Isolation and characterisation of two antimicrobial peptides from haemocytes of the American lobster Homarus americanus

Fish & Shellfish Immunology (2008) 25, 181e187

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Isolation and characterisation of two antimicrobial peptides from haemocytes of the American lobster Homarus americanus Andrea L. Battison a,*, Rachael Summerfield a, Aleksander Patrzykat b a

Atlantic Veterinary College Lobster Science Centre, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island, C1A 4P3, Canada b NRC Institute for Marine Biosciences/Institut des biosciences marines CNRC, 1411 Oxford Street, Halifax, Nova Scotia, B3H 3Z1, Canada Received 21 December 2007; revised 31 March 2008; accepted 9 April 2008 Available online 16 April 2008

KEYWORDS Lobster; Hoa-crustin; Antimicrobial peptide; Homarus americanus; Haemocytes; Homarin

Abstract Two antimicrobial peptides from haemocytes of the American lobster, Homarus americanus H. Milne Edwards 1837, were isolated and partially characterised e the first such description for this species. CAP-2, an approximately 12 kDa peptide, contained amino acid sequences corresponding to the predicted sequence for Hoa-crustin. Crustins are whey acidic protein (WAP) domain - containing peptides isolated from crustacean haemocytes. CAP-2 did not have any activity towards the Gram positive coccus Aerococcus viridans unlike carcinin, a crustin from Carcinus maenas haemocytes, which may partially explain the lobster’s susceptibility to this bacterium. A second peptide, CAP-1, was a multimer composed of 4e6 kDa subunits with similarities to amphibian temporins. CAP-1 may represent a novel group of antimicrobial peptides for marine invertebrates and has been tentatively named ‘homarin’. Homarin had bacteriostatic activity against some Gram negative bacteria and both protozoastatic and protozoacidal activity against two cultured scuticociliate parasites Mesanophrys chesapeakensis and Anophryoides haemophila, the latter a significant pathogen of H. americanus. ª 2008 Elsevier Ltd. All rights reserved.

Introduction Investigation into the antimicrobial properties of the haemolymph of the American lobster, Homarus americanus

* Corresponding author. Tel.:þ1 902 894 2845; fax: þ1 902 894 2885. E-mail address: [email protected] (A.L. Battison).

H. Milne Edwards 1837, began in the late 1960s and 1970s [1e3]. Acton et al. [1] demonstrated increasing titres of antibacterial activity in citrated haemolymph of lobsters injected with formalin-killed bacteria. Stewart and Zwicker [3] examined the antimicrobial properties of different components of haemolymph (haemocytes, plasma, and serum) separately and in combination. They determined that serum, representing a combination of plasma and lysed haemocytes, was the most effective component. This

1050-4648/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.04.005

182 antibacterial activity was also inducible e injection of lobsters with killed bacteria caused a transient increase in activity of the plasma and serum, but not haemocyte lysates [3]. While haemocytes were integral to the antimicrobial activity of serum, the specific nature of this activity was not determined. It is possible that some of this observed activity could be attributed to antimicrobial peptides in H. americanus haemocytes. Antimicrobial peptides are small proteins, usually less than 10 kDa, present in all types of plants, animals and prokaryotes [4,5]. Antimicrobial peptides are an integral component of the innate immune system. Present in most tissues, particularly in secretions on epithelial surfaces (e.g., respiratory, enteric, genitourinary, and cutaneous) and in high concentrations in white blood cells and haemocytes, they provide an immediate defence against potential pathogens. Production of antimicrobial peptides can be constitutive or inducible [6]. Some peptides are cleavage products of larger molecules, e.g., lactoferricin from lactoferrin, buforin derived from histone H2A, and astacidin from haemocyanin [4]. Originally recognised for their ability to kill bacteria, viruses, protozoa, and some neoplastic cells, they have also been ascribed roles as modulators of the immune system, e.g., enhancement of phagocytosis, chemotactic activity [7,8]. Clearly, the scope of the physiological role, or roles, of these peptides has yet to be determined in many cases. An appealing aspect of these peptides is that they offer an alternate approach to antimicrobial therapy once economical, large scale production methods are devised [9,5,7]. In contrast to traditional antibiotics, antimicrobial peptides generally destroy the organisms before they have the opportunity to develop resistance, although mechanisms of resistance do exist [7]. Despite their diverse origins and amino acid composition, most recognised antimicrobial peptides are cationic at physiological pH and often adopt an amphipathic structure [4,5]. Antimicrobial peptides can be classified based on their amino acid content, secondary and tertiary structure, length, and presence of internal disulfide bonds [4,5]. The latter group is represented by the defensins found in vertebrates (where they are subdivided into a- and b- defensins), invertebrates (where they are grouped based on their activity), and plants [4]. Cecropins (arthropods), pleurocidin (fish), magainins (amphibians), and cathelicidins (humans) are examples of antimicrobial peptides that adopt an a- helical conformation [4e6]. While knowledge of the location, production, and secondary and tertiary structure of these peptides is increasing, there is still uncertainty regarding their exact mechanism of action in many cases [5,7]. Combining knowledge of physiological membrane composition and function with experimental systems utilising a few well-studied peptides, e.g., magainin and defensin A, mechanistic models have been proposed [5e7]. These include attachment of peptides to target membranes and subsequent destabilisation of the membrane by forming ‘pores’ or ion channels [5,7]. The peptides may also bind to intracellular proteins, directly leading to disruption of the normal metabolic pathways of the target cell [5,7]. Antimicrobial peptides are well described in the haemolymph of many marine invertebrates [4,9]. These include the tachyplesins and polyphemusins of the Chelicerata,

A.L. Battison et al. penaeidins, crustins, and callinectin in the Crustacea, defensins and mytilins in Mollusca, and styelin and clavalin in tunicates. Recently, a crustin-like peptide, Hoa-crustin, was predicted for H. americanus using a multi-tissue EST library [10]. A sequence closely matching Hoa-crustin has also been identified and studied in the European lobster Homarus gammarus [11,12]. In the present study, two antimicrobial peptides were isolated from H. americanus haemocytes. The first peptide corresponded to the predicted Hoa-crustin. The second peptide had some sequence similarities to amphibian temporins and may represent a new group of antimicrobial peptides for decapod crustaceans. In addition, the antibacterial and antiprotozoal activity of each peptide, in natural and synthetic forms, was partially characterised.

Materials and methods Haemolymph (w15 ml), from either individual lobsters or pooled samples, was centrifuged at 3500  g for 5 min at 4  C. Plasma supernatants were removed and the surface of the packed haemocyte pellets washed with TBS (10 mM Tris, 150 mM NaCl, pH 7.4) and 5 mg/ml aprotinin to remove any residual plasma. Washed haemocyte pellets were homogenized in 3 ml of cold 10% acetic acid and 5 mg/ml aprotinin on ice using a glass piston homogeniser and stirred overnight at 5  C. Lysates were centrifuged at 13,000  g for 30 min at 4  C and the supernatants applied to an Oasis HLB 3 ml reversed phase extraction cartridge (Waters Corporation, Milford, MA) previously conditioned with 3 ml of 100% methanol and washed with 3 ml of ddH2O. The flow-through sample from each cartridge was applied to a second HLB cartridge. Cartridges were washed twice with 3 ml of 5% methanol and eluted with 3 ml of 100% methanol. All flow-through, wash and eluate fractions were retained and the methanol evaporated under nitrogen gas prior to freezing at 80  C and subsequent lyophilisation (Model 75035 freeze dryer, Labconco Corporation, Kansas City, MO). Fractions were reconstituted in 500 ml of ddH2O and tested for antimicrobial activity against a variety of bacteria (Micrococcus luteus ATCC 4698 (NCIMB 9278), Aerocococcus viridans, Vibrio sp. (lobster intestinal isolates), Halomonas sp., Enterobacter aerogenes) representing marine and non-marine, Gram negative and Gram positive, recognised pathogenic and non-pathogenic isolates, and commensal and non-commensal agents. Five ml aliquots of the reconstituted fractions were spotted onto Muellere Hinton agar, supplemented with 3% NaCl, previously coated with a 24-h broth culture (MacFarland standard 0.5) of the test organism. Fractions were also examined by reducing and non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [13], using 17% gels, and acid-urea PAGE (AU-PAGE) modified after Wang et al. [14]. Two antimicrobial peptides, NRC-10 and NRC-14, were used as positive controls [15]. For AU-PAGE, continuous separating mini-gels comprised of 19.5% (w/v) acrylamide/N,N0 -methylenebisacrylamide (37.5:1), 0.4% (v/v) N,N,N0 ,N0 -tetramethylenediamine, 6.5 M urea, 7% (v/v) acetic acid and 0.2% (w/v) ammonium persulfate were polymerised at 37  C. Gels were pre-electrophoresed in reverse polarity at 150 V for 2 h

Antimicrobial peptides from Homarus americanus haemocytes in 5% acetic acid running buffer. Samples were diluted 2:1 with sample buffer (9 M urea, 5% acetic acid, 0.001% Pyronin Y), loaded onto gels in fresh 5% acetic acid running buffer and electrophoresed for w1 h. All gels were stained with Coomassie Brilliant Blue R-350 stain (GE Healthcare BioSciences Inc., Baie d’Urfe ´, Que ´bec). Eluate fractions demonstrating antibacterial activity were applied to a 5 ml CM Sepharose Fast Flow column (GE Healthcare Bio-Sciences Inc., Baie d’Urfe ´, Que ´bec). The column was washed with four column volumes of 100 mM ammonium acetate and eluted with three column volumes of 300 mM ammonium acetate. Fractions were analysed by reduced and non-reduced SDS-PAGE and AU-PAGE and those fractions containing the desired peptide were pooled. Buffer was exchanged in peptide pools by several rounds of concentration and addition of TBS with 10 mM CaCl2 using 1000 MWCO Microsep Centrifugal Devices (Pall Life Sciences, Ann Arbor, MI), prior to final concentration to <500 ml of sample (representing >20-fold concentration). Bands of interest were excised from the SDS-PAGE gels and submitted for in-gel trypsin digestion and amino acid sequencing by mass spectroscopy (nanospray QSTAR XL MALDI QTOF and micromass ESI QTOF, Advanced Protein Technology Centre, The Hospital for Sick Children, Toronto, ON, Canada). Database searches (NCBIBLAST, http://aps. unmc.edu/AP/, http://www.marinegenomics.org) were performed [16]. Selected fragments were synthesised (FMOC synthetic chemistry) at the same facility for use in antimicrobial testing. Purified, concentrated, native peptides (undiluted) and synthetic peptides (1.28 mg/ml) were assessed for antibacterial activity as described above. Peptides were also tested against three ciliated protozoa (Anophryoides haemophila (two isolates) and Mesonophrys chesapeakensis) recognised as pathogens in lobster and crabs [17e19]. Native peptides (diluted serially 1/2e1/16) or synthetic peptides (diluted serially 1280e80 mg/ml) were diluted in enriched ciliate media (10% peptone, 10% tryptone, 10% fetal bovine serum, 1% yeast RNA, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.2  RPMI-1640 vitamins, artificial seawater) [18]. Each peptide dilution (50 ml of synthetic, 25 ml of native) was mixed with an equal volume of ciliate culture in a 96-well plate and incubated at 5  C. Cultures were in a log growth phase, where cell concentrations generally range from w5.23  107 to 1.13  108 cells/l. Wells were assessed visually for the condition and number of ciliates present approximately every 5 days, for a total of 26 days (native peptides) and 35 days (synthetic peptides). Haemolytic activity was evaluated by mixing 180 ml of peptide solution (5e1280 mg/ml) with 20 ml of a 10% suspension of sheep erythrocytes and incubating at room temperature for 1 h. The suspensions were centrifuged at 300  g for 5 min at room temperature and the absorbance of the supernatants read at 540 nm. Absorbances at 540 nm were compared to haemolysis standards prepared with sheep erythrocytes and deionised water.

Results The eluate fraction of the HLB cartridge contained two peptides visible on AU- and SDS-PAGE gels (Figs. 1 and 2).

183

Figure 1 Isolation of two antimicrobial peptides from H. americanus haemocytes using reverse phase chromatography. Acid urea PAGE gel (19.5%) stained with Coomassie Brilliant Blue R-350. Lane 1, positive control antimicrobial peptides NRC-14 (upper) and NRC-10 (lower); lanes 2e4, negative control (flow-through, wash, and elution fractions); lane 5e7, haemocyte extract (flow-through, wash, and elution fractions). Aprotinin in the buffer solution is evident in lane 4.

One peptide, cationic antimicrobial peptide 1 (CAP-1), appeared as a very broad band (w4e6 kDa) on reduced SDS-PAGE gels and as a multimer (dimer or trimer) on non-reduced gels. A second peptide, CAP-2, with a reduced

Figure 2 Isolation of two antimicrobial peptides from H. americanus haemocytes using reverse phase chromatography. Reducing SDS-PAGE gel (17%) stained with Coomassie Brilliant Blue R-350. Lanes 1e3, negative control (flow-through, wash, and elution fractions); lanes 4e6, haemocyte extract (flow through, wash, and elution fractions). MW , molecular weight marker.

184

A.L. Battison et al. Native CAP-1 and its synthetic fragments (sCAP1.1, sCAP1.4) demonstrated bacteriostatic activity against selected Gram negative organisms (Table 1). The peptides also demonstrated protozoastatic or protozoacidal activity against the ciliate parasites (Table 2). Native CAP-2 was ineffective against all bacteria tested while the synthetic CAP-2 fragment (sCAP-2) had mild bacteriostatic activity (Table 1). Antiprotozoal activity was limited to CAP-2 (Table 2). Neither native nor synthetic versions of either peptide had haemolytic activity against sheep erythrocytes.

Figure 3 Separation of two antimicrobial peptides isolated by reverse phase chromatography from the haemocytes of H. americanus using cation exchange chromatography. Reducing SDS-PAGE gel (17%) stained with Coomassie Brilliant Blue R350. Lane 1, reversed phase chromatography elution fraction loaded onto the cation exchange column; lane 2, flow-through fraction containing CAP-2; lane 3, 100 mM ammonium acetate wash; lane 4, 300 mM ammonium acetate wash containing CAP-1. MW, molecular weight marker.

molecular weight of w12 kDa was a monomer on non-reduced gels. Cation exchange chromatography effectively separated the two peptides (Fig. 3). BLAST analysis of CAP-1 sequences revealed that CAP-1 had similarities to temporins (Fig. 4), while CAP-2 likely represented the predicted antimicrobial peptide for H. americanus, Hoa-crustin (Fig. 5) [10].

Discussion Two antimicrobial peptides, CAP-1 and CAP-2, were isolated from haemocytes of the American lobster, Homarus americanus. Initial lyophilisation and resuspension of the haemocyte lysate prior to extraction by reverse phase chromatography enhanced recovery of CAP-1 and CAP-2 (data not shown). CAP-1 was found to have similarities to temporins while CAP-2 was presumptively identified as the predicted H. americanus crustin-like peptide (Hoa-crustin) based on similarities in the amino acid sequences and molecular weights [10]. Hoa-crustin and other crustins in decapod crustaceans belong to a family of peptides characterised by conservation of 12 cysteine residues in the carboxy-terminal region which includes a whey acidic protein (WAP) domain [10,11,20e24]. The WAP domain consists of eight cysteine residues forming a four disulfide core domain (4-DSC) [11,20,24]. Recovery

A Mr (expt)

Peptide Sequence

CAP-1.1

1509.62

QYGNLLSLLNGYR

CAP-1.2

1463.58

XXGNLLSLLZZZX

XX = CM, QY, or KY; ZZZ = QCR (probable)

CAP-1.3

1388.54

XXSSNGGDCVYR

XX = NF, CS, FN, SC, or MN

CAP-1.4

1555.58

YCGNLLNQANGYR

Temporin C

LLPILGNLLNGLL---

B 13

Temporin IPa

FLPIVGKLLSGLL---

13

Temporin K

LLP---NLLKSLL---

10

Temporin B

LLPIVGNLLKSLL---

13

Temporin E

VLPIIGNLLNSLL---

13

CAP 1.1

---QYGNLL-SLLNGYR

13

CAP 1.2

---QYGNLL-SLLQ-CR

12

CAP 1.4

---YCGNLLNQANGYR

13

Figure 4 CAP-1 peptide sequences identified by mass spectroscopy and their relationships to temporins. (A) Expected molecular weights and amino acid sequences of fragments obtained after in-gel trypsin digestion of CAP-1. (B) CLUSTALW alignment of CAP-1 fragments CAP-1.1, CAP-1.2, and CAP-1.4 with various temporins.

Antimicrobial peptides from Homarus americanus haemocytes

185

A

B

C

MR (expt)

Peptide Sequence

1421.76

IVENTSLEPHAGR

1209.4

ENTSLEPHAGR

1190.58

CLLHTMCVK

1085.4

GDFTPPSPIR

IVENTSLEPHAGRCLLHTMCVKGDFTPPSPIR

Figure 5 Relationship between CAP-2 and Hoa-crustin. (A) Predicted sequence of Hoa-crustin indicating signal sequence (italics) and location of CAP-2 fragments (bold). (B) Estimated molecular weights and amino acid sequences of peptide fragments obtained after in-gel trypsin digestion and mass spectroscopy of CAP-2. (C) Composition of sCAP2 used for antimicrobial testing.

and sequencing of the WAP-containing portion of CAP-2 would have been ideal as this is a requirement for identification of the peptide as a crustin. These domains are associated with protease inhibition or antimicrobial activity depending on the type of amino acid residue adjacent to the second cysteine in the 4-DSC [11]. Crustin isoforms are recognised and this may account for the few amino acid sequence inconsistencies noted between CAP-2 and the predicted sequence for Hoa-crustin [20,22,23]. The first crustin, carcinin, was isolated from haemocytes of the European green crab Carcinus maenas (Linnaeus 1758) [25,26]. Carcinin’s antimicrobial activity was specific towards salt-tolerant Gram positive cocci including the lobster pathogen A. viridans. This bacterium, the causative agent of gaffkemia, is of minor consequence to C. maenas which may act as a reservoir host along with other decapod crustaceans [26e28]. Gaffkemia is a generally fatal disease of American lobsters [28]. Early studies demonstrated that A. viridans is able to grow in haemolymph serum, a composite of plasma and lysed haemocyte contents [29]. This serum could potentially contain antimicrobial peptides such as CAP-1 and CAP-2. Thus, the lack of in vitro activity of native CAP-2 against A. viridans is intriguing. Alteration of the peptide during the extraction process, e.g., loss of posttranslational modifications is one possible explanation.

Alternatively, the anti-A. viridans activity of carcinin may reside in the non-carboxy terminal (WAP-containing) region where numerous differences among carcinin, Hoa-crustin, and other crustins occur [10]. This may also explain, in part, the variation in susceptibility of H. americanus and C. maenas to A. viridans and merits further investigation. Conversely, lack of activity of CAP-2 against the nonmarine Gram positive coccus M. luteus ATCC 4698 (NCIMB 9278) is consistent with carcinin [26]. Testing against a wider range of Gram positive bacteria may have revealed additional activity and could be the focus of future studies. Numerous antimicrobial peptides, including many crustins, have been predicted utilising techniques such as EST libraries in recent years; however, the actual production and activity of these peptides is rarely confirmed [20,22,30]. Variance of CAP-2 from its anticipated activity (as representing Hoa-crustin) clearly illustrates the need to isolate, purify, and test these predicted peptides. In the case of crustins, comparison of their anti-A. viridans activity and host susceptibility could shed light on a potential link between the differential pathogenicity of the agent and the mode of action of the peptides. sCAP2 represented only a fraction of the entire predicted sequence for Hoa-crustin and did not include the WAP domain, so lack of activity against A. viridans was therefore not unexpected if the WAP domain is required

Table 1 Summary of antibacterial activity of native peptides CAP-1 and CAP-2 and synthetic fragments sCAP1.1, sCAP1.4, and sCAP-2 tested using a solid phase, spot diffusion method Halomonas sp.b Enterobacter Micrococcus luteus Aerococcu Vibrio sp. Vibrio sp. Vibrio a a ABHa3 parahaemolyticus aerogenes (ATCC 4698) viridans ABHa2 CAP-1 (undiluted) sCAP1.1 (1.28 mg/ml) sCAP1.4 (1.28 mg/ml) CAP-2 (undiluted) sCAP2 (1.28 mg/ml)

    

    

þ þ   þ

ntc   nt 

nt þ þ nt þ

When present, activity was bacteriostatic only. Results represent two or three replicates. a Isolated from lobster intestinal contents. b Previously identified as Pseudomonas perolens (unpublished results). c Not tested.

    

nt þ  nt 

186

A.L. Battison et al.

Table 2 Summary of antiprotozoal activity of native peptides CAP-1 and CAP-2 and synthesised fragments sCAP1.1, sCAP1.4, and sCAP2 in liquid culture media

CAP-1 (1:1 dilution) sCAP1.1 (mg/ml) sCAP1.4 CAP-2 (1:1 dilution) sCAP2

Mesanophrys chesapeakensis

Mesanophrys pugettensis

Anophryoides haemophila I [17]

Anophryoides haemophila II [19]

þ þþ (640) þ (320)   

þ þþ (640) þ (320)   

þþ þþ (640) þ (320)  þ 

þ þþ (640) þ (320)   

, culture growth was unaffected at the concentrations indicated; þ, growth was inhibited; þþ, cultures died. Results represent two replicates for the synthetic peptides and one for the native peptides (due to limited amounts available).

for activity against this organism. The WAP domain has been shown to be required for antibacterial activity in the recombinant product of a crustin-like gene, CruFc, in the shrimp Fenneropenaeus chinensis [31]. The bacterisotatic activity of the sCAP2 fragment against the commensal lobster Vibrio sp. isolates was intriguing as this activity was absent in native CAP-2. While the sCAP2 fragment displayed no antiprotozoal activity, native CAP-2 demonstrated limited activity against the 1993 Maine A. haemophila isolate only. Whether this differential susceptibility reflects an inherent resistance in the 2004 Nova Scotia isolate or the difference in age of the cultures requires further investigation. Activity against A. haemophila is significant as this agent causes a uniformly fatal disease in H. americanus known as bumper car disease [19,32]. CAP-1 showed some amino acid sequence similarities to temporins e short (10e13 amino acids) antimicrobial peptides first isolated from the skin of the European red frog Rana temporaria and also found in many North American and Eurasian ranid frogs [33,34]. Temporins are generally effective against Gram positive bacteria while some also have activity against Gram negative bacteria, Candida, and the chytrid fungus Batrachochytrium dendrobatidis [34]. Temporins A and B are also effective against protozoans, specifically the promastigote and amastigote forms of Leishmania [35] CAP-1, sCAP1.1, and sCAP1.4 had bacteriostatic activity against selected Gram negative organisms. CAP-1 and sCAP1.1 also demonstrated antiprotozoal activity against all three ciliate isolates. Of particular note again, was the activity against the A. haemophila cultures. To confirm their efficacy, it will be important to also test these peptides against organisms isolated from active infections, rather than cultures which have undergone numerous in vitro passages. Pooled haemocyte samples were used for some peptide extractions. The possibility that the similarities noted in the fragments obtained from the CAP-1 amino acid sequence data could represent individual variation among lobsters was considered. However, similar results were obtained when sequencing was performed on CAP-1 samples obtained from a single lobster (data not shown) suggesting that multiple isoforms of the peptide, a homoor heteromultimer, are expressed in individual lobsters. As the H. americanus EST library is expanded, identification of the complete sequence and probable structure of CAP-1 is anticipated.

In summary, two antimicrobial peptides from H. americanus haemocytes were isolated and their biological activity confirmed. One presumably represents the predicted Hoa-crustin. The second, unrelated to any others presently described for marine invertebrates, may represent a novel class of antimicrobial peptide for decapod crustaceans proposed as homarins. Further investigations into the structure, function, and physiological role of both peptides is warranted.

Acknowledgements This project was sponsored by the AVC Lobster Science Centre which receives funding from a consortium of public and private sources representing all aspects of the lobster industry in Atlantic Canada. The authors wish to thank S. Greenwood for critical review of the manuscript.

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