Biochimica et Biophysica Acta 1834 (2013) 546–558

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Biochimica et Biophysica Acta

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Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin:Interactions with human β2 integrins and erythrocytes

Jesper Reinholdt a,⁎, Knud Poulsen b, Christel R. Brinkmann b, Søren V. Hoffmann c,Romualdas Stapulionis b,c, Jan J. Enghild d,e,f, Uffe B. Jensen g, Thomas Boesen f, Thomas Vorup-Jensen b,e,h,⁎⁎a Department of Dentistry, Aarhus University, Aarhus, Denmarkb Department of Biomedicine, Aarhus University, Aarhus, Denmarkc ISA, Department of Physics and Astronomy, Aarhus University, Aarhus, Denmarkd Center for Insoluble Protein Structures (inSPIN), Aarhus University, Aarhus, Denmarke Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmarkf Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmarkg Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmarkh The Lundbeck Foundation Nanomedicine Center for Individualized Management of Tissue Damage and Regeneration (LUNA), Aarhus University, Aarhus, Denmark

⁎ Correspondence to: J. Reinholdt, Department of DThe Bartholin Building, Wilhelm Meyers Allé 4, DK-⁎⁎ Correspondence to: T. Vorup-Jensen, Department of BThe Bartholin Building, Wilhelm Meyers Allé 4, DK-80008716 8153; fax: +45 8619 6128.

E-mail addresses: jr@microbiology.au.dk (J. Reinholdvorup-jensen@microbiology.au.dk (T. Vorup-Jensen).

1570-9639/$ – see front matter © 2012 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.bbapap.2012.12.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 May 2012Received in revised form 15 November 2012Accepted 3 December 2012Available online 9 December 2012

Keywords:Bacterial toxinLeukotoxinIntegrinSynchrotron radiation circular dichroismspectroscopy

Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a fre-quent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin(RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. Theknown receptor for LtxA on leukocytes is integrin αLβ2 (LFA-1 or CD11a/CD18). However, the molecularmechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has provendifficult to prepare for experiments as free of contaminants and with its native structure. Here, we describea protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturingprocedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotronradiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content,and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonaswith a known three dimensional structure supports the presence of a so-called beta-ladder domain in theC-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express β2 integrin. Cells express-ing αMβ2 (CD11b/CD18) or αXβ2 (CD11c/CD18) were killed as efficiently as cells expressing αLβ2. Erythro-cytes, which do not express β2 integrins, were lysed more slowly. In ligand blotting experiments, LtxAbound only to the β2 chain (CD18). These data support a previous suggestion that CD18 harbors the majorbinding site for LtxA as well as identifies integrins αMβ2 and αXβ2 as novel receptors for LtxA.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Aggregatibacter actinomycetemcomitans is a gram-negative, fac-ultatively anaerobic cocco-bacillus, which is a frequent member ofthe human oral flora primarily located in dental plaque [1]. It occa-sionally causes endocarditis and brain abscesses [2] but is knownmainly for its association with aggressive periodontitis in adolescents[3,4].

entistry, Aarhus University,8000 Aarhus C, Denmark.iomedicine, Aarhus University,Aarhus C, Denmark. Tel.: +45

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l rights reserved.

A. actinomycetemcomitans produces a number of virulence factorsthe best studied of which is a leukotoxin denoted LtxA1. This proteinis a member of the repeats-in-toxin (RTX) [2] family of bacterial cy-totoxins [5,6]. In support of a role of LtxA in periodontitis, coloniza-tion by a particular clone of A. actinomycetemcomitans named JP2that produces up to 20 fold more LtxA than other genotypes impliesa high risk of developing aggressive periodontitis [7]. This clone col-onizes almost exclusively humans of North and North-West Africanethnicity [4,8].

The ltx operon of A. actinomycetemcomitans consists of fouropen reading frames where ltxA is the 3165 bp structural gene,

1 The abbreviations used are: Ab, antibody; ELISA, Enzyme-linked immunosorbentassay; GPC, gel permeation chromatography; LFA-1, Lymphocyte function-associatedantigen-1; LtxA, Leukotoxin; Mr, relative molecular mass; RTX, Repeats-in-toxin;SRCD, synchrotron radiation circular dichroism spectroscopy.

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ltxC encodes a protein that modifies and thereby activates the toxinby acylation of two lysyl residues, and ltxB and ltxD are required forits secretion [6,9]. Genetic and biochemical analyses have revealedstructural elements of LtxA (Mr ~114 k) shared with other RTXfamily toxins including N-terminal hydrophobic alpha helices, acyl-ation at two lysyl residues and Ca2+-binding glycine-rich repeats[6,10,11]. The molecule carries a positive charge with an isoelectricpoint at pH~9.0. Such highly charged proteins are often associatedwith a relatively unfolded structure typical of proteins and pep-tides with cytotoxicity to prokaryotic cells [12,13]. However, ex-perimental insight into the higher order structure of LtxA is notavailable.

LtxA efficiently destroys human leukocytes, particularly neutrophilsand mononuclear phagocytes [14]. Phagocyte activities are crucial forthe defense and homeostasis of periodontal tissues [15]. Before killingphagocytes, the toxin may cause degranulation and abundant secretionof inflammatory cytokines by these cells, which may contribute largelyto the tissue-destructive potential of the infection [16–19]. Besides, atelevated concentrations, LtxA has a slow hemolytic activity [20]. LtxAexerts its effects on leukocytes through molecular mechanisms, whichare only partly known. Itwas shown that LtxAuses a humanβ2 integrin,αLβ2 (also named LFA-1 or CD11a/CD18) as a cell surface receptor. Theintegrin β2 chain (CD18) and possibly a part of the αL (CD11a) chainwere suggested to be involved in the interaction with the toxin[10,21,22]. By contrast, the roles as LtxA receptors of the three otherknown human β2 integins αMβ2 (Mac-1, complement receptor 3, orCD11b/CD18), αXβ2 (p150,95, complement receptor 4, or CD11c/CD18),and αDβ2 (CD11d/CD18) have not been systematically examined.This is an important lacuna in our knowledge on the receptor usageof LtxA since the biology of these integrins, in spite of their sharedbeta-2 chain, is fundamentally different. Notably, the integrinsαMβ2 and αXβ2 play important roles in the biology of myeloid cellswith a regulation on their cell surface expression through releasefrom pools of intracellularly-stored receptors, which is not foundfor integrin αLβ2 [23]. Unlike the situation for β2 integrin-mediatedcell killing, the hemolytic activity of LtxA is not species restricted in-dicating that erythrocytes are lysed by reaction schemes differentfrom those involved in the killing of leukocytes [20]. Notably, eryth-rocytes do not express β2 integrin [21]. An important concernin these analyses is the purity and structural integrity of the appliedLtxA. In cultures of fresh isolates of A. actinomycetemcomitans of therough colony phenotype, almost all LtxA is bound non-covalentlyto the bacterial surface. Hence, early protocols for the preparationof LtxA involved extraction of the toxin from the bacterial cells[14,24–26]. LtxA isolated in this way may or may not be contaminat-ed with lipopolysaccharide (LPS) from the bacteria [27,28]. Contam-ination by LPS is a problem if LtxA is used for studies of toxin-mediated inflammatory effects and host immune response to thetoxin [27,29]. In strains with a smooth colony morphology, whichis caused mainly by mutation in the tad locus [30], a large fractionof the toxin is released to the culture medium from where it maybe isolated [25,31]. However, also in this case contamination withLPS appears as a problem.

Here, we describe a convenient protocol for the isolation ofLtxA from culture supernatant produced by a smooth variant ofthe JP2 clone. The purified toxin was essentially monodisperseand free of LPS as determined by Western blotting or a sensitiveLimulus-based assay. Determination of the secondary structurecontent by synchrotron radiation circular dichroism spectroscopy(SRCD) and comparison with the secondary structure predictedin silico showed that the protein is likely to be folded in a wayquantitatively comparable to its native structure. Using such pu-rified LtxA, we examine the affinity of the toxin to componentchains of three different β2 integrins and compare its effects ontarget cells expressing different β2 integrin species and on puresuspensions of erythrocytes.

2. Materials and methods

2.1. Bacterial strains, cell lines and monoclonal antibody

A. actinomycetemcomitans strain HK975 is of serotype b withsmooth colony type and was originally received as strain Y4 fromB. Hammond, Temple University School of Dentistry, Philadelphia,PA. A. actinomycetemcomitans strain HK921 is of serotype b withsmooth colony type and was received as strain JP2 from E.T. Lally,School of Dental Medicine, University of Pensylvania, Philadelphia,PA. K562 human erythroleukemia cells, unmodified or cotransfectedwith cDNAs encoding the α and β subunits of either integrin αLβ2,αMβ2, or αXβ2, were obtained from T. A. Springer, Harvard MedicalSchool, Boston, MA. The three recombinant cells had been obtainedby homologous transfection protocols using the CDM8 expressionvector [32].

Hybridoma cells producing the monoclonal antibody (Ab) KIM127,which reacts with an epitope of human CD18 [33], were purchasedfrom American Type Culture Collection and the Ab was purified fromculture supernatant as described [34].

2.2. Anaerobic and aerobic culture

To examine the potential regulation of LtxA production by oxy-gen, parallel cultures of A. actinomycetemcomitans strains HK 921(= JP2) and HK975 (= Y4) in 10 ml 2×YT medium [35] were incu-bated anaerobically in an atmosphere with 85% (v/v) N2, 10% (v/v)CO2, and 5% (v/v) H2 or aerobically in ambient air supplementedwith 5% (v/v) CO2. Themedia were pre-equilibrated in the respectiveatmospheres and inoculated from anerobic or aerobic overnightcultures on chocolate agar as appropriate. After incubation for 20 hat 37 °C, bacteria were separated from supernatant by centrifuga-tion, the bacteria re-suspended in water to the original volume,and equal volumes of bacteria and supernatant were analyzed forcontent of LtxA by SDS-PAGE and Western blotting as describedbelow.

2.3. Production and purification of leukotoxin

Fifteen litres of 2×YT medium distributed in 1-litre bottles werepre-warmed to 37 °C and inoculated with A. actinomycetemcomitansstrain HK921 from an overnight culture on chocolate agar. The bottles,with loose-fitting caps, were incubated in air plus 5% (v/v) CO2 at37 °C for 20 h corresponding to early stationary phase. The density ofthe culture was followed by direct counting using a Helber countingchamber. The culture was centrifuged at 12,500 ×g for 30 min at 4 °Cand the supernatant was passed through a 0.45 μm pore-size filter(Millipore). The supernatant was supplied with 2 mM of sodium azide.

Using an Äcta 900 Purifier system (GE Healthcare) operated at4 °C and equipped with a sample pump (GE Healthcare, code S960),the supernatant was pumped at 2.5 ml min−1 through a Source S®(GE Healthcare) cation exchange column (10×1 cm), equilibratedin 20 mM Tris, 40 mM NaCl, 0.2 mM CaCl2, 2 mM NaN3, pH 7.1(buffer A). After washing with 15 column volumes of buffer A, mate-rial was eluted from the column with a linear gradient of NaCl(40 mM to 1 M over 100 min) in buffer A at 0.5 ml min−1. Elutedproteins were monitored spectrophotometrically at 280 nm and elu-ent fractions (0.5 ml) were analyzed for content of LtxA by SDS-PAGEand Western blotting (see below). LtxA-containing fractions werepooled and further fractionated on a gel permeation chromatography(GPC) column (0.8×30 cm) of Superose 12 HR (GE Healthcare)equilibrated in 20 mM Tris, 250 mM NaCl, 0.2 mM CaCl2, pH 7.1(LtxA buffer), and calibrated with standard proteins (GE Healthcare).Proteins eluted at 0.5 ml min−1 were monitored at 280 nm and0.5 ml eluent fractions were analyzed by SDS-PAGE/Western blotting.LtxA-containing fractionswere pooled and concentrated approximately

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10 fold using a Centriprep YM 10 filtration unit (Millipore). In some ex-periments the LtxA purification protocol was extended by passing thepreparation through a 1 by 15 cm column of Polymyxin B activated aga-rose (Sigma-Aldrich) at 0.3 ml min−1 to removepotential contaminatingLPS. The concentration of purified LtxA was determined spectrophoto-metrically using E280nm (1 cm, 1 mg ml−1)=0.726 as determined fromthe amino acid sequence using the ExPASy software (www.expasy.org).In certain experiments, LtxA-containing eluent fractions were suppliedwith a protease inhibitor cocktail (Complete, EDTA free, RocheDiagnostics) at a concentration corresponding to one tablet per 50 mlas recommended by the manufacturer.

For comparison we also applied the protocol described by Kachlanyet al. [31] involving ammoniumsulfate precipitation to purify LtxA. Theprecipitation was followed by dialysis of the re-dissolved precipitateand finally ultracentrifugation to eliminate a potential contaminationwith lipid vesicles.

2.4. Bioinformatical analyses and synchrotron radiation circular dichroismspectroscopy

The primary structure of LtxA [9] was analyzed for the content ofhigh-order structure by use of PSIPRED (version 2.6) [36] at http://bioinf.cs.ucl.ac.uk/psipred/ for the prediction of the secondary structureand with DisProt algorithm (release 5.8) [37] to predict unfolded re-gions at http://dis.embl.de/ using a window size of 11 residues. To sup-port these predictions, structural homologues to LtxAwere identified byuse of HHpred (release 2.17.0) [38] at http://hhpred.tuebingen.mpg.de/hhpred.

Synchrotron radiation circular dichroism (SRCD) spectra werecollected on Beamline CD1 at the ASTRID storage ring (ISA, AarhusUniversity, Denmark). The beam from CD1 [39,40] was polarizedwith a MgF2 Rochon polarizer (B-Halle GmbH, Berlin), and a photoelastic modulator (Hinds, USA) produced alternating left and righthanded circular polarized light. The light was passed though the sam-ple and detected by a photo multiplier tube (Type 9406B, ETL, UK).LtxA was measured at a concentration of 0.9 mg/ml in buffer with10 mMTris/H2SO4, 40 mMNa2SO4, and 0.2 mMCaCl2 at pH 7.4. Spectraof the plain bufferwere recorded for baseline subtraction. Samplesweremeasured in a 100 μmpath-length Suprasil cell (Hellma, GmbH). To testthe effect of calciumon the secondary structure, LtxAwas dialyzed over-night at a concentration of 1.4 mg/ml against 20 mMTris–H2SO4 pH 7.4,40 mM Na2SO4. SRCD recordings were done on LtxA at 1.26 mg/ml inthis buffer or with addition of 1 mMEDTA or 2 mM CaCl2. Temperaturescans of LtxA were made in the presence of either 1 mM EDTA or 2 mMCaCl2 going from 25 °C to 77 °C in steps with 5 min equilibration aftereach step before the SRCD scan. All sample and baseline spectra werecollected 3 times with 1 nm step size and 2 s dwell time. The spec-tra were averaged, baseline subtracted, and mildly smoothed with aSavitzky–Golay filter using the CDtool software [41].The secondarystructure content was calculated from the spectra by fitting the experi-mental data using the CDSSTR algorithm [42] and the SP175 protein CDspectra (175–260 nm) reference set [43].

2.5. Amino acid sequence and MALDI-MS analyses

Samples were denatured in SDS sample buffer at 80 °C in the pres-ence of 50 mM dithiothreitol. SDS-PAGE was performed in a 10% gel(10 cm×10 cm×0.15 cm) using the glycine/2-amino-2-methyl-1,3-propanediol/HCl system described previously [44]. The gel waselectroblotted to Immobilon-P membrane (Millipore) in 10 mM 3-[cyclohexylamino]-1-propane sulfonic acid (CAPS) 10% methanol,pH 11 [45] and stained for protein using Coomassie Brilliant Blue.Bands of interest were excised and placed directly onto Polybrene-treated glass filters. Samples were analyzed by automated Edmandegradation using an Applied Biosystems Procise 494HT sequencer

with on-line phenylthiohydantoin HPLC analysis. The instrumentswere operated according to instructions by the manufacturer.

Bands derived from SDS-PAGE gels were excised and washed ex-tensively with water and acetonitril. Trypsin (Promega) in 50 mMNH4HCO3 (12.5 ng/μl) was added to the gel plugs and incubatedat 37 °C for 16–17 h. Peptides were isolated using C-18 ZipTips(Millipore) and mixed with α-cyano-4-hydroxy-cinnamic acid at2 mg/mL in 50% (v/v) acetonitril/0.3% (v/v) trifluoroacetic acid. Ap-proximately 0.5 μl was spotted on MALDI targets and analyzed byMALDI-MS and MALDI-MS/MS using a quadrupole/time-of-flightUltima Global mass spectrometer (Micromass). Data was submittedto a local Mascot server as well as manually interpreted.

2.6. Production of polyclonal Ab to LtxA

The primers 5′-CCATGGGTTATGATGGCGATGATCG-3′ and 5′-GGATCCAGCAGTAGTTGCTAACGAAT-3′ were used to amplify a fragmentof the lktA gene from A. actinomycetemcomitans strain HK921 (GenBankaccession number X16829) encoding 310 amino acid residues in theC-terminal part of the LtxA protein [9]. The primers added an NcoI anda BamHI site at opposite ends. The PCR fragment was cloned usingthe TA cloning kit (Invitrogen), and the NcoI/BamHI fragment of theresulting recombinant plasmid was subcloned into the expressionvector pQE60 (QIAGEN) cleaved with the same restriction enzymesand transformed into E. coli BL21. The construct adds the sequenceGSRSHHHHHH to the C-terminus of the LtxA fragment and providesthe His-tag that facilitates purification using affinity chromatographyon Ni-NTA agarose. Expression of the recombinant proteinwas inducedby adding IPTG, and the protein was purified using denaturing condi-tions as described (The QIAexpressionist, QIAGEN). Immunization ofrabbits with the LtxA fragment was done at the animal facility of theDAKO Corporation. Immunoglobulin from immune serumwas purifiedas described [46]. Purified immunoglobulin was biotinylated usingbiotinamidocaproate N-hydroxysuccinimide ester (Sigma-Aldrich) asdescribed [47].

2.7. Western blotting of LtxA

SDS-PAGE was done either in 4–20% gradient gels as previouslydescribed [48] or in 7% Tris-acetate buffered gels (Nu-PAGETM,Invitrogen) as described by the manufacturer. Proteins were stainedwith Coomassie Brilliant Blue R250 or, afterWestern transfer at 5 V/cmovernight to a polyvinylidene difluoride membrane (Millipore), by re-action with specific ligands. Gels with non-LtxA proteins were blottedat pH 8.4 (27) whereas gels containing LtxA were blotted in a bufferof 10 mM CAPS, pH 11.0, containing 10% (v/v) methanol. Pilot experi-ments demonstrated that LtxA, having a pI of 9.0 as calculated fromthe amino acid sequence using the ExPASy software (www.expasy.org), was incompletely transferred when using the pH 8.4 buffer origi-nally recommended for Western blotting of proteins [49]. Blocking ofmembranes, incubation with Ab and alkaline phosphatase conjugates aswell as washing were done in 20 mM Tris, pH 7.5, containing 250 mMNaCl, 0.15% (v/v) Tween 20, 1% (w/v) of gelatine, and 5 mM sodiumazide (incubation buffer) and blots were developed with a chromogenicsubstrate of bromo-chloro-indolylphosphate/nitroblue tetrazolium.

In some experiments, Western blots of LtxA preparations were ex-amined for the presence of bacterial LPS by incubation in a solution ofperoxidase-conjugated Polymyxin B, prepared essentially as described[50] and kindly provided by Uffe S. Sørensen, Dept. of Biomedicine,Aarhus University, Denmark. Polymyxin B binds selectively to the lipidA portion of lipopolysaccharide [51]. In these experiments sodiumazide was omitted from the incubation and washing buffer. Prior toincubation with conjugated Polymyxin B, some blots were incubatedwith 50 mM sodium meta-periodate in PBS, pH 7.4, for 1 h at roomtemperature to selectively inactivate carbohydratemoieties. PolymyxinB-binding bands were visualized by chemiluminescense using

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SuperSignal substrate (Thermo Scientific, Rockford, IL) and a LAS 3000analyzer (Fujifilm, Woodbridge, CT). In later purification experimentsprepared LtxA was examined for potential contaminating LPS by asensitive Limulus amoebocyte lysate (LAL) based assay (kineticturbidimetric version) at Scan Dia Laboratories Aps, Borkop, Denmark(www.scandialabs.com).

2.8. Enzyme-linked immunosorbent assay for detection of LtxA

To detect LtxA by enzyme-linked immunosorbent assay (ELISA),polystyrene microplates (cat.no. 269620, Nunc, Roskilde, Denmark)were coated overnight with rabbit Ab to LtxA in 10 mM phosphate-buffered saline, pH 7.4 (PBS). After washing and blocking the platewith washing solution (PBS containing 0.25 M NaCl and 0.15%Tween 20), test samples appropriately diluted in washing solutionwere incubated in wells for 2 h. Serial three-fold dilutions of a prep-aration of purified LtxA starting at 0.1 μg ml−1 were included on theplate to provide for quantification of LtxA in gravimetric units. BoundLtxAwas detected by sequential incubations with biotinylated rabbitAb to LtxA and alkaline phosphatase-conjugated streptavidine(DAKO). The assay was developed with a chromogenic substrateof p-nitrophenylenephosphate in diethanolamine buffer, pH 9.0.Plates were read at 405 nm by a Multiscan RC reader (Labsystems).

2.9. Binding specificity of LtxA by ligand blotting

Cell membrane proteins from K562 cells and from K562 cellstransfected with DNA encoding β2 integrin in the form of αLβ2,αMβ2, or αXβ2, respectively, were prepared as described by Li et al.[52]. Briefly, 109 cells were broken using a Potter-Elvenhjem grinderin 50 mM 3-(N-morpholino)-propanesulphonate (MOPS) buffer, pH7.0, and cell membranes were separated from intact cell and nucleiby differential centrifugation. Cell membranes were extracted with 1%(v/v) Triton X100 in MOPS buffer. Extracts were mixed with non-reducing SDS-PAGE sample buffer, separated in 4–20% (w/v) polyacryl-amide gels, and either stained with Coomassie Blue or electroblotted toa PVDF membrane. Duplicate blots both of which included proteinsrepresenting each of the four cell types in separate lanes were probedwith monoclonal Ab (KIM127) to the β2 chain (CD18), or with purifiedLtxA, 2 μg ml−1, in blocking buffer supplied with 5 mM CaCl2. Theformer blot was developed with alkaline phosphatase-conjugatedrabbit Ab to mouse Ig (DAKO), the latter blot with biotinylated rabbitAb to LtxA followed by alkaline phosphatase-conjugated Streptavidin(DAKO). Blots were stained with bromo-chloro-indolylphosphate/nitroblue tetrazolium as chromogenic substrate. Secondary reagentcontrols were included, in which first layer Ab was substituted withmouse Ig isotype control or preimmune rabbit Ig.

2.10. Cytolytic activity of LtxA determined by luminescent cell viabilityassay

The cytolytic activity of LtxA was analyzed by the use of K562 cellswith recombinant expression of integrin αLβ2, αMβ2, or αXβ2. Theparent K562 cell line devoid of β2 integrin served as control. Cellswere cultured in RPMI 1640 medium (containing 0.5 mM Ca2+ and5 mM phosphate) supplemented with glutamine, 10 mM HEPES,10% (v/v) fetal calf serum, and antibiotics. The effect of LtxA was de-termined by titration using the CellTiter-Glo™ luminescent cell via-bility assay as described by the manufacturer (Promega). The assayestimates the number of viable cells in culture based on quantifica-tion of ATP in metabolically active cells. In a pilot experiment, lumi-nescent signals from serial two-fold dilutions of a suspension ofK562 cells in exponential growth phase were measured to ascertainthat the signal was directly proportional to the number of viablecells as employed in the assay. In assays of LtxA-mediated cytol-ysis, 50 μl volumes of serial two-fold dilutions of LtxA starting

at 10 μg ml−1 in supplemented medium were dispensed in duplicatein wells of opaque microplates. Each well then received 2×104 cells in50 μl of supplemented medium. The plate was incubated at 37 °C for1.0 h, processed for detection of viable cells, and luminescence wasread in a Victor-3 reader (PerkinElmer). Controls included wells withcells in medium without LtxA (spontaneous lysis, S0) and in mediumwith detergent causing lysis of all cells during the incubation (maximallysis, SMax). The relative cell viability, Vrel, was estimated from thesignals from wells incubated with LtxA (SLtx) and the signals from thecontrol wells according to the equation:

Vrel ¼ 100%⋅ SLtx−SMax

S0−SMax

2.11. Hemolysis assay

Hemolytic activity was measured as light absorbance by hemoglo-bin released from lysed erythrocytes. Cells from 5 ml of heparin-stabilized blood from each of two healthy donors were washed 10times in 15 ml of a solution with sodium chloride, adenine, glucose,and mannitol (SAGM; Suru International, Mumbai, India). After eachwash the sample was centrifuged at 200×g for 15 min and thebuffy coat discarded. The final suspensions contained from 17 to 26nucleated cells per 100,000 erythrocytes as revealed by flow cytometryupon DNA-specific stainingwith Vybrant Dye Cycle Green (Invitrogen).Hemolysis reactionswere performed in 1 ml of a 1% (v/v) suspension oferythrocytes in SAGM suppliedwith 3 mMCaCl2 and 100 μg ampicillin.Purified LtxA to a final concentration of 5 μg ml−1 was added in avolume of 10 μl. Reactionmixtures were incubated at 37 °Cwith gentleshaking and samples of 150 μl were withdrawn after 0, 2, 4, 6, and 23 h.Intact erythrocyteswere removed by centrifugation and the absorbanceat 450 nm of the supernatant was measured using a microplate reader.Four controls were included where (i) erythrocytes were incubated inwater (maximal lysis, HMax), (ii) erythrocytes were incubated in bufferwithout LtxA (spontaneous lysis, H0), (iii) erythrocytes were incubatedwith toxin inactivated by incubation with 1 mg ml−1 proteinase K for1 h at 55 °C followed by incubation at 95 °C for 10 min to inactivateproteinase K, or (iv) the reaction mixture was supplemented with10 μl 1% (v/v) of polyclonal rabbit Ab to LtxA. The relative hemolysis,Hrel, was estimated by comparison of the signal from wells incubatedwith LtxA (HLtxA) to the signals from the control wells according tothe equation:

Hrel ¼ 100%⋅HLtxA−H0

HMax−H0

Each reaction was run in triplicate wells.

3. Results

3.1. Production of LtxA during anaerobic and aerobic culture

Analysis of parallel anaerobic and aerobic cultures of strain HK921and strain HK975 demonstrated oxygen-dependent variations in theprotein profiles of the bacterial cells (Fig. 1A). In none of the strains,however, did these variations involve LtxA as verified by Westernblotting (Fig. 1B).

LtxA in culture medium of A. actinomycetemcomitans strain HK921reached its maximal level at early stationary phase (more than109bacteria ml−1) after 17–20 h with the toxin being distributedequally between bacterial cells and supernatant, and with no signsof degradation (Fig. 2). In the supernatant the concentration of LtxAwas 1.5 to 2.0 μg ml−1 as determined by ELISA. The pH was 5.8increasing to 6.0 during the process of sterile filtration which wasdriven by vacuum suction.

Fig. 1. Comparison of LtxA production under anaerobic and aerobic conditions by A. actinomycetemcomitans strains HK 921 (=JP2) and HK975 (=Y4), respectively. Culture supernatantsfrom samples collected after 20 h were isolated by centrifugation and the bacterial pellet was resuspended to the original volume in water. (A) Coomassie-stained SDS-PAGE profilesof proteins in bacterial cells and corresponding supernatants. (B) Western blot of a gel corresponding to (A) developed with biotinylated Ab to LtxA followed by alkalinephosphatase-conjugated streptavidin. Note that whereas oxygen-regulated expression of a ~85 kD protein was evident (A), this does not apply to LtxA.

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3.2. Purification of LtxA from culture supernatant using ion exchangechromatography

The purification method described by Kachlany et al. [31] involv-ing ammonium sulphate precipitation and ultracentrifugation of theredissolved product, was adopted initially. Upon gel permeationchromatography and analysis of eluent fractions for LtxA by ELISA,it was evident that approximately half of the LtxA remained in aggre-gated form after dissolution in buffer (Fig. 3A). The largest aggre-gates eluted with the void volume of the calibrated Superose 6 HRcolumn, i.e., corresponding to Mr≥2×106 (Fig. 3A).

With the alternative protocol LtxAwas immobilized directly fromthe culture supernatant onto a cation exchange resin. Peptides orother constituents of the medium did not interfere significantlywith the binding since a 10 ml column of packed resin quantitativelyretained LtxA from 15 l of culture supernatant (corresponding to~25 mg protein) as revealed by SDS-PAGE of supernatant samplescollected at the column outlet (not shown). An attempt to performbatch immobilization of LtxA by suspending the same amount ofcation exchange resin in 6 l of culture supernatant with gentle agita-tion for 16 h at 5 °C resulted in only 22% recovery of the toxin asdetermined by ELISA.

LtxA was eluted from the cation exchanger as a distinct peak at0.4 M NaCl (Fig. 3B). Most other proteins co-eluting with LtxA were ofsmaller molecular size as they were eliminated by the subsequentGPC step (Fig. 3C). LtxAwas eluted from the calibrated GPC as a distinctpeak with an elution volume approximately corresponding to non-aggregated toxinmolecules of the expected size (Mr~114 k) as demon-strated by the protein elution profile (Fig. 3C, D) and Western blot-ting analysis of eluent fractions (Fig. 3E). A hydrodynamic diameter of

Fig. 2. Accumulation of LtxA produced by A. actinomycetemcomitans strain HK921 duringaerobical cultivation in 2×YTmedium. Culture supernatants from samples collected after13, 17, and 20 h were isolated and the bacterial pellets were resuspended to the originalvolume in water. Corresponding samples of bacteria and supernatant were analyzed forcontent of LtxA by SDS-PAGE/Western blotting. Note that LtxA was equally distributedbetween bacteria and supernatant and reached its maximal level after 17–20 h.

4.5 nm for the purified Ltx was calculated by reference to diameters ofproteins used for calibration of the column.

3.3. Structural folding of LtxA

With the indication that at least certain protein purificationmethodsare able to affect the structural integrity of purified LtxA, we consideredthe possibilities for assessing the structural content of the protein puri-fied according to our protocol. However, in the absence of any informa-tion on the LtxA structure with atomic resolution, we explored thepossibilities for using bioinformatical tools for creating a reference forcomparison.

The secondary structure was predicted using the PSIPRED [36] algo-rithm and the over-all folding was predicted by the use of the DisProtalgorithm [37]. Interestingly, the PSIPRED analysis predicted that LtxAhas an almost entirely alpha helical region in the 460 N-terminal resi-dues, followed by a long region (residues 461–1055), which includesthe RTX repeats, exclusively containing beta strands. Most of thesebeta strands were predicted to be short (2–4 residues) as typicallyfound in parallel beta roll, also named beta ladder, structures [53](Table 1).

As expected, the HHpred algorithm [38] identified a number of pro-teins with sequence similar to the region of LtxA spanning residues610–899 and with known three dimensional structure. The proteinwith the highest score in the HHpred analysis was a family I.3 lipasefrom Pseudomonas sp. MIS38 (PML) [54]. To validate the accuracy ofthe PSIPRED prediction we aligned the matching sequence of PMLwith LtxA and compared the structural elements found in PML withthose predicted for the LtxA sequence. The primary structure of LtxAcorresponding to residues ~600–900 and the residues ~250–580 inPML show a considerable structural similarity (Fig. 4A). The predictedsecondary structure of LtxA in this region had several similarities withPML, notably a number of shared beta sheets. From the known tertiaryorganization of PML and other similar proteins [54,55] these betastrands in LtxA are likely to be organized in a parallel beta roll. However,in a few positions there seemed to be differences between the second-ary structure of PML and the predicted structure of LtxA, notably inthe segment with the residues ~785–830 (LtxA numbering). Here,the analysis by the DisProt algorithm predicted the segment to beunordered, consistent with a relative reduction in secondary LtxAstructure content according to the prediction by the PSIPRED algorithm(Fig. 4A). Taken together, these findings suggest that the PSIPRED algo-rithmoffers a reasonably accurate estimate of the total secondary struc-ture content of LtxA.

The secondary structure content of LtxA purified according to ourprocedurewas investigated by SRCD. From recently developed softwarewe found that the structure contentwas distributed as 29% alpha helicalsegment, 24% beta sheets, and a total content of unordered or beta sheetturns of 47% when the recording of the spectrum was made in bufferwith 10 mM Tris/H2SO4, 40 mM Na2SO4, and 0.2 mM CaCl2 at pH 7.4

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(Table 1). These findings agreed well with the prediction made byPSIPRED suggesting that the LtxA preparation was folded according tothe expectations for the native structure.

The average length and number of secondary structure elements inLtxA was analyzed using the CDSSTR algorithm [42]. The SP175 dataset [43] optimized for the wavelength range of 175–240 nm corre-sponding to our data range was used for optimal results. The analysis(Table 1) revealed an average beta strand length of 5 residues and atotal number of beta strands of 45 in LtxA. This corresponds well withthe PSIPRED prediction giving an average beta strand length of 3.5 res-idues and a total number of beta strands of 47. An average alpha helixlength of 8.2 residues and a total number of helices of 38 was the resultof fitting the experimental data using CDSSTR. In comparison, PSIPREDpredicted an average helix length of 15.0 and a total number of helicesof 20. As the average helix length derived from PSIPRED was twicethat revealed by CDSSTR analysis of the recorded spectra and the num-ber of helices was half, PSIPRED on the average predicted a long helixinstead of two helices of approximately half the length as observedexperimentally.

As the repeats-in-toxin motif in LtxA has been suggested to be acalcium-binding motif, we tested the effect of calcium on the second-ary structure of LtxA. Following careful dialysis, the SRCD spectrawere recorded in the presence of 1 mM EDTA or 2 mM CaCl2 addedto test the effect of no calcium and high calcium, respectively. At25 °C the spectra were identical and thus showed no effect of calcium(Fig. 4B, C). To test if calcium had an effect on the stability of LtxA,temperature scans were made in the presence of 1 mM EDTA or2 mM CaCl2 (Fig. 4B, C). The temperature scans revealed a significanteffect of calcium on the stability of LtxA. The melting temperaturescalculated from the curves in Fig. 4B and C were 49.8 °C and 45.2 °Cin the presence of 2 mM CaCl2 or 1 mM EDTA data, respectively.This indicates that calcium has a stabilising effect on the structureof LtxA. Interestingly, the thermal destabilization appeared to followquite different routes under these two conditions. As shown in Panel4B, the SRCD signal at nearly all wavelengths was essentially lost athigh temperatures in buffer with calcium showing that the thermalunfolding caused a concomitant precipitation of LtxA. By contrast,thermal unfolding in buffer with EDTA preserved a considerablyhigher dichroism (Fig. 4C). A comparison of the raw SRCD signals atλ=194 nm as a function of the temperature further illustrates thisdifference (Fig. 4D).

3.4. Storage-dependent degradation and purity of Ltx

The purified LtxA was stable when stored at −80 °C. However, ifkept unfrozen for extended periods limited degradation occurred.Sequential analysis during storage at 4 °C indicated that LtxA wasgradually degraded to a stable product over a period of 120 dayswith an apparent Mr of the truncated protein at ~105 k (Fig. 5A).The rate of degradation varied between batches of purified LtxA,was slightly faster during storage at room temperature or at 37 °C,

Fig. 3. Comparison of two LtxA preparations purified from A. actinomycetemcomitans cul-ture supernatant. (A) GPC of LtxA purified according to a protocol involving ammoniumsulfate precipitation and ultracentrifugation [31]. Eluted protein was monitored by UVabsorbance (A280nm) (solid line). LtxA in collected fractions was quantified by ELISA andexpressed in units of absorbance at 405 nm (hatched line). The void (V0) and total (Vt)volumes for the Superose 6® column are indicated together with elution volumesfor three of the calibration markers (thyroglobulin[669 kDa], aldolase [158 kDa] andovalbumine [43 kDa]). (The peak in the LtxA profile corresponding to monomeric Ltx islabeled with the estimated hydrodynamic diameter). (B,C) Purification of LtxA by ion ex-change chromatography (B) followed by GPC (C). Protein eluted from the ion exchangecolumnbya gradient of NaCl (hatched line)wasmonitored byA280 nm (solid line). Collect-ed fractions containing LtxA (bar) were pooled and subjected to GPC on a Superose 12®column. Calibration markers (and the estimated hydrodynamic diameter) are shownand LtxA-containing fractions are indicated. (D,E) SDS-PAGE followed by Coomassiestaining (D) or Western blotting (E) of the fractions from the GPC shown in Panel C. Mr

standards are indicated.

Table 1Secondary structure content of LtxA predicted by the PSIPRED and CD spectroscopy.

α helixa β sheeta Unstruc.a

% Avg. length (res.) N % Avg. length (res.) N %

PSIPREDb 28.4 15.0 20 15.1 3.5 47 56.5SRCDc 29 8.2 38 24 5.4 45 35 (12)d

a The distribution of secondary structure was given as over-all percentages secondary structure elements, average length of the structure segments, and number of segments.b Distribution determined by PSIPRED from simple counting of the identified secondary structure segments.c The CD spectrum was analyzed by the CDSSTR algorithm returning numbers for over-all percentage of structural content as well as the segmental distribution (average length

and number [N] of segment).d The unstructured content of LtxA was returned as the percentage of coils with the percentage of turns stated in brackets.

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and could not be inhibited by the addition of a mixture of proteinaseinhibitors with or without EDTA (not shown). Degradation occurredalso in batches of LtxA purified after renewal of column materialsand tubing.

To characterize the intact and truncated forms of LtxA, individualprotein bands were subjected to N-terminal sequencing as wellas MALDI-TOF MS. The same sequence of N-terminal amino acids(ATTTLPNTKQQ), equivalent to the N-terminus of the open readingframe of ltxA except for lack of the start methionine [9], was obtainedfrom both forms of LtxA, indicating that degradation was in theC-terminal part. From MALDI-TOF MS data, estimated molecularmasses of 114,350 Da and 109,300 Da were calculated for the intactand the truncated form, respectively. By reference to the amino acidsequence of LtxA [9], the eliminated 5050 Da corresponded to resi-dues L-1006 to A-1055 (5064.51 Da), A-1055 being the C-terminus.In silico analysis of the LtxA sequence by the DisProt algorithm [37]revealed that the C-terminal part subjected to degradation was rela-tively unstructured and of low polarity (Fig. 5B).

Fig. 4. Comparison of the secondary structure predicted by the PSIPRED algorithm and experimPsiPred [36,88] and from a comparisonwith the X-ray crystallographic structure of PML [54] (PDsequence alignment of LtxA and PML by the Clustal W2 algorithm [89] is outlined by horizonidentity or similarity between aligned residues. Alpha helical or beta sheet segments for LtxAwas calculated with the DisProt algorithm and indicated with a color code. A value higher thantemperatures of LtxA in the presence of 2 mM CaCl2 and 1 mM EDTA, respectively. (D) The CD

LtxA is modified by acylation of two lysyl residues, Lys-562 andLys-687 [56]. The estimated mass of intact LtxA (114,350 Da) com-pared to the mass of the LtxA polypeptide determined from theamino acid sequence (113,723 Da), leaves ~627 Da to be accountedfor by the modifications. The fatty acids of LtxA have recently beenfound to be heterogeneous [18].

To examine for contaminationby LPSweprobedWesternblots of LtxApreparations with peroxidase-conjugated polymyxin B. LtxA preparedaccording to Kachlany et al. [31] displayed polymyxin-binding substancesmigrating as a blurred band with mobility corresponding to proteinswith Mr≤25 k, which were not separated by the 7% polyacrylamidegel employed (Fig. 5D). Also, in this preparation, protein-containingsubstances co-migrating with the polymyxin-binding substances wereobserved (Fig. 5C). Polymyxin B-reactive substances of similar mobilitywere obtained from cultivated A. actinomycetemcomitans bacteria and,barely visible, from the corresponding culture supernatant (Fig. 5D).In contrast, no polymyxin B-reactive substances, and no co-migratingprotein, were detected in any of two batches of LtxA purified as described

ental analysis by use of SRCD. (A) Prediction of the secondary structure in LtxA by use ofB #2Z8X). For clarity only the C-terminal part of LtxAwith similarity to PML is shown. Thetal black lines with the sequence numbering indicated. Black dots are shown to indicateand PML are indicated with cylinders and arrows, respectively. The disorder probability0.5 suggests that the sequence is unfolded. (B,C) SRCD spectra for successively increasingsignal of the main positive peak at 194 nm as a function of temperature.

Fig. 5. Storage-dependent proteolysis and purity of LtxA. (A) SDS-PAGE of purified LtxAincubated at 4 °C for 1, 20, 87, 103, or 120 days. (B) In silico analysis of the structuralcontent and polarity of the LtxA sequence. The disorder probability (solid line) wascalculated with the DisProt algorithm as mentioned in the legend to Fig. 4. The polarityindex (hatched line) was estimated according to the method of Zimmerman et al. [90]using a window size of 11 residues in the ProtScale algorithm at http://www.expasy.org/.The cleavage site in LtxA at the COOH-terminal side of Met-1005 identified bymass spectrometry is indicated (arrow). (C–E) Analysis of LtxA samples for contentof LPS. Proteins extracted from cultured HK 921 bacteria, the corresponding culturesupernatant, and LtxA prepared according to Kachlany [31] [LtxA (1)] or according tothe protocol presented here [two different preparations: LtxA(2) and LtxA(3)] wereanalyzed by SDS-PAGE and Western blotting (C). Blots were probed with peroxidase-conjugated Polymyxin B directly (D) or after treatment with sodium meta-periodate(E). Lanes received equal amounts of sample. Fig. 6. Comparison of newly purified, intact LtxA (A) and LtxA truncated during storage

for 120 days (B) with respect to cytolytic activity against K562 cells with recombinantexpression of β2 integrin in the form of either αLβ2, αMβ2 or αXβ2. Recombinant K562cells and K562 control cells devoid of β2 integrin were incubated in triplicate with LtxAat 37 °C for 1 h upon which cell viability (median, SD) was determined by theCellTiter-GloTM assay. The data suggest moderate differences in the susceptibility ofthe three integrin-expressing target cells to LtxA. Note also that the truncated LtxAmaintained lytic activity but at a reduced level compared to the intact toxin. Datafrom one out of two sets of experiments with similar results are shown.

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in this report (Fig. 5C, D). Treatment of blotted substances fromA. actinomycetemcomitans cultures and LtxA preparations with sodi-um meta-periodate inhibited reactivity with enzyme-conjugated

polymyxin B (Fig. 5D, E), indicating that the polymyxin B-bindingmolecular structures were of carbohydrate nature.

In spite of these results analysis of our LtxA preparation with anLAL based assay revealed a content of LPS of 22 EU (i.e. 2.2 ng) permg of LtxA, whereas LtxA prepared according to Kachlany et al.[29] was estimated at 21,434 EU of LPS per mg of LtxA protein.After passing our LtxA preparation through a column of PolymyxinB activated agarose, LPS was estimated at 0.5 EU per mg of LtxA byLAL based analysis. This estimate was not significantly differentfrom that obtained for the buffer control.

3.5. Cytolytic specificity of LtxA determined by the CellTiter-Glo™ assay

The interaction of purified LtxA with different human β2 integrinsas reflected in cytotoxicity was compared using three lines of K562cells as targets, each transfected to express a single variant of β2

integrin (αLβ2, αMβ2, or αXβ2). Dose-dependent cytotoxicity of LtxAwas observed irrespective of which of the β2 integrins was expressedby the target cells whereas K562 parent cells were left intact (Fig. 6A).Moderate differences in the toxicity of LtxA against the three integrin-expressing target cells were observed (Fig. 6A). However, moderateand partly corresponding differences were apparent also in the expres-sion ofβ2 integrins by the three transfected cell lines (Fig. 7B). Thus, theexperiment suggested the three β2 integrins to be of comparable effi-ciency in transferring LtxA-mediated cytotoxicity.

We compared the toxicity of intact LtxA to that of LtxA withstorage-induced degradation of the carboxy-terminal. The degradedtoxin maintained toxicity towards all three β2 integrin-expressingtarget cells although at a reduced level compared to the intact toxin(Fig. 6B).

Fig. 7. Interaction of LtxA with β2 integrin component chains as studied by ligand blotting. Proteins extracted from cell membranes of K562 cells and from K562 cells transfected toexpress individual β2 integrins were separated by non-reducing SDS-PAGE (panel A), electroblotted to PVDF membranes and probed either with the monoclonal Ab KIM127 [33] tothe β2 chain (B) or with purified LtxA (panel C). Blots were developed with alkaline phosphatase-conjugated rabbit Ab to mouse Ig (panel B) or with biotinylated rabbit Ab to LtxAfollowed by alkaline phosphatase-conjugated Streptavidin (panel C) and stained with BCIP/NBT as chromogenic substrate. The specificity of primary Ab reactions were controlledby substituting KIM127with a mouse Ig isotype control (panel D) and anti-LtxA with preimmune rabbit Ig (panel E). The experiment shows that among the denatured β2 integrincomponent chains only the β2 chain (Mr 90 k) binds LtxA. In addition, the blot stained with KIM127 (panel B) indicates that the three transfected cell lines express their respectiveβ2 integrins at approximately equal levels.

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3.6. Binding specificity of LtxA

Cell membrane proteins extracted from equal numbers of theparental K562 cells and K562 cells expressing either αLβ2, αMβ2, orαXβ2 were separated by non-reducing SDS-PAGE (Fig. 7A), and pro-teins capable of binding LtxA were identified by ligand blotting.LtxA was bound by a membrane component with an apparent Mr of90 k extracted from each of the three transfected cells lines but notfrom K562 parent cells (Fig. 7C). In duplicate lanes this componentwas identified as β2 chain (CD18) by its binding of CD18-specific

Fig. 8. Hemolysis of erythrocytes from two normal human volunteers (A and B). Hemolysifunction-blocking Ab (LtxA+anti-LtxA), or inactivated by proteolytic treatment (Inactiv. L±SD of triplicate determinations is shown. In panel A the lines representing the lysis in pla

monoclonal Ab (Fig. 7B). No binding of LtxA was detected at a positioncorresponding to the Mr of each of the three α chains (Mr≥145 k).Furthermore, application of relevant control Ab showed no staining atthe position of the β2 chain (Fig. 7D, E).

3.7. LtxA-induced hemolysis

Contrary to the brisk effect of LtxA on leukocytes, LtxA-inducedhemolysis required hours. LtxA at 5 μg ml−1, which lysed 80% of β2

integrin-expressingK562 cells during 1 h, caused less than10%hemolysis

s was induced by LtxA at 5 μg ml−1 either as native protein (LtxA), in the presence oftxA). A control was included in which SAGM buffer substituted for LtxA. The medianin buffer and in buffer with inactivated LtxA are overlapping.

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during 2 h and 30 to 50% hemolysis only after 24 h, and erythrocytesfrom two different donors showed a difference in sensitivity to thetoxin (Fig. 8A, B). The findings that no hemolysis was observedwith LtxA inactivated by incubation with proteinase K and thathemolysis could be inhibited with Ab to LtxA (Fig. 8A, B) confirmedthat the hemolysis observed was caused by LtxA.

4. Discussion

LtxA is anticipated to be an important virulence factor ofA. actinomycetemcomitans [1,7] and the toxic as well as stimulatoryeffects of LtxA on leukocytes have been studied for long in vitro. Morerecently, it was shown that LtxA is also responsible for the hemolyticactivity of A. actinomycetemcomitans [20]. However, preparations ofLtxA may be contaminated by LPS [27,29], a substance whichmay itselfcause strong reactions in leukocytes [57,58]. Also, conceivably, LtxA–LPS heterocomplex formation may change the reactivity of LtxA withtarget cells. Thus, experiments with LtxA contaminated with LPS maylead to misinterpretation of LtxA-mediated effects as demonstratedfor a closely related leukotoxin [59]. Here, we describe a protocol forthe preparation of LtxA in pure and non-aggregated form. An LtxApreparation containing only trace amounts of LPS was obtainedfrom bacterial culture supernatant by non-denaturing chromato-graphic procedures. Using such LtxA, we have examined the effectsof the toxin on cells carrying different human β2 integrins and onpure preparations of human erythrocytes.

Complete removal of LPS as verified by a sensitive LAL based assaywas obtained by final filtration through Polymyxin B activated agarose.

4.1. Purification of LtxA

Strain HK921 (=JP2) of A. actinomycetemcomitans was chosen assource of LtxA because of its elevated level of LtxA production. 50%of which was released to the medium. The 2×YT medium does notcontain large-sized proteins, which might be difficult to separatefrom LtxA by GPC. Accordingly, LtxA was the vastly dominant macro-molecular polypeptide in HK 921 culture supernatant. The productionor secretion of LtxA during culture has been found to depend on fer-mentable sugars [60] and limitation of iron [61]. However, because2×YT medium supported LtxA production by strain HK 921 to a con-centration of up to 2 μg ml−1 in culture supernatant, we did not at-tempt to optimize these factors in the medium. It has been reportedthat LtxA production in all A. actinomycetemcomitans [62], or in non-JP2 variants only [63], is suppressed by oxygen. In the case of JP2[62], this result was obtained with a strain modified to contain anintegrated plasmid carrying a leukotoxin-β-galactosidase fusion con-struct, where β-galactosidase functioned as a reporter. We examinedthe significance of oxygen for LtxA production by HK921 and thenon-JP2 genotype HK975, both genetically unmodified, by measuringLtxA directly in cells and supernatant and found no oxygen depen-dence of LtxA production in either of the strains. The initial quantita-tive immobilization of LtxA directly from the culture supernatantonto cation exchange resin may be explained by the high pI of LtxA(9.0) relative to the pH of 6.0 for the culture supernatant. Duringthe final GPC, LtxA was eluted in the form of monodisperse proteinmolecules as indicated by the volume of elution for LtxA relative tothose for standard proteins. By contrast, we found that LtxA preparedby a protocol described by Kachlany et al. [31], which was also basedon JP2 clone culture supernatant but included ammonium sulphateprecipitation of LtxA, consisted partly of aggregates. Thus, we suggestthat this method may induce aggregation, either in the form of LtxAhomoaggregates or heteroaggregates consisting of LtxA and anothercomponent not detectable by staining with Coomassie blue. Contraryto Kachlany et al. [31], we found that LtxA prepared as describedby these authors contained an unidentified protein material with an

apparent Mr~25 k or less. Theoretically, also this material may havebeen part of complexes with LtxA.

4.2. Purity of the LtxA preparation

LPS has been previously identified in preparations of RTX toxinsincluding LtxA [27,29,52] and found to be removable only by denatur-ing procedures like SDS-PAGE [27,52], suggesting heterocomplex for-mation. Non-covalent interactions between LPS and other substancesoften involve electrostatic as well as hydrophobic interactions [64].This is likely to apply to an LtxA-LPS complex as LtxA and LPS haveopposite overall electrical charges at the pH of the culture superna-tant and LtxA, like LPS, has a hydrophobic region within the molecule[65]. However, our results indicate that interactions between LtxAand LPS occurred only very sparsely in the culture supernatant unlessammonium sulphate was added. Presumably, addition of ammoniumsulphate stimulated aggregation through a salting-out effect. Ion ex-change chromatography has been previously found to separate LPSfrom protein if the pH is such that the two components have oppositecharges like in the present case [64,66].

The purified LtxA was subject to slow proteolytic truncation ifstored unfrozen for prolonged time by a protease not inhibited bytraditional protease inhibitors. The vicinity of the site ultimatelycleaved, 1005M–L1006, is rich in hydrophobic residues and the regionappears as unstructured. These characteristics are in agreement withthe cleavage profile of the ATP-dependent Lon proteases. Lon recog-nizes and degrades misfolded or unstructured regions of proteinswith non-globular conformation and is not dependent on a specificsubstrate peptide consensus sequence. However, it shows preferencefor hydrophobic residues such as leucine adjacent to the scissile peptidebond [67–69]. The genome of A. actinomycetemcomitans strain HK1651,a JP2 clone strain, has been sequenced at the Actinobacillus GenomeSequencing Project at University of Oklahoma (http://www.genome.ou.edu/act.html) and two open reading frames predicted by the LANLoral gene project (http://www.oralgen.lanl.gov/_index.html) are anno-tated as Lon protease genes. The two lon genes, AA02270 and AA02395,encode proteinswithMrs of 66 k and 90 k and predicted pI values of 4.9and 8.0, respectively. The Lon proteases function as oligomeric as-semblies and we suggest that very small amounts of such structuresco-purified with the LtxA preparation and caused the observed proteo-lytic degradation. Degradation of LtxA during prolonged culture ofA. actinomycetemcomitans has been previously observed [25,31], insome cases detectable merely by LtxA migrating as a double band inSDS-PAGE [28]. In these cases the proteolytic activity and the patternof degradation were not characterized and might have involved Lonproteases. Interestingly, a similar phenomenonwas reported for anoth-er RTX toxin, namely the adenylate cyclase-hemolysin toxin fromBordetella pertussis. In this case, a recombinant fragment containingthe RTX repeats was unusually susceptible to proteolytic cleavage.[70]. The truncated LtxA toxin displayed reduced leukotoxicitycompared to the intact form. This indicates that the unstructuredC-terminal part of the molecule is significant but not essential forLtxA-mediated leukotoxicity.

4.3. Structural integrity and properties of LtxA

The structural integrity of the purified LtxA was analyzed by com-paring the theoretically predicted secondary structure content withexperimental measurement of this property by SRCD. Compared tomore conventional methods for circular dichroism spectroscopy, syn-chrotron radiation is a considerably brighter light source in the lowwavelength regiment permitting a more robust estimate of the con-tent and distribution of secondary structure. The agreement betweenthe in silico analysis of the structure and the data from SRCD clearlysuggests that LtxA purified according to our protocol is well folded.With the problems in achieving such material from other protocols

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as also demonstrated in our paper, this structural enquiry is impor-tant and contributes a methodological aspect concerning the com-bined use of SRCD and algorithms for probing structural integrity bypredicting the secondary structure. Such an application of SRCD is,of course, greatly aided by the recent progress in analysing spectra,which now permits a quite detailed insight into properties of thesolution structure of proteins [39,71].

While our analysis with SRCD and the PSIPRED algorithm [36] donot enable an unambiguous insight into the secondary structure ofthe protein, it is noteworthy that nearly all beta-sheeted structure isfound in the C-terminal part of the molecule while alpha helices arefound in the N-terminal part (data not shown). The alpha helices pre-dicted by PSIPRED appeared in general to be longer than averagelength estimated from SRCD. Algorithms for predicting secondarystructure content have a particular challenge in localizing the bound-aries of alpha helices [72]. This was also reflected in PSIPRED analysisof LtxA, where these boundaries were established with less confi-dence than central parts (data not shown). In consequence, kinks inthe helical regions are easily missed in the predictions while SRCDwould identify these as an interruption of the helix, hence reducingthe calculated average length of the helices compared with PSIPRED.While the beta-sheeted structure is likely to form a parallel beta-roll-like structure resembling a part of the PML protein fromPseudomonas, it should be noted that the LtxA appears to containat least one large unfolded region inserted between the beta sheets,which is not found in the PML protein. It is an often-made observationthat such unfolded loop regions play important roles in forming con-tacts with other macromolecular partners for complex formation.Based on SRCD temperature scans calcium was found to stabilize thestructure of LtxA as was expected from the homology to the calciumbinding beta-roll domain of PML and other evidence that calcium is im-portant formaintaining the structural integrity of LtxA. However, unlikethe RTX repeats in adenylate cyclase toxin which are of unorderedstructure in the absence of calcium [73], it appears that the structureof LtxA is more resilient to removal of calcium since no structuraldiffrences were noted between the apo and holo forms when SRCDmeasurements were made at 25 °C. An unexpected finding was the ob-servation that the thermal unfolding of LtxA precipitated the protein inthe presence of calcium while in EDTA-containing buffer the proteinremained in solution, most likely with unfolded structure. The precisephysicochemical explanation for this behavior remains to be explored,but it seems reasonable to suggest that the presence of calcium mayinfluence the unfolding pathway, e.g., by affecting refolding kinetics aswas recently reported in a careful study over the thermal denaturationof von Willebrand Factor [74]. In the case of LtxA this may lead to afavored formation of precipitating folding intermediates.

In database searches it was not possible to find proteins similar tothe N-terminal part of LtxA and with a known three-dimensionalstructure. However, taken together with the suggested domain struc-ture of the C-terminal part, and the distinct alpha helical structure ofthe N terminal part, it encourages the suggestion that LtxA contains asa minimum two domains.

4.4. Interaction of purified LtxA with different β2 integrins

Our results with target cells expressing different β2 integrins com-plement previous data by others. Lally and coworkers [10]first observedthat human LFA-1 functions as a cell surface receptor for RTX toxins inthe form of LtxA from A. actinomycetemcomitans and HlyA producedby E. coli. In search of the exact receptor structure within LFA-1, exper-iments with target cells expressing bovine/human chimericβ2 integrinsindicated that susceptibility to LtxA-induced cytotoxicity is maintainedif humanαL (CD11a) is substituted with its bovine homologuewhereasβ2 chain (CD18) encompassing the so-called integrin-epidermal growthfactor-like domains 2, 3, and 4 of human CD18 is necessary for confer-ring susceptibility to LtxA-induced biological effects [21]. However,

other experiments with target cells expressingmurine/human chimericαL (CD11a) chains together with human CD18 showed that LtxA-induced cytotoxicity required the N-terminal beta-sheets 1 and 2 ofthe CD11a beta-propeller (comprising 128 aa residues) to be of thehuman form whereas all-murine CD11a conferred resistance to thetoxin [22]. Collectively, this would suggest that a part of CD11a is eitherrecognized by LtxA together with structures in CD18 or is critical for theoccurrence of LtxA-binding structures in CD18.

Amino acid sequences of mammalian β2 integrin componentchains are retrievable from the NCBI database. BLAST-alignment[75] of human CD11a against the bovine and murine homologuesreveals ~77% identity (0% gaps) and ~73% identity (1% gaps), respec-tively, and these figures do not change significantly when the analysisis restricted to the 128 N-terminal amino acids of the α chains. Incomparison, alignment of human αL against the three other potentialα chains of human β2 integrins, αM, αX, and αD, reveals sequenceidentities of only 34–35% with 6–9% gaps. Other studies suggest thatthe evolutionary change of the αM chain is faster than that of the β2

chain [76]. Notably, human alpha chains with the latter low degreeof identity to the αL sequence do not necessarily confer resistanceto LtxA since a recombinant cell line, P/5, expressing human αXβ2

was previously shown to be susceptible to the toxin although appar-ently less so than the human HL-60 cell line [21]. HL-60 expresses β2

integrin only in the form of αLβ2 unless stimulated by cytokines orother stimuli [32]. The different susceptibilities of P/5 and HL-60 toLtxA, however, need not reflect the different β2 integrin speciesexpressed by the cells; they might also be due to differences in levelsof expression. Another recombinant cell line, KL/4, expressing humanαLβ2 like HL-60 was found to be less susceptible to LtxA than HL-60cells [10,21]. KL/4 and P/5 had both been generated by genetic trans-fection of K562 cells by way of a vector different from the CDM8 vec-tor used for transfection of the K562 cells serving as targets in thepresent study [77]. However, in none of the studies mentionedwere the levels of integrin expression by the target cells assessedor compared.The three lines of transfected K562 cells used in thisstudy, by expressing roughly similar levels of the individual β2

integrin species on a common cellular background and by showingonly moderately different susceptibility to the toxin, suggested thatthe three β2 integrins are of comparable efficiency in transferringLtxA-mediated cytotoxicity. The finding that LtxA binds exclusivelyto the β2 chain in ligand blotting analysis is in accordance with theobservation by Dileepan et al. [21] that the EGF-like repeats 2–4 ofthe human β2 chain are critical for LtxA binding.

The expression ofβ2 integrins in leukocytes in vivo is subject to com-plex regulation. In monocytes, when they extravasate from the bloodvessel to differentiate into tissue macrophages, αXβ2 is upregulatedwhereas expression ofαLβ2 decreases [32,78]. If extravasation is stimu-lated by chemoattractants such as fMLP, C5a and leukotriene B4 whichare generated during bacterial infection, monocytes and neutrophilsrapidly upregulate αMβ2 as well as αXβ2 ten-fold on the surface bymobilization from intracellular pools, whereas αLβ2 expression in-creases only two-fold inmonocytes and does not change in neutrophils[23,32]. In particular integrinαXβ2 has a striking ability to bind structur-ally decayed proteins [79,80]. Expressed on the surface of neutrophilsand other myeloid cells, this receptor binds well to proteins followingincomplete proteolytical digestion, probably as a consequence of thedestruction of higher-order protein structure [79]. With the stronginflammatory response associated with aggressive periodontitis, theextracellularmatrix of the inflamed tissue is targeted by several proteo-lytic mechanisms of endogenous and microbial origin [81,82]. Bypresenting multiple ligands for αXβ2 this environment would presum-ably act to entrap myeloid leukocytes and render them easier targetfor the cytolytic effects of LtxA. In this way, the targeting of LtxAto αXβ2 as well as αLβ2 is not merely an incremental addition ofleukotoxicity, but an intricate molecular mechanism providing ben-efit to microbial survival through several levels of the biochemistry

557J. Reinholdt et al. / Biochimica et Biophysica Acta 1834 (2013) 546–558

associated with tissue inflammation. Our observation that αMβ2 and,in particular, αXβ2 bind—and even may surpass αLβ2 in ability tofunction as receptor and transfer the cytotoxic effect of LtxA—pointsto LtxA as a virulence factor functionally well-adapted to meet thechanges in host cell β2 integrin expression during periodontitis.

Balashova et al. [20], like originally Tsai et al. [14], noted thathemolytic activity was not detectable in LtxA preparations made byextraction from A. actinomycetemcomitans bacteria and speculatedthat in such preparations contaminating LPS might inhibit hemolyticactivity [20]. If so, our LtxA preparation free of LPS might provide areliable source for measuring LtxA-mediated hemolysis. Using anexperimental setup similar to that of Balashova et al. [20], however,our LtxA preparation produced time-response curves essentially likethose obtained for LtxA prepared by the protocol of Kachlany et al.[31]. More studies are required to explain the mechanism of hemoly-sis by LtxA and other RTX toxins, which may not involve toxin-specific cell surface receptors [83,84].

LtxA appears to be an important virulence factor of A.actinomycetemcomitans in relation to aggressive periodontitis [7].The toxin may destroy phagocytes which are indispensable for the con-trol of subgingival bacteria and stimulate the production of inflammato-ry cytokines by these cells, leading to increased degradation of fibersand bone [85,86]. The hemolytic capacity of LtxA, though moderate,may also be important [29]. The role of β2 integrins as toxin receptorson leukocytes iswell documentedwhile the signaling pathways leadingto cell death or cytokine response are not clear. Difficulties in preparingLtxA for experiments in its native molecular configuration and free ofLPS, which by itself may stimulate some of the cellular reactions as-cribed to LtxA, may have complicated research in LtxA-mediated effects[29]. Other complicationswere the lack of LtxA appropriate for structur-al studies and for use as antigen in antibody-based assays [27,87]. Theprotocol for LtxApreparation described heremay facilitate such studies.

Acknowledgements

We wish to thank Nykola Jones for assistance with calculations onthe CD spectra and Bettina W. Grumsen for excellent technical assis-tance. This study received support from the Novo Nordisk Fonden tocover the experimental cost (5614) and from The Lundbeck Foundationand Carlsberg Foundation for support to cover the construction the UVbeamline CD1 in Institute for Storage Ring Facilities Aarhus.

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