Journal of Microbiological Methods 83 (2010) 8–12

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Journal of Microbiological Methods

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Analysis of antimicrobial peptides from porcine neutrophils

Joanna Wessely-Szponder a,⁎, Barbara Majer-Dziedzic b,1, Anna Smolira c,2

a Department of Pathophysiology, Chair of Preclinical Veterinary Sciences, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033 Lublin, Polandb Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12,20-033 Lublin, Polandc Institute of Physics, Division of Molecular Physics, Maria Curie Sklodowska University 20-031 Lublin, Poland

⁎ Corresponding author. Tel.: +48 814456774; fax: +E-mail address: joanna.wessely@up.lublin.pl (J. Wes

1 Tel.: +48 814456016.2 Tel.: +48 815376130.

0167-7012/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.mimet.2010.07.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 March 2010Received in revised form 2 July 2010Accepted 5 July 2010Available online 17 July 2010

Keywords:Antimicrobial peptidesCathelicidinsNeutrophilsPig

Cationic host defence peptides are important components of innate immunity in pigs and othermammalians. Most of these peptides have a direct antimicrobial activity and they also have a broadspectrum of effects on the host immune system, which may be taken into account in the introduction ofnovel therapeutics. Our method permits simultaneous isolation of six antibacterial peptides, i.e. prophenin-1,prophenin-2, PR-39, and protegrins 1-3 from a porcine neutrophil crude extract and characterisation ofthem. Among the obtained peptides the greatest bactericidal activity expressed as MBC was seen inprotegrins (10 μg/ml), whereas in the other studied peptides MBC was on the level of 20 μg/ml. Minimalinhibitory concentrations (MIC) reached 10 μg/ml for protegrins 1–3 and 20 μg/ml for prophenins, and PR-39. Within the bactericidal range all isolated peptides didn't show cytotoxicity on cell lines used in ourexperiment.

48 814456024.sely-Szponder).

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The increasing bacterial resistance to commercial antibioticsinduces a search for new sources of antibacterial agents. Among thepromising reservoirs of such efficient antibacterial agents currentlyunder study are antimicrobial peptides derived from neutrophils(Cheung et al., 2008; Hancock and Sahl, 2006; Ramanathan et al.,2002; Wieczorek et al., 2010).

Previous studies on mammalian antimicrobial peptides establishedthat pigs have themost diverse collection of cathelicidins of any species.Apart from three myeloid antimicrobial peptides (PMAP-23, PMAP-36,and PMAP-37) there are porcine cathelicidins purified from neutrophils(PMN), i.e. proline–phenylalanine-rich prophenin-1 (PF-1) and pro-phenin-2 (PF-2), proline-arginine-rich 39-amino-acid peptide (PR-39),and cysteine-rich protegrin-1 to 5 (PG-1 to PG-5)(Sang and Blecha,2009).

Prophenins 1 and 2 are two cathelicidins purified from porcineleukocytes (Zang et al., 2000). They were also isolated from porcinelung tissue (Wang et al., 2004). These 79-residue cathelicidins areeffective against Gram-negative bacteria (Zang et al., 2000). They haveextended-helical structure and high contents of proline (53%) andphenylalanine (19%) (Ramanathan et al., 2002).

PR-39 manifests antibacterial activity against Gram-negativebacteria, and some Gram-positive (Ramanathan et al., 2002). It is

known that PR-39 stops DNA and protein synthesis in Escherichia coli(Vunnam et al., 1997). Moreover, it participates in wound repairmechanisms, has chemoattractant properties and interacts with LPS.It also inhibits the NADPH oxidase reactivity, which has the directeffect of reducing local tissue injury by reactive oxygen species (ROS)(Ramanathan et al., 2002; Zang et al., 2000).

Protegrins (PG-1 to PG-5), as active and abundant porcinecathelicidins were found at bactericidal concentrations in neutrophilsecretions and the abscess fluid (Han et al., 2007). They sharecommon features such as small size (16–18 amino acids), β-sheetstructure, cationic charge and an amphipathic nature (Steinberg et al.,1997). The best studied protegrin 1 an 18-amino-acid peptide isolatedfrom porcine leukocytes displays a broad-range antimicrobial activityspectrum and has potential for therapeutic use. The cationic nature ofthis peptide allows its interaction with the lipid matrix of bacterialmembranes containing negatively charged lipids (Han et al., 2007).

Apart from synthetic peptides also naturally obtained cathelicidinsfrom the neutrophil extract may be used for analysis and possibletherapeutic usage (Anderson et al., 2004; Anderson and Yu, 2008).Although some papers describing methods of isolation of ovinecathelicidins have appeared recently (Anderson and Yu, 2003, 2008),there are few publications about the isolation of porcine cathelicidins.Most of the available reports concern studies of synthetically obtainedporcine peptides (Jacobsen et al., 2007; Mangoni et al., 1996;Robinson et al., 2005; Shi et al., 1996). Others describe methods thatallow obtaining only a single kind of peptides. For example,Kokryakov et al. (1993) described the analysis of protegrin isolatedwith ultrafiltration and vacuum centrifugation andwith the use of fastatom bombardment (FAB)-mass spectrometric analyses. Prophenin

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was obtained with ultrafiltration and characterised by LC-ESI-MS(Hartwig et al., 1995). The method of separation of PR-39, with AU-PAGE for identification, used by Shi et al. (1994) is not sufficient forpeptides with lowmolecular masses (Hortin, 2006; Shi et al., 1994). Inthis paper we describe a simple and effective method for isolation ofantibacterial peptides from a neutrophil extract. It is a combination ofpreviously reported techniques used for neutrophils from differentspecies. This method permits isolation of cathelicidins without thepreliminary step of ultrafiltration, therefore the method is simple andcost-effective. Moreover, we have used matrix assisted laser desorp-tion/ionization time of flight mass spectrometry (MALDI-TOF MS)without previous trypsin digestion for identification of antibacterialpeptides on the basis of molecular weight. Our modified methodallows for simultaneous isolation of six different bactericidal peptidesfrom porcine neutrophil extraction.

2. Materials and methods

2.1. Crude extraction

Fresh porcine blood was collected with anticoagulant 3.8% citrateat an abattoir. A crude extraction from blood neutrophils was obtainedafter red blood cells were lysed by the addition of 0.83% ammoniumchloride at the ratio of 3:1. White blood cells (75–85% PMN) werecollected by centrifugation (700×g, 15 min, 4 °C) and resuspended inmodified phosphate buffer saline (PBSX) buffer (137 mM NaCl,2.7 mM KCl, 0.5 mM MgCl2, 8.1 mM Na2HPO4, 1.5 mM KH2 PO4, pH7.4)(Treffers et al., 2005). The cells were then homogenisedwith DIAX900 Heidolph (12.5 rpm, 15 min) to release the neutrophil granules.These granules were collected (25,000×g, 40 min, 4 °C), suspended in10% acetic acid and stirred overnight at 4 °C to extract theantimicrobial peptides. Total protein was assessed by Lowry's methodwith bovine albumin as a standard (Romeo et al., 1988). The solutioncontaining the peptides was separated from the granules (25,000×g,20 min, 4 °C) lyophilised and stored at −20 °C.

2.2. Peptide purification

Gel filtration chromatography was used to separate the compo-nents present in the crude extraction according to their sizes. Theextract was passed through a Sephadex G-50 (Fine, Sigma-Aldrich)column, using a running buffer of 5% acetic acid at 0.5 ml/min. Theabsorbance of the eluate (every 0.5 ml) was monitored at 280 nm andfractions were pooled of 5.0 ml (Anderson and Yu, 2003). Theconcentration of protegrins and other cathelicidins in every fractionwas assessed using the extinction coefficient (Kokryakov et al., 1993).

The active fractions were lyophilised, resuspended in a solvent(0.1% trifluoroacetic acid [TFA] in water v/v) and were loaded onto aC18 RP-HPLC column (250×4 mm, particle size 5 μm, LiChrospiner100, Merck, Germany) with the flow rate of 0.7 ml/min. Peptides wereeluted with gradient 0–60% acetonitrile in 0.1% TFA andmonitored forabsorbance at 225 nm.

2.3. MALDI-TOF analysis

MALDI-TOF analysis was performed on the reflectron time of flightmass spectrometer (RTOF MS — built in the Institute of Physics,Division of Molecular Physics, UMCS Lublin, Poland, with an ionsource MALDI) with parameters described in detail previously (Głuchet al., 2001; Gruszecka et al., 2008).

A matrix, α-cyano-4-hydroxycinnamic acid (CCA, 189.2 Da, Sigma-Aldrich), used without further purification, was diluted in portion of0.03 g in 3 ml of mixture of acetonitrile and 0.1% TFA (1:1,v/v). Next, anequal volume of the peptide solution (from 1 ml of total volume of 20–50 μg dissolved in 0.1% TFA, 60% acetonitrile) and of thematrix solutionwere put on a stainless steel sample holder and dried in air at room

temperature. After evaporation of solvents a visible and apparentlyhomogenous mixture spot of ~1 mm diameter appeared. The massspectra of the investigated peptideswere obtained in the linearmode ofthe spectrometer, collected at the nitrogen laser (337 nm) in thepositive polarity of the ion source.

2.4. Bacterial strain

A strain of E. coli (25922; obtained from the American Type CultureCollection — ATCC) was used for evaluation of the antibacterialactivity of the isolated peptides. It was stored in 10% glycerol at−80 °C. The bacterial cultures were cultivated in Mueller–Hintonbroth (MHB) and Mueller–Hinton agar (MHA) (Biocorp WarsawPoland)(Shi et al., 1994).

2.5. Minimal inhibitory concentrations

The method used was based on the broth dilution assay. Briefly,bacteria were grown in MHB (one colony in 10 ml) pH 7.2, for 20 h at37 °C. The culture for the assaywas prepared by diluting the overnightE. coli culture 1:100 in fresh MHB, and then 1 ml of diluted suspensionof bacteria in 20 ml MHBwas stirred for 1 h at 37 °C. Equal volumes of4.6×105 CFU/ml bacteria (150 μl) were mixed with a crude extractionor with each obtained gel filtration fraction (150 μl). All samples wereincubated for 20 h at 37 °C. Then the mixtures were diluted 100-foldin PBS and 0.1 ml of every dilution was plated in triplicate on MHA.After incubation for 20 h at 37 °C, the colonies were counted and thenumbers of CFU/ml were calculated and compared with the CFUvalues of controls of E. coli prepared in the same conditions. The activesamples were used for determination of minimal inhibitory concen-tration (MIC). The lyophilised peptides were diluted in two-fold serialdilutions in MHB from160 to 2.5 μg/ml and standardised suspensionsof 4.6×105CFU/ml E. coli were added and the mixture was incubatedfor 20 h at 37 °C. Then the inhibitory properties of the examinedsamples were estimated by visual comparison with the controlsuspension. The smallest concentrations of peptides causing inhibi-tion of E. coli growth (the broth remained clear) were determined asMIC (μg/ml)(Shi et al., 1994; Skerlavaj et al., 1996).

2.6. Evaluation of bactericidal activity (MBC)

For the bactericidal activity (MBC) determination 100 μl ofsolution from the last tube where the growth of bacteria wasobserved and from subsequent four clear tubes were transferredonto the plates with MHA. After incubation for 20 h at 37 °C theminimal bactericidal concentration was identified as the lowestconcentration of peptides that did not permit any visible growth onthe surface of the agar. Every assay was done in triplicate (Shi et al.,1994; Steinberg et al., 1997).

2.7. Cytotoxicity assay

Minimal Essential Medium (MEM), Medium 199, and Hams F-12(PAA-Immuniq) were used for evaluation of cytotoxicity of activepeptides on the cell culture in vitro. The assay was carried out on twocell lines, i.e. a feline lung cell line tissue culture (FLF-3) and a rathepatocyte cell line (ECACC Salisbury, Wiltshire). MEM with 10%neonatal calf serum and penicillin (100 U/ml) with streptomycin(100 μg/ml) was used as the growing medium. Medium 199 withantibiotics was used as medium maintenance. These peptides werediluted 1:10 and 1:20, and 0.2 ml of each dilution was added to 6tubes (for dilution) with a tissue culture. The tubes with cell lineswithout addition of peptides were used as a control. The tubes werekept for 10 days at 37 °C and were controlled microscopically everyday (Kowalska-Pyłka et al., 2001; Shi et al., 1994).

Table 1Antibacterial activity of studied antimicrobial peptides against E. coli (strain 25922ATCC) tested with the broth dilution technique.

Fraction Peptide MIC (μg/ml) MBC (μg/ml)

F 3 Prophenin 1 20 20F 4 Prophenin 2 20 20F11 PR-39 20 20F13 Protegrin (1-3) 10 10

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3. Results

3.1. Purification of porcine neutrophil cathelicidins

The crude extraction obtained from each batch by overnightextraction of porcine neutrophil granules in 10% acetic acid waslyophilised in portions of about 250 μg, as indicated by Lowry'smethod. One representative portion from each batch was resus-pended in MHB and subjected to the initial antibacterial assay. Alltested crude extractions were bactericidal. Subsequently, portions ofcrude extractions from active batches containing peptides werepassed through a Sephadex G-50 column in concentrations of about500 μg/ml. Every fraction of 0.5 ml was monitored spectrometricallyat 280 nm and these fractions were pooled of 5.0 ml. The initialpurification of porcine antibacterial peptide extract from SephadexG-50 column is shown in Fig. 1.

Every 5.0 ml fraction was tested for antibacterial action against E.coli and fractions F3, F4, F11 and F13 appeared active. All activefractions were assessed by HPLC analysis, which permitted theresolution of several distinct peaks.

3.2. Peptide characterisation

Fractions which showed antibacterial activity against E. coli wereassessed byMALDI-TOFMS analysis. Molecular weightwas analyzed bythe ExPASy MW/pI tool (http://www.expasy.ch/tools/pi_tool.htlm).

3.3. Characterisation of prophenin 1

The molecular mass of peptide obtained in fraction F3 was 8683 Daand was consistent with the predicted PF-1 molecular mass. Thispeptide was active against E. coli 25922 ATCC at concentration of 20 μg/ml (Table 1). There were no cytotoxic effects on any cell line in anystudied concentration up to 20 μg/ml.We didn't observe changes in cellsize and shape. The cytoplasmic vacuoles and dying cells didn't appearduringobservationperiod. Themicroscopic picturewas the same for thecontrol cells and cells with addition of peptide. After 10 days thedegeneration of the cells appeared both in the control and treated lines.

3.4. Characterisation of prophenin 2

Using MALDI TOF technique we estimated that an antimicrobialpeptidewithMIC of 20 μg/ml andwithMBC equal 100% of MIC against

Fig. 1. Gelfiltration chromatogram for crude extraction applied on a SephadexG-50 column in 5

E. coli (Table 1) was present in fraction F4 and was recognised as PF-2with a molecular mass of 8807 Da (Fig. 2). We didn't observe anymorphological changes and degenerative alterations in either the FLF-3 tissue culture or the rat hepatocyte cell line during the 10 days of theexperimental period caused by these peptides at examined concen-trations up to 20 μg/ml.

3.5. Characterisation of PR-39

Fig. 3 shows the resulting chromatograph of fraction F11. The pickshown at this chromatogram had the molecular mass of 4716 Da, asrevealed in MALDI TOF analysis and corresponded to PR-39 (Fig. 4).PR-39 had antimicrobial activity against E. coli with MIC of 20 μg/mland with the same MBC (Table 1). The highest dose of PR-39 that wastested (20 μg/ml) was not cytotoxic to the FLF-3 tissue culture or therat hepatocyte cell line. The microscopic picture was the same for thecontrol and tested cells.

3.6. Characterisation of protegrins PG- 1 to PG- 3

Protegrins PG-1 to PG-3 were present in fraction F13. Theirobtained masses were as follows: 2154.5 Da for PG-1, 1955.6 Da forPG-2, and 2055.5 Da for PG-3 (Fig. 5). Moreover, we noticed apeptide with the molecular mass of 1307 Da in this fraction butwithout antibacterial and cytotoxic properties. The antibacterialactivity of PG-1 to PG-3 was described as MIC 10 μg/ml and MBC atthe same level (Table 1). We didn't observe any morphologicalchanges and degenerative alterations in either the FLF-3 tissueculture or the rat hepatocyte cell line during the 10 days of theobservation of these peptides at concentration up to 10 μg/ml. Afterthis time degenerative changes of the cells in all groups wereobserved.

% acetic acid andmonitored at 280 nm. Fractionswere collected in tubes (0.5 ml per tube).

Fig. 2. Positive ion MALDI time of flight mass spectrum of prophenin 2 (8807 Da)obtained in linear mode of the spectrometer.

Fig. 4. Positive-ion MALDI mass spectrum of PR-39 with α-cyano-4-hydroxycinnamicacid (CCA) used as a matrix. The spectrum is an average of 256 laser shots.

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4. Discussion

The increasing number of virulent, antibiotic-resistant strains ofbacteria has created a pressing need for alternative therapies forinfection control (Jacobsen et al., 2007). Moreover, the possibility ofusing the immunomodulatory properties of antibacterial peptides as abasis for therapy has appeared recently (McPhee and Hancock, 2005).

Our experimental method permits isolation of PF-1, PF-2, PR-39,and PG-1 to PG-3 in an active bactericidal form in quantities sufficientfor further analysis. Porcine neutrophils lack defensins and cathe-licidins are the only antibacterial peptides there (Sang and Blecha,2009). These cathelicidin peptides from neutrophil secretionremained stable in inflammatory fluids for days in contrast to thereactive oxygen and nitrogen intermediates (Shi and Ganz, 1998).Among porcine cathelicidins protegrins were identified as the majorstable antimicrobial substances released by neutrophils. The imma-ture forms of peptides are stored in porcine neutrophils as inactiveproprotegrins and neutrophil elastase is required for in vitroactivation of protegrins (Cole et al., 2001). However, as in the studyconducted by Anderson and Yu (2003), an extraction process used inour experiment did not involve the addition of elastase, so thecleavage was probably carried out by neutrophil elastase naturallypresent in the neutrophil extraction.

Fig. 3. HPLC chromatogram of fraction F11 obtained in gel filtration. The fractionresuspended in 0.1% trifluoroacetic acid [TFA] in water (v/v) was loaded onto a C18 RP-HPLC column (250×4 mm, particle size 5 μm, LiChrospiner 100, Merck, Germany) witha flow rate of 0.7 ml/min. Peptides were eluted with gradient 0–60% acetonitrile in 0.1%TFA and monitored for absorbance at 225 nm.

As in experiment described by Anderson and Yu (2003) we alsoused gel filtration method for initial separation of peptides accordingto decreasing molecular weight (Fig. 1). However, in our study thecrude extract was passed through a Sephadex G-50 column, whichwas previously used by Wang et al. (2008) instead of Biogel P10column. Treffers et al. (2005), in turn, applied ion-exchangechromatography for evaluation of antimicrobial peptides from deerneutrophils.

Polypeptides smaller than 7000 Da are not detected by the usualtechniques of 2-dimensional electrophoresis because they are belowthe limits of size resolution, and small componentsmay not be fixed ingels and produce lower staining intensity per mole of peptide.Consequently MALDI-TOF MS has served as a tool to open uppeptidomic analysis in the mass range from 1000 to 7000 (Hortin,2006). In our study the MALDI TOF technique permitted assessing theobtained porcine cathelicidins as PF-1, PF-2, PR-39, and PG-1 to PG-3with molecular masses which are consistent with the predictedmolecular masses.

Prophenin 1 with the molecular mass of 8683 Da was activeagainst E. coli 25922 ATCC at concentration of 20 μg/ml. According toHartwig et al. (1995) PF-1 demonstrated potent bactericidal activityagainst E. coli. In our experiment PR-39 had antimicrobial activityagainst E. coliwith MIC of 20 μg/ml and with the same MBC. Sang andBlecha (2009) showed significant efficacy of this peptide against

Fig. 5. Positive-ion MALDI mass spectrum of protegrins obtained at 17 kV acceleratingvoltage in linear mode of the time of flight mass spectrometer.

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Gram-negative bacteriawithMIC about 20 μg/ml. Protegrins belong toelastase-activated polypeptides, which are the predominant antibac-terial factors in porcine neutrophil secretions generated duringphagocytosis (Shi and Ganz, 1998). In our study the bactericidalactivity of PGs against E. coli was on the level of 10 μg/ml. Cole et al.(2001) estimated that two concentrations (20 and 200 μg/ml) of PG-1completely cleared the wound of S. epidermidis in pigs. According toRamanathan et al. (2002) protegrins kill many bacteria at concentra-tions of 1–5 μg/ml. Sang and Blecha (2009) found that PG-1 is effectiveagainst 14 Gram-negative bacteria with most MIC less than 5 mM.Apart from these we also established the presence of a peptide withthe molecular mass of 1307 Da. It would be the result of enzymaticcleavage of prophenin (Hartwig et al., 1995). We didn't observeantibacterial activity of this peptide.

We didn't observe cytotoxic effect within tested concentrations ofall studied peptides. The cytotoxicity assay was based on theoccurrence of morphological changes. Although it is not a quantitativemethod, it allows a relatively fast cytological screening of testedsubstances. The use of culture tubes increases precision because itgives the possibility of obtaining a greater number of cells, enablingmore accurate determinations (Kowalska-Pyłka et al., 2001).

The use of cationic peptides mostly involved topical treatment ofdifficult infections because of the potential toxicity associated withsystemic drug usage. Unfortunately, the majority of infections that arelife threatening, especially those due to multidrug resistant bacteria,require systemic treatment (McPhee and Hancock, 2005). Althoughour studies revealed that the studied cathelicidins didn't showcytotoxicity within bactericidal range, literature data underline thatthe systemic usage of cathelicidins is restricted because of theircytotoxic potential, especially in the case of protegrin 1. As Robinsonet al. (2005) estimated, PG-1 is toxic to the mammalian cell atconcentration of 100 μg/ml. On the other hand, according to Shi et al.(1996) PR-39 is not cytotoxic to Madin–Darby bovine kidney (MDBK)cells up to 50 μM.

An alternative to the microbicidal effect approach would involveutilising the immunomodulatory activity of cationic peptides as abasis for therapy. The stimulation of innate immunity while notinducing or even suppressing harmful proinflammatory responses isan attractive alternative because of the fact that such immunomodu-lators would act on the host cells rather than attack bacteria directly.This approach should prevent any resistance. Therefore it may be aneffective treatment for the increasing number of individuals withmultidrug resistant bacterial infections (Hancock and Sahl, 2006;McPhee and Hancock, 2005).

As mentioned by Anderson and Yu (2008), in the case of ovineantibacterial peptides they are significantly more active in combina-tion. Therefore it is suggested that better therapeutic results could beobtained if isolated peptides were used synergistically. Our method ofseparation leads to obtaining six different antibacterial peptides fromfresh porcine blood with activity maintained.

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