Engineered covalent leucotoxin heterodimers form functional pores: insights into S–F interactions

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Engineered covalent leucotoxin heterodimers form functional pores: insights into S–F interactions
  Biochem. J. (2006)  396 , 381–389 (Printed in Great Britain) doi:10.1042/BJ20051878  381 Engineered covalent leucotoxin heterodimers form functional pores:insights into S–F interactions Olivier JOUBERT*, Gabriella VIERO † , Daniel KELLER*, Eric MARTINEZ*, Didier A. COLIN*, Henri MONTEIL*, Lionel MOUREY ‡ ,Mauro DALLA SERRA †  and Gilles PR´EVOST* 1 *Laboratoire de Physiopathologie et d’Antibiologie Microbiennes, EA 3432, Institut de Bact´eriologie de la Facult´e de M´edecine (Universit´e Louis Pasteur), Hˆopitaux Universitaires deStrasbourg, 3 rue Koeberl´e, F-67000 Strasbourg, France,  † ITC and CNR-IBF Unit at Trento, 18 Via Sommarive, I-38050 Povo (Trento), Italy, and  ‡ Groupe de Biophysique Structurale,D´epartement M´ecanismes Mol´eculaires des Infections Mycobact´eriennes, CNRS-IPBS UMR 5089, 205 route de Narbonne, F-31077 Toulouse Cedex, France The staphylococcal  α -toxin and bipartite leucotoxins belong to asingle family of pore-forming toxins that are rich in  β -strands,although the stoichiometry and electrophysiological character-istics of their pores are different. The different known structuresshow a common  β -sandwich domain that plays a key role in sub-unit–subunitinteractions,whichcouldbetargetedtoinhibitoligo-merization of these toxins. We used several cysteine mutants of bothHlgA( γ  -haemolysinA)andHlgB( γ  -haemolysinB)tochal-lenge 20 heterodimers linked by disulphide bridges. A new strat-egy was developed in order to obtain a good yield for S-S bondformation and dimer stabilization. Functions of the pores formedby14purifieddimerswereinvestigatedonmodelmembranes,i.e.planar lipid bilayers and large unilamellar vesicles, and on targetcells, i.e. rabbit and human red blood cells and polymorpho-nuclear neutrophils. We observed that dimers HlgA T28C–HlgBN156C and HlgA T21C–HlgB T157C form pores with similar characteristics as the wild-type toxin, thus suggesting that themutatedresiduesarefacingoneanother,allowingporeformation.Ourresultsalsoconfirmtheoctamericstoichiometryoftheleuco-toxin pores, as well as the parity of the two monomers in the pore.Correctly assembled heterodimers thus constitute the minimalfunctional unit of leucotoxins. We propose amino acids involvedin interactions at one of the two interfaces for an assembledleucotoxin.Key words: assembly intermediate,  β -barrel, intermolecular di-sulphide bond, pore-forming toxin, protein–protein interaction, Staphylococcus aureus . INTRODUCTION PVL (Panton–Valentine leucocidin) and Hlg ( γ  -haemolysin) arebicomponent leucotoxins of   Staphylococcus aureus  which formpores in blood cell and purely lipid membranes. Components of a leucotoxin sequentially bind to target cells: a class S protein(LukS-PV or HlgA, 32 kDa) first binds the surface of leucocytesand then, a class F protein (LukF-PV or HlgB, 34 kDa) interactsto develop a bipartite oligomeric and cytolytic pore [1].  S. aureus α -toxin and leucotoxins belong to the family of the β -barrel pore-forming toxins. This family includes other members, such as theaerolysin from  Aeromonas hydrophila , the  Vibrio cholerae  cyto-lysin, leucotoxins from  Clostridium septicum  and cholesterol-dependent cytolysins, such as PFO (perfringolysin O) from Clostridiumperfringens [2].Thisfamilyalsoincludescompoundsthat have a role as translocation subunits, such as the anthrax pro-tective antigen from  Bacillus anthracis  [3], and the iota-toxinfrom  Clostridium perfringens  [4]. All of these toxins have threecommon features. They are rich in β -strands, they have no hydro-phobic stretch and full oligomerization is a prerequisite.  S. aureus α -toxin forms non-lytic prepores before undergoing conform-ational changes leading to pore formation [5 – 8], as do several related toxins [9 – 14]. Oligomerization occurs near to or on the membrane surface before insertion, rather than by lateral additionof monomers already inserted [15].Astheyoftenareassociatedwithpathologies[1],thecharacter-ization of protein–protein interactions mainly constitutes animportant step towards the inhibition of oligomerization of pore-forming toxins and the understanding of the molecular dynamicsleading to pore formation. In the case of PFO, the monomer bind-ing to cholesterol-containing membranes, it induces some allo-steric modifications occurring at a structural element (strand  β 5)to move and to expose the edge of a previously hidden  β -strand( β 4) that participates in the protomer–protomer interaction viahydrogen bonding of strand  β 1 of a given monomer to the now-exposed strand  β 4 in the srcinal PFO dimer  [16]. The oligomer assembly is obtained through productive collisions of the pre-viously inserted monomers. Up to 50 PFO molecules assemble ina circular prepore complex [12]. Finally, there is a co-operativeinsertion of the transmembrane  β -hairpins into the membrane toform a large pore [17]. HlgA and HlgB bind to membranes asmonomers [18]. Until now, the stoichiometry of the leucotoxinpore has been controversial, with heptamers suggested by elec-tron microscopy [19], or octamers identified by the analysis of labelled or covalently linked proteins [20,21], but hexamers were also proposed [22]. An HlgA–HlgB dimer contains an equimolar ratio of the two proteins [22], and FRET (fluorescence resonanceenergy transfer) experiments have demonstrated the alternativelocation of the two proteins in the pore [23]. Furthermore, slightmodifications occur in the secondary structure during the preporeformation [22] that may favour protein–protein interactions [24]. Valeva et al. [25] demonstrated that a co-operative assemblybetween  α -toxin protomers occurs during prepore formation. Inthe present study, several purified heterodimers of HlgA–HlgBwere obtained by disulphide bonding via mutagenesis. BiologicalactivitiesandcharacteristicsoftheporesfordimersandWT(wild-type) toxin were compared using different membrane systems togain insights into monomer S–monomer F interactions. Abbreviations used: DTDP, 2,2  -dithiodipyridine; DTT, dithiothreitol; GST, glutathione S-transferase; Hlg,  γ  -haemolysin; HRBC, human red blood cell;LUV, large unilamellar vesicle; PEG, poly(ethylene glycol); PFO, perfringolysin O; PMN, polymorphonuclear neutrophil; PVL, Panton–Valentine leucocidin;RRBC, rabbit red blood cell; WT, wild-type. 1 To whom correspondence should be addressed (email c   2006 Biochemical Society  382  O. Joubert and others MATERIALS AND METHODSBacterial strains, vector and site-directed mutagenesis  Escherichia coli  XL1 Blue cells  { recA1 endA1 gyrA96 thi1hsdR17 supE44 relA1 lac  [F’  proAB lacI  q  Z   M15  Tn 10 ( tet  r  )] } (Stratagene) were used as recipient cells for transformation withrecombinant pGEX-6P-1 ® control plasmids or following site-directed mutagenesis.  E. coli  BL21 [F − ,  ompT  ,  hsdS  ( rB − ,  mB − ), gal ] was used for overexpression of the GST (glutathione S-transferase)–leucotoxin fusion  HlgA  and  HlgB  genes (GenBank ® accession numbers L01055 and X81586), according to the manu-facturer’s instructions (Amersham Biosciences) [26,27]. The dif-ferent mutantswere obtainedbyusingdedicatedoligonucleotidesinatwo-stepmutagenesisproceduresimilartoQuikChange ® mu-tagenesis (Stratagene), except that Tfu DNA polymerase ® (Finnzymes)andT4GP32protein ® (Qbiogene)wereusedinsteadof Pfu Turbo ® DNA polymerase. Protein purification Recombinant HlgA was affinity-purified by glutathione–Sepha-rose 4B chromatography, followed, after cleaving the GST tagwith PreScission ® protease (Amersham Biosciences), by cation-exchange MonoS ® FPLC chromatography (Amersham Bio-sciences) using a NaCl gradient from 0.36 to 0.6 M [26]. It waseluted at approx. 0.51 M NaCl. Recombinant HlgB was purifiedby hydrophobic interaction chromatography (Resource ISO ® ;AmershamBiosciences)usinga(NH 4 ) 2 SO 4  gradientrangingfrom0.96 to 0.36 M [27], and it was eluted at 0.6 M (NH 4 ) 2 SO 4 . Alldimer preparations were first adjusted to 1.2 M (NH 4 ) 2 SO 4 ,beforebeingappliedtoahydrophobicinteractionchromatographycolumn using the (NH 4 ) 2 SO 4  gradient as above. Materials thatwereelutedat0.6 Mwerediluted1:6in0.05 MNa 2 HPO 4 ,pH 7.0,todecreasethe(NH 4 ) 2 SO 4  concentrationandwerepurifiedfurther using cation-exchange chromatography as described above. Pro-teins were then dialysed at 4 ◦ C overnight against 0.02 M Hepesand 0.5 M NaCl, pH 7.5. Controls for homogeneity were per-formed using SDS/10–15 %  PAGE, and the proteins werethen stored in the presence of 1 mM DTT (dithiothreitol) at − 80 ◦ C. Dimer synthesis and purification In order to limit any oxidation of the thiol groups of the cysteinemutants, all buffers were flushed with nitrogen for 30 min. Cys-teinemutantsofHlgBwerefirstreducedinthepresenceof20 mMDTT, desalted by gel filtration on PD10 ® columns (AmershamBiosciences) against 0.05 M Hepes, 0.5 M NaCl, 1 mM sodiumEDTA and 0.25 mM DTT, pH 7.5, before being activated with2.5 mMDTDP(2,2  -dithiodipyridine)for10 minatroomtemper-ature (25 ◦ C). To remove excess DTDP, the activated toxins weredesalted for a second time under the same conditions as above.Concurrently, cysteine mutants of HlgA were first desalted bygel filtration on PD10 ® columns against 0.02 M sodium acetate,0.5 M NaCl and 1 mM sodium EDTA, pH 5.0. The two partnerswere then mixed for 3 h at 23 ◦ C at a ratio of HlgB Cys–TP/HlgACys of 1.5, after the pH was adjusted to 7.5 using 0.5 M Bicine,pH 9.0 (15 mM final concentration). Finally, mixtures were ad- justedto0.5 mMDTDPtoblocktheresidualfreethiolgroups,andbeforefurtherpurificationofheterodimersasindicatedabove.For testing the residual accessibility of free thiols, approx. 30 nmolof proteins in 0.5 ml were pelleted by centrifugation at 5000  g for 10 min in 5 % (w/v) trichloroacetic acid and left for 5 min at0 ◦ C,beforebeingwashedthreetimeswiththesamesolution.Theprecipitate was dissolved in 400 µ l of N 2 -saturated 0.2 M Hepes,0.2 M NaCl and 1 mM sodium EDTA, pH 8.0, and was used for a direct Ellman’s titration [28]. Planar lipid bilayer experiments Planar lipid bilayers were prepared by the application on bothsides of a 0.1 mm hole in a 12 µ m Teflon foil (pre-treated with n-hexadecane) of two monolayers of 99 % pure diphytanoyl phos-phatidylcholine (Avanti Polar Lipids) spread from a 5 mg · ml − 1 solution in pentane. Toxins were added on one side ( cis ) to stablepre-formed bilayer. All experiments were started in symmetricalsolutions (10 mM Hepes, 100 mM KCl, and 0.1 mM sodiumEDTA, pH 7.0). Macroscopic currents were recorded on a patchclampamplifier(Axopatch200 ® ;AxonInstruments).Acomputer equippedwithaDigiData1200A/Dconverter(AxonInstruments)was used for data acquisition. The current traces were filtered at0.1 kHz and acquired by computer assistance using Axoscope 8software (Axon Instruments). Measurements were performed atroom temperature as described previously [29]. Permeabilization of lipid vesicles by leucotoxins Forcalcein(Sigma)releaseexperiments,LUVs(largeunilamellar vesicles) were prepared by extrusion of 3 mg/ml phosphatidyl-choline/choline at a 1:1 molar ratio, as described previously[30]. LUVs were washed on Sephadex G-50 medium pre-equili-brated with 20 mM Tris/HCl, 20 mM NaCl and 0.1 mM EDTA,pH 7.0.Permeabilizationwasassayedusingafluorescencemicro-plate reader. Each well contained LUVs (7 µ M lipid) and vari-ous leucotoxin dilutions. The two components were applied atequimolar concentration in 200 µ l of 20 mM Tris/HCl, 20 mMNaCl and 0.1 mM EDTA, pH 7.0. Maximal protein concentrationwas 5 nM with HlgB plus HlgA, 50 nM with heterodimers. Human PMNs (polymorphonuclear neutrophils) and flowcytometry measurements Human PMNs from healthy and anonymous donors were purifiedfrombuffycoatsobtainedfromabloodbank[EFS(EtablissementFranc¸aisduSang),Strasbourg,France]asreportedpreviously[31]and were resuspended in 10 mM Hepes, 140 mM NaCl, 5 mMKCl, 10 mM glucose and 0.1 mM EGTA, pH 7.3, at 5 × 10 5 cells/ ml. Flow cytometry was carried out using a FacSort ® flow cyto-meter (Becton-Dickinson) equipped with an argon laser tuned to488 nm [27]. Intracellular calcium was evaluated using flow cyto-metry of cells loaded previously with 5 µ M Fluo-3 (Molecular Probes) in the presence of 1.1 mM extracellular Ca 2 + . Pore form-ation and univalent cation influx were revealed by the penetrationof ethidium bromide into the pores; cells were incubated for 30 min with 4 µ M ethidium bromide before toxin addition in theabsenceofextracellularCa 2 + .Fluo-3andethidiumbromidefluor-escence was measured using Cell Quest Pro TM software (Becton-Dickinson) [27,31]. Results from at least four different donorswere averaged and expressed as percentages of control of humanPMNs treated with HlgA–HlgB. Base level values were obtainedfor each series of data from a control without addition of toxin.These were systematically subtracted from the other assays. S.D.values never exceeded 10 %  of the obtained values and wereremoved from the Figures for clarity.The dissociation constant ( k  D [ S ]) of HlgA for the PMN mem-brane and that of HlgB for the PMN membrane-bound HlgB( k  D [ F ]) were reported previously to be 2 nM and 0.04 nM respect-ively [32]. WT and mutants of HlgA were applied at 20 nM,whereas WT and mutants of HlgB were applied at 0.4 nM. Theheterodimers were applied at 20 nM. c   2006 Biochemical Society  Functional heterodimers of staphylococcal leucotoxins  383Determination of pore radii Theradiiofporesformedbynativeandmodifiedleucotoxinswereassessed using flow cytometry by determining the relative abilityof PEG [poly(ethylene glycol)] molecules of various sizes toprotect cells from osmotic leakage as described previously [33].HlgA and HlgB (each at 20 nM), or 20 nM of heterodimers weremixed with 30 mM PEG polymers of different aqueous pore radii(0.94, 1.12, 1.22 and 1.44 nm), incubated for 40 min, before for-ward side scatter values were collected at 0, 10, 20 and 30 minafter toxin application [26]. Haemolysis assays HRBCs (human red blood cells) were retrieved from buffy coatsusedforPMNpreparations(seeabove).Theywerefirstpelletedbycentrifugationat1000  g for5 min,thenwashedthreetimesinPBS(10 mM NaH 2 PO 4 , 1.5 mM Na 2 HPO 4  and 0.15 M NaCl, pH 7.0),and finally resuspended in this buffer at 1 % (v/v) concentration.Kinetics of haemolysis were carried out at room temperature in a0.1 % (v/v) suspension of HRBCs, which corresponds to an  A 650 of 1.Wemeasuredthe decayof   A 650  every30 s over12 min. Com-plete haemolysis was obtained after 10 min of incubation of 20 nM HlgA and 20 nM HlgB. Measurements were carried outusing a Camspec M330 spectrophotometer. The activities of theheterodimers were tested also on RRBCs (rabbit red blood cells)obtained from fresh rabbit blood. Haemolytic activity was deter-mined following the turbidity at 650 nm in a 96-well microplatereader (UVmax; Molecular Devices) for 45 min as described pre-viously [22]. Identification of oligomers Oligomers formed in solution or on to human PMN membranesby WT HlgA and HlgB and heterodimers were examined usingSDS/3–8 %  PAGE and immunoblotting. For investigations of oligomers in solution, 4 ng of each toxin component was in-cubatedwith0.3or3 mMglutaraldehydeasdescribedbelow.Pre-parations of 5 × 10 7 cells/ml in 10 mM Hepes, 140 mM NaCl,5 mM KCl, 10 mM glucose and 0.1 mM EGTA, pH 7.3, wereincubatedwith100 nMofLukS-PV,LukF-PVorderivativesinthepresence of 10 µ l/ml mammalian cell-tissue antiprotease cocktail(Sigma). After a 45 min incubation at 22 ◦ C, biological activitywas evaluated by optical microscopy as swelling of the cells androunding of the nuclei. The cells were washed twice and then re-suspended in 1 ml of the same buffer and 1 µ l/ml antiproteasecocktail as above. The cells were ground using a FastPrep ® ap-paratus (Qbiogene) in FastPrep Blue ® tubes with orbital centri-fugation at 3500  g  for 10 s at room temperature. The membraneswere harvested by ultracentrifugation at 22000 rev./min for 20 minat4 ◦ CinaTLArotor.Membranepelletswereresuspendedin100 µ lofthesamebuffercontaining1 % (w/v)saponin(Sigma)and 2 µ l of antiprotease, incubated for 30 min at room temper-ature and then centrifuged at 25000 rev./min for 30 min in a TLArotor.Thesupernatantswereadjustedto1 mMglutaraldehydeandincubated for 10 min at 50 ◦ C. Loading buffer [0.5 M Tris/HCl,pH 8.5, 2 % (w/v) SDS, 0.04 % (w/v) Bromophenol Blue, 30 % (v/v) glycerol and 100 mM ethanolamine] was added to block thecross-linking reaction, and assay mixtures were heated to 100 ◦ C.A 10 µ l sample of the solution was loaded on to Tris/acetate,pH 8.1, 3–8 % (w/v) polyacrylamide gels (Invitrogen) and waselectrophoresed for 75 min at 150 V at room temperature in50 mMTris,50 mMTricine,pH 8.2,and0.1 % (w/v)SDS,beforebeingtransferredontonitrocellulosemembranesfor1 hat30 Vin25 mM Tris, 192 mM glycine, pH 9.3, and 20 % (v/v) methanol.Leucotoxin complexes were characterized by immunoblottingusing affinity-purified anti-rabbit polyclonal antibodies and ahorseradish-peroxidase-labelled goat anti-rabbit antibody usingECL ® (enhancedchemiluminescence)detection(AmershamBio-sciences). Apparent molecular masses of proteins were estimatedaccording to Precision Plus ® protein standards (Bio-Rad). RESULTSChoice of mutations Biologically active cross-combinations of leucotoxins and recentadvances have demonstrated the alternating locations of class SandclassFcomponentswithintheporeforthestructurallyrelatedleucotoxin component sequences (Figure 1A) [23,33 – 35]. Exa- mination of the crystal structures of HlgB and LukS-PV [34,35]revealed that residues Tyr-99, Asn-103, Gln-104, Arg-150, Thr-152, Ser-154, Arg-155, Asn-156, Thr-157 and Asn-158 are ex-posed to solvent and may be involved in protein–protein inter-actions within the functional pore (Figure 1A). All of theseresidues, as well as the solvent-exposed HlgA Thr-21 and Thr-28,werecysteine-substituted.ResiduesHlgBTyr-99andGln-104arelocated on a loop connecting strands 6 and 7, and residues HlgBArg-150 and Asn-158 are located on a loop connecting strands 9and 10 [34]. HlgA Thr-28, located on  β -strand 3, was shown toplay a key role in monomer oligomerization [18]. This residue of leucotoxin class S components aligns with His-35 of a given  α -toxinmonomerA(Figure1B),whichinteractswith Tyr-101,Thr-161 and Asp-162 of monomer G. HlgB Tyr-99 aligns perfectlywith Tyr-101 of   α -haemolysin and was shown to be involved incontacts between monomers [36 – 38]. In parallel, HlgA Thr-21 ( β -strand 1) might be close to HlgB Thr-157 and HlgB Asn-158respectively [34,35]. Heterodimer construction and purification Figure 2 illustrates the purification steps of the covalent hetero-dimer HlgA T28C–HlgB N156C, which is representative of thehighly purified dimers that were investigated in the present study.Lanes 2 and 6 of Figure 2 show HlgA T28C and HlgB N156ChavingsimilarapparentmolecularmassesasthoseofrecombinantHlgA and HlgB (Figure 2, lane 7). These mutants may also have atendency to form homodimers (64 or 74 kDa) in oxidizing condi-tions (Figure 2, lanes 3 and 5 respectively). Blocking the cysteineof one mutated component by DTDP favoured the thiol-exchangereactionofthelabelledproteinwiththepartnercomponent,givingrise to a heterodimer with an apparent molecular mass of 68 kDa(Figure 2, lane 4). In contrast, when WT HlgA and HlgB weremixed together for 90 min at 23 ◦ C, no dimerization appeared(Figure 2, lane 7). The heterodimer was detected by immuno-blotting with both anti-HlgA and anti-HlgB antibodies (resultsnot shown). Thiol-titrations with DTNB on to SDS-denaturatedproteins always gave significant yields (25–65 % ) of dimers in-cludingheterodimersusefulfortheirpurification,exceptforHlgBY99C, HlgB R150C and HlgB N152C mutants challenged witheither HlgA T21C or HlgA T28C, where one accessible cysteinewasdeterminedperprotein(Lowrytitration).Thusonlysome,butnotall,ofthecysteineresiduesremainedaccessiblefordisulphidebridge formation and failed to promote heterodimers. Purificationof most heterodimers led to  > 99 % pure complexes (Figure 2,lane 8). Cell-biological activities of heterodimers The presence of cysteine mutations was first checked to be notdeleterious for both haemolytic activities (HRBCs and RRBCs)compared with WT HlgA and HlgB (results not shown). In fact,HlgA T28C combined with HlgB was slightly less active than c   2006 Biochemical Society  384  O. Joubert and others Figure 1 Basic three-dimensional structures and sequence alignment of leucotoxins ( A ) Three-dimensional structures of LukS-PV (PDB code 1T5R; right-rear view) that might be comparable with that of HlgA and HlgB (PDB code 1LKF; left-rear view) and the location of thecysteine-substituted amino acids. Amino acids thought to interact each other are underlined and in italics respectively. ( B ) Sequence alignment of the  S. aureus   Hlg (GenBank ® accession numberX81586), PVL (GenBank ® accession number X72700), LukE and LukD (GenBank ® accession number Y13225) and  α -toxin (Hla, GenBank ® accession number M90536). Conserved residues areshown in white on a black background, while common residues to bipartite leucotoxins are shown in bold; numbering of the amino acids is given on the basis of HlgA and HlgB respectively. WT, and HlgB N103C combined with HlgA was approx. 10-foldless active on HRBC.The lytic activities of heterodimers (20 nM) on to HRBCs,RRBCs and human PMNs are detailed in Table 1, and Figures 3and 4. Pore-formation and ethidium bromide entry promoted intoPMNs by heterodimers show that these cells are globally moresensitive than HRBCs. Dimer HlgA T21C–B N103C displays nolytic activity upon any cells tested. HlgA T21C–HlgB Q104C,HlgA T21C–HlgB R155C and HlgA T21C–HlgB N156C dis-played decreased activities, judged to be approx. 20-fold greater than that of WT (see values in Table 1 for 1 nM WT) on HRBCs,RRBCs and human PMNs (Table 1, and Figures 3A and 4A).More curiously, HlgA T21C–HlgB S154C also had intermediatehaemolytic activities (Table 1 and Figure 3A), but its leucocyto-lytic activity was comparable with that of the WT on PMNs (Fig-ure 4A), indicating that behaviour against each cell was not uni-form. Similarly, whereas HlgA T21C–HlgB N158C needs18 minto lyse half of the HRBCs, its activity on RRBCs and PMNs wascloser to that of WT (Table 1, and Figures 3A and 4A). HlgAT21C–HlgB T157C and HlgA T21C–HlgB N158C seemed fullyefficient against all of these PMNs.Again, HlgA T28C–HlgB N103C had no lytic activity on cellsand HlgA T28C–HlgB Q104C, HlgA T28C–HlgB S154C hetero-dimers harboured weak potentials to disrupt membranes of HRBCs,RRBCsandhumanPMNs,withbothlongerlagtimesandtimecoursestoreach100 % lyticactivity(Table1,andFigures3Band4B).HlgAT28C–HlgBR155CandHlgAT28C–HlgBT157Cwere haemolytic, but they take more than 60 min to start haemo-lysis on HRBCs or RRBCs (Figure 3B and Table 1). The half-haemolysis time of HlgA T28C–HlgB N158C was 20 min,whereasHlgAT28C–HlgBN156CandHlgAT28C–HlgBN158Cshowed activity on HRBCs closer to that of the control, but with a4 minlagtime.OnhumanPMNs,HlgAT28C–HlgBR155Cgene-rated poresasrapidly astheWT,while HlgA T28C–HlgBN158Crevealedadecreasedactivity(Figure4B).OnlyHlgAT28C–HlgBN156CandHlgAT28C–HlgBT157Chadpore-formingactivitiesbetween those of the WT and those of HlgA T28C combined withHlgB. c   2006 Biochemical Society  Functional heterodimers of staphylococcal leucotoxins  385 Figure2 EngineeringandpurificationoftheHlgcovalentheterodimerHlgAT28C–HlgB N156C SDS/10–15% PAGE and silver staining. Lane 1, molecular ladder; lanes 2 and 6, HlgA T28C(0.3 µ g) and HlgB N156C (0.5 µ g) respectively, under reducing conditions; lanes 3 and5, HlgA T28C (1 µ g) and HlgB N156C (0.8 µ g) respectively under oxidizing conditions;lane 4, HlgA T28C–HlgB N156C (1 µ g of the coupling reaction); lane 7, mixture of recombinantWT HlgA (0.2 µ g) and WT HlgB (0.15 µ g); lane 8, FPLC-purified HlgA T28C–HlgB N156C.Molecular-mass sizes are given in kDa. Table 1 Cytotoxic activities of Hlg covalent heterodimers Results( t  50 )forHRBCsandRRBCsaretimestoreachhalf-haemolysisusing20 nMtoxin;thosefor PMNs are times to reach 50% activity using 20 nM toxin. nd, not determined; na, not active. t  50  (min)Toxin HRBCs RRBCs PMNsWT 2.5 2.9/4.3 14.6WT (1 nM) 24.3 nd 27.1HlgA T21C–HlgB N103C na na naHlgA T21C–HlgB Q104C 9.8 9.4 20.0HlgA T21C–HlgB S154C 10.8 7.9 16.3HlgA T21C–HlgB R155C  > 60 23.0 22.7HlgA T21C–HlgB N156C  > 60 13.7 20.0HlgA T21C–HlgB T157C 8.8 3.1 14.6HlgA T21C–HlgB N158C 18.0 4.9 17.3HlgA T21C–HlgB N103C na na naHlgA T21C–HlgB Q104C  > 60 na naHlgA T21C–HlgB S154C  > 60 na 32.1HlgA T21C–HlgB R155C  > 60 12.5 17.5HlgA T21C–HlgB N156C 6.8 3.6 13.8HlgA T21C–HlgB T157C  > 60 44.4 15.0HlgA T21C–HlgB N158C 23.0 3.6 20.5 Comparable results were obtained for the calcium influx pro-moted by these toxins and heterodimers, which is known to occur beforeporefunction[26,35].HlgAT21Cmutantsmaybeinactive orpartiallyactive,butcalciumchannelopeninginducedbydimersHlgA T21C–HlgB T157C and HlgA T21C–HlgB N158C wascomparable with that of WT Hlg (Figure 5). Calcium influx in-duced by HlgA T28C–HlgB N103C and HlgA T28C–HlgBQ104C were null or weak (Figure 5). HlgA T28C–HlgB S154Cinduced an intermediate activity, and the other heterodimers ap-peared closer to control (Figure 5B), which is likely to be becauseof the higher sensitivity of the Fluo-3 probe compared withethidium bromide fluorescence. In fact, heterodimers engagingpositions far from Hlg N156C became less biologically active(Figures 3–5 and Table 1). Characteristics of ion channels formed by the heterodimers All of the heterodimers able to permeabilize RBCs and LUVs(Tables 1 and 2) also opened similar ion conductive pores (Fig-ure 6) as HlgA–HlgB does in planar lipid bilayers [22,26,39]. Heterodimers display similar electrophysiological pore proper-ties, i.e. channel conductance and current voltage characteristics(  I   –V)(Figure6andTable2),whichareveryclosetothoseofWT.In 100 mM KCl, pH 7.0, at  + 40 mV applied voltage, all of thepores normally stay open for most of the time (Figure 6), as dothose formed by HlgA–HlgB [39] and  α -toxin [40]. Under suchconditions, all of the dimers have similar mean conductance,ranging from 118 to 162 pS, similar to that of the WT, i.e. 128 pS(Figure 6 and Table 2). Some of them show a different propensityto open pores, as evidenced by the different rate of channel inser-tion,e.g.HlgAT28C–HlgBT157Cwasevenmoreactive.Further-more, HlgA T21C–HlgB S154C shows a clear increase in currentnoise, possibly due to an increase in instability of the  β -barrel[41],whichmaybeduetoalongerdistanceofinteractionbetweenkey residues [24]. The  I   –V curve of the WT is markedly asym-metrical, i.e. there is a larger current flow at negative voltages[39]. An estimate of the extent of this non-linearity is given bythe ratio  I  −  /   I  + measured at 120 mV (Table 2). This ratio is iden-tical for the Hlg and the heterodimers, indicating a similar chargedistributionalongthelumenofthechannel.Allofthetoxinstestedare slightly cation-selective as is the WT [23]. The cation/anionpermeability ratio was determined as in Comai et al. [39] with a20 mM  cis  –200 mM  trans  KCl gradient into 10 mM Hepes and0.1 mM EDTA, pH 7.0. Thereafter, the concentration of the  trans chamber was increased stepwise up to 200 mM KCl and the per-meabilityvaluesP +  /P − weregivenbytheratioofcationandanionpotentialmobilitiesrespectively.Undertheseconditions,WTHlghad a cationic selectivity with a P +  /P − of 1.4. The most activeheterodimers tested, HlgA T21C–HlgB N156C and HlgA T28C– HlgB T157C were similar to WT or HlgA T28C–HlgB T157Cpointed at 1.5, or HlgA T28C–HlgB N156C to 1.6. However,the pores formed by HlgA T28C–HlgB N158C and HlgA T21C– HlgB N158C harboured a higher cationic selectivity of 2.0 and2.1 respectively. Oligomer analysis To screen the amount and stoichiometry of oligomers formed byheterodimers and inserted into cell membranes, these oligomerswere recovered from membranes and stabilized by a chemicalcross-linkingtoescapetheirdegradationwhenreleasedfrommem-branes. As shown in Figure 7 (lane 3), 4 ng of purified HlgA andHlgB at nanomolar concentrations which were mixed together and incubated for 10 min with 0.3 mM glutaraldehyde producedconcatemers of the two proteins where at least dodecamers couldbe distinguished on SDS/3–8 % (w/v) PAGE. The use of 3 mMglutaraldehyde in such assays alters the signal by producingaggregates(Figure7,lane4).Nomaterialcross-reactingwithanti-bodies were detected from lysed PMNs (Figure 7, lane 1). HlgAand HlgB in solution produce low amounts of homodimers, butthose produced are stable to either SDS treatment (lane 2) or application to PMNs and retrieval after saponin treatment (lane 5)andboiling.BoilingisalsonecessarywhenoligomerscontainedinPMNs lysates are treated with 3 mM glutaraldehyde. Absence of boiling actually reveals a bulky signal of probable non-denaturedoligomers (octamers?) (Figure 7, lane 6). In fact, even if a Schiff reaction may also be promoted by glutaraldehyde, cross-linking c   2006 Biochemical Society
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