ⁄ PAGE of many of the active fractions (data not shown), some of those are low molecular weight antimicrobial pep- tides similar to those previously identified in other organisms [12], including fish [5]. Therefore, the frac- tions used for isolation of antimicrobial peptides⁄ poly- peptides were selected both according to the intensity of their antimicrobial activity as well as their pep- tide ⁄ polypeptide composition. Fractions containing a Table 2. Homology of isolated antimicrobial polypeptides with proteins from other Teleostei species. The degree of conservation of observed sequences is expressed as identical amino acids in all sequences in the alignment (*); conserved substitutions (:); and semicon- served substitutions (.). ND, not determined. Identified protein N-terminal sequence C-terminal sequence Molecular mass (Da) Histone H2B protein a PEVAKPAAKKGSKKAVSKVA. SK 13 565 *: **.*.********:* * ** Ribosomal protein L40 b IIEPSLRMLAQKYNCDKMIXRXXYARLHPR. . . ND 6397 ******* *******:*** * ******* ND Ribosomal protein L36A c VNVPKTRRTYCKK. ND 12 340 ************* ND Ribosomal protein L35 d AKIKARDLRGKKKEELLKQLDDLKNELSQL. . . AVKA 14 215 ******************::**** ***** **** By alignment with: a S. trutta13464.59 Da (HSSB22), O. mykiss 13595.79 Da (CAA26673), I. punctatus13495 Da (P81903); b T. nigroviridis 61198.57 Da (CAG00768.1), O. mykiss 6195.51 Da (BAA88568.1), I. punctatus 6209.54 Da (AAK95168.1), P. major 6195.51 (AAP20221.1), S. schlegli 4448.37 Da (AAV68176); c D. rerio 12396.75 Da (NP_775369.1), I. punctatus 12396.75 Da (AAK95164.1), T. rubripes 12396.75 Da (CAC44627.1), P. flesus 12527.94 Da (CAE53391.1); d T. nigroviridis 14460.41 Da (CAF90126), H. comes 14444.53 Da (AAQ63320), D. rerio 14421.47 Da (NP_775340), I. punctatus 14432.41 Da (AAK95161). Antimicrobial components of cod mucus G. Bergsson et al. 4964 FEBS Journal 272 (2005) 4960–4969 ª 2005 FEBS large proportion of smaller peptides were picked before the fractions containing mainly large peptides. The difficulty in isolating antimicrobial peptides ⁄ poly- peptides may be due to several factors, e.g. pH or salt concentration in the antimicrobial assays and the decrease of activity as a result of interactions. Another possible reason is the low levels of antimicrobial pep- tides from healthy cod where bacterial challenge prior to sample collection might induce peptide expression to levels where isolation becomes more plausible. In addition, several antimicrobial factors are known only to exhibit activity by interacting together with other factors in the same tissue. These activity interactions are easily lost during the purification procedure where interacting components are separated. These reasons can also explain the apparent absence of lysozyme activity which is a significant contribution to host defence of other aquatic organisms [24]. Histones are small, abundant basic proteins most commonly found in association with DNA in the chro- matin of eukaryotes. Four histones, H2A, H2B, H3 and H4 are important for chromosome organization in the nucleosome. Previous studies have suggested that histones have additional functions, including hormone activity [25], activation of leucocytes in salmon [26] and as part of the antimicrobial defence in mammals [27–29]. Even if the antimicrobial effect of histones has been known for decades [30], they were just recently linked to the innate immune system of frog [31], fish [2,3,15,16,32–36] and mammals [29,37,38]. In the study by Robinette et al. [15], a histone 2B-like protein was shown to inhibit important bacterial and fungal patho- gens of fish, e.g. Aeromonas hydrophila and Saprolegnia spp. A further study of channel catfish skin suggests that the levels of histone-like proteins are suppressed during early stages of stress [39]. The same study states that histone-like protein levels in channel catfish skin are reduced in the absence of disease. In addition to their antimicrobial activity, histones have also been suggested to exhibit endotoxin-neutralizing activities in the human placenta [40]. Histone fragments with antimicrobial properties have been isolated and identified in human wound fluid together with a-defensins, lysozyme and LL-37 [41], as well as in fish tissues [2–4,42], where N-ter- minal segments of catfish H2A were shown to be induced in the epidermal mucus upon stimulation [2]. Intact histone H2B is found in an extracellular com- plex together with DNA in bovine milk and serum [43], and complexes consisting of histones, elastase and DNA are released by activated neutrophils [27] through an unknown mechanism. These complexes have been named neutrophil extracellular traps and are highly bactericidal. By using immunohistochemical analysis, it was reported that histone H1 in human ter- minal ileal mucosa is not only localized to the nucleus but also in the cytoplasm [29]. Histones H2A and H2B were also shown to be present in the cytoplasm of syncytiotrophoblasts and amnion epithelial cells. Unlike histones, many fewer reports describe antimi- crobial properties of ribosomal proteins or of frag- ments thereof. Hiemstra et al. [44] isolated a small (6654-Da) antimicrobial cationic protein from the cyto- sol of interferon (IFN)-c-activated mouse macrophag- es, designated ubiquicidin and found highly similar to ribosomal protein S30. Ubiquicidin was also isolated from human colon mucosa because of its antimicrobial activity [38]. An additional antibacterial peptide shar- ing similarity with the 40S ribosomal protein S30 was isolated from the skin of the rainbow trout [6]. Ribo- somal protein S19, also a monocyte chemoattractant [45], and ribosomal protein L30, were isolated from the human colonic epithelium [37]. Furthermore, Tollin et al. [38] isolated the ribosomal protein L39 with bac- tericidal properties from human colon mucosa. Finally, antibacterial cecropin-like peptides in Helicobacter pylori have been suggested to be derived from the ribosomal protein L1 [46,47]. Combined, all these data show that ribosomal proteins have a role in immu- nity, ascribing them to a second function, and suggest- ing that also the ribosomal proteins have multiple functions. Prominent antimicrobial activity suggests that the mucus layer of the Atlantic cod is an important tissue in surface defences of cod, and most likely protects the fish from infections caused by pathogenic microbes. We have demonstrated that the acidic extract of cod mucus contains the antimicrobial polypeptides histone H2B, and ribosomal proteins L40, L36A and L35. Experimental procedures Experimental animals and sample collection Healthy female and male cod (Gadus morhua) were grown for 3 years in an outdoor seawater aquarium at the Marine Research Institute of Iceland and 50 specimens were caught randomly. After killing the fish by a concussion of the brain by striking of the cranium 2 , mucus samples were collected by scraping the skin and then were immediately frozen on dry ice. EC guidelines were followed for all animal experiments. Extraction of proteins from cod mucus The material was extracted by shaking overnight at room temperature in 60% (v ⁄ v) acetonitrile containing 1% G. Bergsson et al. Antimicrobial components of cod mucus FEBS Journal 272 (2005) 4960–4969 ª 2005 FEBS 4965 (v ⁄ v) TFA. After centrifugation twice at 5000 g for 10 min, the supernatants were transferred into fresh Epp- endorf tubes and centrifuged again at 3600 g for 10 min. After lyophilization the material was dissolved in 0.1% (v ⁄ v) TFA and loaded onto OASIS hydrophilic-lipophilic balance cartridges (Waters, Milford, MA, USA), equili- brated in 0.1% (v ⁄ v) aqueous TFA. After loading the sample, the cartridges were washed with 0.1% (v ⁄ v) aqueous TFA, and 20% (v ⁄ v) acetonitrile in 0.1% (v ⁄ v) TFA. Bound proteins were eluted with 80%, and 100% (v ⁄ v) acetonitrile in 0.1% (v ⁄ v) TFA, and eluates were lyophilized. Protein concentrations were determined at 595 nm using a Bradford assay [48] after addition of Bio-Rad Protein Assay solution (Bio-Rad, Sundbyberg, Sweden). Microbial strains Bacillus megaterium strain (Bm11), E. coli strain (D21), and C. albicans strain (ATCC 14043) were used to analyse the antimicrobial activity in the mucus. For each antimicrobial experiment, bacterial colonies were seeded from frozen stocks and grown on Luria–Bertani (LB) agar (GibcoBRL, Life technologies, Paisley, Scotland) plates containing strep- tomycin (100 lgÆmL )1 ). Yeast cultures were prepared from frozen stocks and grown on agar plates containing YM medium (Difco laboratories, Detroit, MI, USA). The plates were incubated at 37 °C for 24 h. Colonies were picked from the agar plates and the bacteria suspended in 20 mL LB broth, or YM broth for yeast cells, and incubated at 37 °C with shaking until the desired cell density was reached (D 590 ¼ 0.6). Inhibition zone assay Agarose (1%) in LB broth with and without salt solution (medium E: 0.8 mm MgSO 4 , 9.5 mm citric acid, 57.5 mm K 2 HPO 4 , 16.7 mm NaNH 4 HPO 4 ) [49] was mixed with bacterial cultures to achieve a final density of 6 · 10 4 cellsÆmL )1 . This mixture was poured into Petri dishes to make a 1-mm layer of agarose. Wells 3 mm in diameter were punched in the agarose layer and 3 lL samples, dis- solved in 0.1% aqueous TFA, were loaded into each well. LL-37 dissolved in 0.1% aqueous TFA (1 gÆL )1 ) was used as positive and 0.1% TFA alone as negative control. The assay for C. albicans was performed in the same manner but with YM broth, and nystatin dissolved in 0.1% aqueous TFA (1 gÆL )1 ) was used as positive con- trol. After incubation overnight at 30 °C the diameters of inhibition zones were recorded. The activity of extracts, with and without medium E, was analysed by method for multiple unplanned comparisons among pairs of means (The GT2 method) [50]. The differences in activity were deemed significant when the probability was less than 0.01. Determination of the inhibitory concentration Serial twofold dilutions of mucus extract (0.024–100 gÆL )1 ) in water were prepared and 10 lL added into each well of 96-well tissue culture plates (FALCON, Becton Dickinson and Company, Franklin Lakes, NJ, USA). Addition of water alone was used as a positive control. Luria–Bertani or YM broth (45 lL), containing the appropriate NaCl concentrations (0–4.0 m), were then added to the wells. Finally, 45 lL of inoculate, i.e. E. coli and B. megaterium in LB broth or C. albicans in YM broth, containing 10 4 colony forming units were added to the mixture resulting in a final concentration of NaCl ranging from 0 to 2.0 m. Wells without bacteria were used as a negative control. The plates were incubated overnight with shaking (90 r.p.m.) at 37 °C and bacterial growth was monitored by visual inspec- tion and by measuring the change in absorbance of each well at 600 nm using a microtiterplate reader. The inhibi- tory concentrations were expressed as an interval of the highest concentration of extract at which microbes were able to grow and the lowest concentration that inhibited microbial growth completely [51]. Pepsin digestion To determine whether peptides ⁄ proteins were responsible for the antimicrobial activity, the enriched extracts were digested with pepsin. Incubation of 50 lg mucus extract was carried out in 5% formic acid for 5 h at 37 °C with 6, 8 and 10 lg pepsin (Sigma, St. Louis, MO, USA) dissolved in 5% (v ⁄ v) formic acid. The same amount of untreated extract was used as control. After incubation and lyophili- zation, the digests were redissolved in 3 lL 0.1% TFA, and assayed for antibacterial activity against B. megaterium (above), with and without medium E. Detection of lysozyme The presence of lysozyme was investigated by the inhibi- tion zone assay, where lyophilized cells (1 mgÆmL )1 )of Micrococcus lysodeikticus (ATCC no. 4698) (Sigma), were mixed with 1% agarose in LB medium. Isolation of antimicrobial polypeptides An A ¨ KTA purifier system (Amersham Pharmacia Biotech, Uppsala, Sweden) was used for HPLC. The protein extract was first fractionated by WCEX chromatography, utilizing an Ultropac TSK CM-3SW 7.5 · 150-mm (LKB-Produkter AB, Bromma, Sweden). The column was equilibrated in 0.2 m acetic acid (buffer A), and fractions were eluted with a linear gradient of 1 m or 1.5 m ammonium acetate in 0.2 m acetic acid (buffer B) at a flow rate of 1 mLÆmin )1 . The effluent was monitored at 280 nm. Two different gradi- Antimicrobial components of cod mucus G. Bergsson et al. 4966 FEBS Journal 272 (2005) 4960–4969 ª 2005 FEBS ents were utilized, one 0–50% in 50 min, then 50–100% in 5 min, the other 0–50% in 30 min then 50–100% for 50 min. Fractions containing antimicrobial material were further purified using two steps of RP-HPLC on an Vydac C18 column (5 lm; 2.1 · 150 mm, Separations Group, Hes- peria, CA, USA). In the initial step, the column was equili- brated in 0.1% (v ⁄ v) TFA, and elution was with a linear gradient of acetonitrile in 0.1% TFA at a flow rate of 0.2 mLÆmin )1 . In the second step, the column was equili- brated in 0.1% heptafluorobutyric acid (HFBA), and the gradient was linear with acetonitrile in 0.1% HFBA at 0.2 mLÆmin )1 . The fractions in HFBA were re-lyophilized in water before tests of the antibacterial activity. SDS ⁄ PAGE HPLC fractions containing antimicrobial activity were mixed 1 : 1 with loading buffer (Invitrogen, Carlsbad, CA, USA), incubated for 1 h at 56 °C and for 5 min at 95 °C, and submitted to SDS ⁄ PAGE in 10–20% Tricine gels (Invitrogen). The proteins were stained with SilverXpress (Invitrogen). MALDI MS Aliquots of HPLC fractions were mixed (1 : 1) with matrix (saturated a-cyano-4-hydroxy-cinnamic-acid in acetonitrile containing 0.1% TFA) (CAS number 28166-41-8) (Aldrich Chemical Company, Milwaukee, WI, USA) on a target plate and left to dry, before analysis by MALDI-MS in an Applied Biosystems Voyager DE-PRO instrument (Foster City, CA, USA). The mass scale of the instrument was externally calibrated using calibration mixture 3 [i.e. insulin (bovine), thioredoxin (E. coli) and apomyoglobin (horse)] of the Sequazyme TM Peptide Mass Standards Kit (PE Bio- systems, Foster City, CA, USA). ES ionization MS HPLC fractions were lyophilized, redissolved in 60% aceto- nitrile, containing 1% acetic acid, and analysed using gold- coated nano-ES needles (Proxeon Biosystems A ⁄ S, Odense, Denmark) in a quadrupole time-of-flight mass spectrometer (QTOF, Waters, Milford, MA, USA) equipped with a stand- ard Z-spray ES source. The instrument was calibrated using the multiple charged ions of horse heart myoglobin, operated in the positive ion mode with a capillary voltage of 1100 V and a cone voltage of 40 eV. Data were analysed using the MassLynx 4.0 software supplied by the manufacturer. Amino acid sequence analysis For N-terminal sequence analysis, Applied Biosystems Pro- cice instruments (Foster City, CA, USA) were used. For C-terminal analyses, the Applied Biosystems 494C instru- ment was used as described [52]. Alignments and homology analyses Protein sequences obtained were aligned with homologous sequences from the National Center for Biotechnology Information databases, using blast programs and searching for short, nearly exact matches [53]. Multiple sequence alignments were performed using clustal w (1.82) [54]. Acknowledgements Mucus from cod skin surface was kindly given by Dr Matthias Oddgeirsson at the Marine Research Institute, Stað, Grindavik, Iceland. We thank Ernir Snorrason and Eirikur Steingrimsson for help with the sample collection. This work was supported by The Icelandic Research Fund for Graduate Students, The Swedish Foundation for International Cooper- ation in Research and Higher Education (STINT), The Swedish Research Council and AVS R & D Fund of Ministry of Fisheries in Iceland. References 1 Ellis AE (2001) Innate host defense mechanisms of fish against viruses and bacteria. 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