Cloning and Expression of a Transferrin-Binding Protein from Actinobacillus pleuropneumoniaet

Transcription

1 INFECTION AND IMMUNITY, Mr. 1992, p /92/ $02.00/0 Copyright X) 1992, Americn Society for Microbiology Vol. 60, No. 3 Cloning nd Expression of Trnsferrin-Binding Protein from Actinobcillus pleuropneumoniet GERALD F. GERLACH,* CAROL ANDERSON, ANDREW A. PO1T-ER, SANDY KLASHINSKY, AND PHILIP J. WILLSON Veterinry Infectious Disese Orgniztion, University of Ssktchewn, 124 Veterinry Rod, Ssktoon, Ssktchewn, Cnd S7N OWO Received 30 April 1991/Accepted 4 December 1991 An expression librry ws constructed from ActinobciUlus pleuropneumonie serotype 7. Escherichi coli trnsformnts expressing recombinnt proteins were identified by immunoscreening with porcine convlescent serum. One trnsformnt expressing 60-kD protein (60K protein) in ggregted form ws identified. Serum rised ginst the recombinnt protein recognized polypeptide with n indistinguishble electrophoretic mobility in thea. pleuopneumonie wild type fter iron-restricted growth only. The recombinnt protein bound trnsferrin fter blotting onto nitrocellulose. Using competitive enzyme-linked immunosorbent ssy (ELISA), the specificity of this binding for the mino-terminl hlf of iron-sturted porcine trnsferrin ws estblished. Also, the 60K wild-type protein bound hemin s ssessed by hemin-grose chromtogrphy. Hemin could inhibit trnsferrin binding of the recombinnt protein in the competitive ELISA, wheres hemoglobin nd synthetic iron cheltors filed to do so. Southern blot nlysis of severl other A. pkuropneumonie strins indicted tht highly homologous sequence is present in eight of eight isoltes of serotype 7 nd in some isoltes of serotypes 2, 3, nd 4. The bility of microorgnisms to bind nd utilize trnsferrin s sole iron source s well s the correltion between virulence nd the bility to scvenge iron from the host hs long been estblished (2, 3, 11, 29). The species specificity of bcteril trnsferrin-binding bility in vivo ws shown by Holbein (12, 13), who could enhnce Neisseri meningitidis infection in mice by the injection of humn trnsferrin. It ws lso observed tht, despite their lck of siderophore production (20, 30), Neissen spp. expressed new outer membrne proteins in response to iron limittion (19). Also, Simonson et l. (25) showed tht the bility of N. meningitidis to tke up iron from trnsferrin is dependent on its previous cultivtion under iron-limiting conditions. Subsequently, Schryvers nd Morris (23, 24) demonstrted the binding of trnsferrin nd lctoferrin to iron-regulted outer membrne proteins in this species. In ddition, the specificity of bcteril trnsferrin binding for the proteins of the nturl host hs been confirmed for other species by n in vitro binding ssy (7, 18, 21). Actinobcillus pleuropneumonie is porcine respirtory trct pthogen which cuses cute fibrinous pneumoni or chronic lung lesions in infected nimls. A. pleuropneumonie, under iron-restricted growth conditions, cn use porcine trnsferrin, hemoglobin, nd vrious porphyrins s its sole iron source, but it cnnot utilize bovine or humn trnsferrin (5, 7, 18). Iron limittion induces the expression of severl new outer membrne proteins. These enble the cells to bind hemin nd Congo red in competitive fshion (5), nd in ddition, two of the iron-regulted proteins hve been shown to bind trnsferrin in n in vitro binding ssy (7) Ṫhe moleculr mechnisms of the porphyrin nd trnsferrin binding, s well s the metbolic pthwy(s) involved in utilizing the iron, hve not yet been elucidted. To investi- * Corresponding uthor. t VIDO Journl Series publiction gte these questions, we cloned trnsferrin- nd heminbinding protein from A. pleuropneumonie serotype 7. We describe the expression of the ntigeniclly ctive protein in Escherichi coli nd show tht the recombinnt protein hs binding chrcteristics indistinguishble from those described for A. pleuropneumonie cells. MATERIALS AND METHODS Bcteril strins, plsmids, nd medi. A. pleuropneumonie serotype 7 strin AP205 is Nebrsk clinicl isolte kindly provided by M. L. Chepok, Modern Veterinry Products, Omh, Nebr. One A. pleuropneumonie serotype 2 strin nd the serotype 4 strin were obtined from the Americn Type Culture Collection (ATCC nd ATCC 33378). The other A. pleuropneumonie strins re field isoltes from herds in Ssktchewn. E. coli HB101 (16) nd JM105 (16) were used in ll trnsformtion experiments. Plsmids pgh432 nd pgh433 re expression vectors contining tc promoter, trnsltionl strt site with restriction enzyme sites llowing ligtion in ll three reding frmes, nd stop codons in ll reding frmes (1). A. pleuropneumonie strins were grown on PPLO medium (Difco Lbortories, Detroit, Mich.) supplemented with IsoVitleX (1% [vol/vol]; BBL Microbiology Systems, Becton Dickinson & Co., Cockeysville, Md.). Iron restriction ws obtined by dding 2,2'-dipyridyl to finl concentrtion of 100,uM. Het stress ws induced by trnsferring cultures to 45 C for 2 h. Ethnol stress ws exerted by the ddition of 10% (vol/vol, finl concentrtion) of bsolute ethnol to cultures in the mid-log phse. Oxidtive stress ws induced by the ddition of 1% (vol/vol, finl concentrtion) of 30% H202 to the cultures. E. coli trnsformnts were grown in Luri medium (16) supplemented with mpicillin (100 mg/liter) nd induced to form ggregtes by the ddition of isopropylthioglctoside (IPTG, 1 mm finl concentrtion). To detect Congo red or hemin binding, we supplemented the mpicil-

2 VOL. 60, 1992 lin-contining Luri gr with 1 to 10,M IPTG nd 0.003% Congo red or 0.02% hemin. Preprtion nd nlysis of culture superntnts, outer membrnes, nd protein ggregtes. Culture superntnts were obtined by pelletinga. pleuropneumonie cells grown to the mid-log phse t 4,000 x g for 20 min nd further seprted by ultrcentrifugtion t 100,000 x g for 30 min. The superntnts fter low- or high-speed centrifugtion were concentrted by precipittion with 2 volumes of bsolute ethnol nd kept t -20'C for 1 h. Precipittes were recovered by centrifugtion nd resuspended in wter. Totl membrnes were prepred by French press tretment nd subsequent centrifugtion t 100,000 x g (9). Outer membrnes were prepred by sucrose grdient centrifugtion with two-step grdient (8) nd by Srkosyl solubiliztion (5). The integrity of bcteril cells t the mid-log phse of growth ws scertined by,b-glctosidse ssy (16) of cultures nd culture superntnts obtined by low-speed centrifugtion. For the preprtion of protein ggregtes, broth cultures (50 ml) in the mid-log phse were induced by the ddition of 1 mm IPTG (finl concentrtion). After 2 h of vigorous shking, cells were hrvested by centrifugtion, resuspended in 2 ml of 25% sucrose-50 mm Tris-HCl buffer (ph 8), nd frozen t -70 C. Lysis ws chieved by the ddition of 5,ug of lysozyme in 250 mm Tris-HCl buffer (ph 8), 5 min of incubtion on ice, ddition of 10 ml of detergent mix (5 prts of 20 mm Tris-HCl buffer [ph 7.4]-300 mm NCl-2% deoxycholic cid-2% Nonidet P-40 nd 4 prts of 100 mm Tris-HCl buffer [ph 8]-50 mm EDTA-2% Triton X-100), nd soniction. Protein ggregtes were hrvested by centrifugtion for 30 min t 15,000 x g. Aggregte protein ws resuspended in H20 to concentrtion of 5 to 10 mg/ml nd dissolved by the ddition of n equl volume of 7 M gunidine hydrochloride. Proteins were nlyzed by discontinuous sodium dodecyl sulfte-polycrylmide gel electrophoresis (SDS-PAGE) by the method of Lemmli (14). The protein concentrtion ws estimted by compring the intensity of the Coomssie blue-stined bnds to bovine serum lbumin stndrd (Pierce Chemicl Co., Rockford, Ill.). Electrophoretic trnsfer onto nitrocellulose membrnes ws performed essentilly s described by Towbin et l. (27). Nonspecific binding ws blocked by incubtion in 0.5% geltin in wshing buffer (150 mm NCl, 30 mm Tris-HCl [ph 8], 0.05% Triton X-100). Antibody nd lkline phosphtse conjugte (Kirkegrd & Perry Lbortories, Inc., Githersburg, Md.) were dded in wshing buffer nd ech ws incubted for 1 h t room temperture. Blots were developed with substrte contining 5-bromo-4-chloro-3-indolylphosphte (BCIP) nd nitroblue tetrzolium (ImmunoSelect; Bethesd Reserch Lbortories, Inc., Githersburg, Md.) in 100 mm Tris/HCl buffer (ph 9.5), 50 mm NCl, 5 mm MgCl2. Preprtion of ntiser. Convlescent serum ws obtined by exposing pigs in n erosol chmber (22) to 1 x % lethl dose (LD50) (1 x 103 CFU/ml of erosolized bcteril suspension) of A. pleuropneumonie serotype 7 nd chllenging them 2 weeks lter with 2 LD50. Serum ginst the recombinnt protein ws rised in mice by intrperitonel injection of 30,ug of dissolved ggregte in complete Freund's djuvnt nd subcutneous boost with 30,ug of protein in incomplete Freund's djuvnt. Iron compounds. Trnsferrins from different species were obtined commercilly (porcine trnsferrin from The Binding Site, Birminghm, United Kingdom; humn nd bovine trnsferrin from Sigm Chemicl Co., St. Louis, Mo.). TRANSFERRIN-BINDING PROTEIN 893 Porcine trnsferrin ws iron depleted s described by Mzurier nd Spik (17). The resulting porcine potrnsferrin s well s the commercilly obtined bovine nd humn potrnsferrins were iron sturted s described by Herrington nd Sprling (11). The mino-terminl frgment of porcine trnsferrin ws prepred by the method of Linebck-Zins nd Brew (15). Trnsferrin nd hemin binding ssys. To ssess the possible trnsferrin-binding bility of recombinnt proteins, we performed Western blot-like (immunoblot) trnsferrin-binding ssy essentilly s described by Morton nd Willims (18). Briefly, the protein ws resuspended in nonreducing smple buffer, nd during the entire procedure the temperture ws kept below 37 C. Blots were developed with biotinylted trnsferrin (Biotin-XX-NHS ester Lbeling Kit; Clontech Lbortories, Plo Alto, Clif.) coupled to streptvidin-phosphtse nd purified by gel filtrtion with G-100 column. To detect hemin-binding protein(s) of A. pleuropneumonie, sucrose grdient-purified outer membrnes from cells grown under iron-restricted conditions were suspended in TM buffer (50 mm Tris [ph 8.0], 5 mm MgCl2, 0.1% [vol/vol] Triton X-100), incubted for 2 h t room temperture, nd centrifuged t 100,000 x g. The superntnt ws loded onto hemin-grose column s described by Hnson nd Hnsen (10). The hemin-binding protein ws eluted with TM buffer contining 0.5, 1, nd 2 M NCl or 2 M gunidine hydrochloride. Hemin binding ws further ssessed by using hemin s the competitive gent in the enzyme-linked immunosorbent ssy (ELISA) described below. To exclude n effect of nonspecific hemin binding to trnsferrin, n excess of bovine trnsferrin (1 mg/ml) ws included in the incubtion. To determine species specificity of trnsferrin binding, we developed competitive ELISA. ELISA pltes (Immulon 2; Dyntech Lbortories, Inc., Alexndri, V.) were coted with 100,ul of porcine trnsferrin t concentrtion of 100,ug/ml in crbonte buffer t 4'C overnight. All subsequent steps were performed t room temperture. Pltes were blocked with 0.5% geltin in wshing buffer. Dissolved protein t concentrtion of 2,ug/ml in wshing buffer contining 0.5% geltin ws incubted for 1 h with n equl volume of seril twofold dilutions of porcine, bovine, nd humn trnsferrin s well s hemin nd other possible inhibitors in wshing buffer. Subsequently, 200 pd of this solution ws dded to the coted nd wshed wells nd incubted for 1 h. The ssy ws developed with the mouse serum rised ginst the recombinnt 60-kD protein (60K protein), n lkline phosphtse-lbeled conjugte, nd p-nitrophenyl phosphte in 1 M diethnolmine (ph 9.5)-5 mm MgCl2 s the substrte. The pltes were red t 405 nm, nd the inhibition vlues stte the percentge by which certin concentrtion of competitor in the preincubtion step inhibits the rection between the recombinnt 60K protein nd the coting trnsferrin. Cloning of the gene encoding trnsferrin-binding protein. All restriction enzyme digests were done in T4 DNA polymerse buffer (16) contining 1 mm dithiothreitol nd 3 mm spermidine. Genomic DNA ws prepred s previously described (26) nd prtilly digested with the restriction endonuclese Su3AI. Frgments of 1,500 to 2,500 bp were isolted by sucrose density grdient centrifugtion (16) nd ligted into pgh432 nd pgh433. E. coli HB101 trnsformnts were replic plted onto nitrocellulose discs (Bio-Rd Lbortories, Richmond, Clif.) nd induced for 2 h on pltes contining 1 mm IPTG. Trnsformnts expressing A.

3 894 GERLACH ET AL. INFEcr. IMMUN. b ptf205/el =: E E 9in co z *t AO :il snll z XY co,..,/ mmommm*-,.--._&w.. ~~~~~~~~~~~~~~~~~~~~~~~~J R.dtl t; =co CD ptf205/e2 X,. ptf205/e9 i#_] ptf205/e1 1 1 kb FIG. 1. Physicl mp nd trnsltionl ctivity of plsmid ptf205/e1 nd its deletion derivtives ptf205/e2, ptf205/e9, nd ptf205/e11. () The thick line represents DNA of the cloning vehicle (pgh433), P,c indictes the loction of the tc promoter, nd the sterisk indictes stop codons in ll three reding frmes. The horizontl rrow indictes the loction nd direction of trnscription of the encoding gene designted tjba. The EcoRV-BglII frgment ws used s probe. (b) SDS-polycrylmide gel of the IPTG-induced ggregte proteins produced by ptf205/e1 (lne 1), ptf205/e2 (lne 2), ptf205/e9 (lne 3), nd ptf205/ell (lne 4); lne 5 depicts moleculr weight stndrd contining phosphorylse b (97,400), bovine serum lbumin (66,200), ovlbumin (45,000), crbonic nhydrse (31,000), nd soyben trypsin inhibitor (21,500). pleuropneumonie ntigens were detected by immunoblotting with pig convlescent serum, conjugte, nd substrte s described bove. Positive trnsformnts were replted, induced, nd nlyzed by Western blot, using s control whole-cell lyste of A. pleuropneumonie grown under iron-limiting conditions. The identity of the encoding DNA ws confirmed by Southern blot nlysis s described by Mnitis et l. (16). The wshing conditions were 0.1 x SSC (lx SSC is 0.15 M NCl plus M sodium citrte)-0.5% SDS nd 68 C. RESULTS Identifiction of the 60K protein-expressing E. coli trnsformnt. Of pproximtely 6,000 trnsformnts screened by the immunoblot method, 22 rected with the convlescent serum nd showed n immunorective bnd in the Western blot nlysis. One trnsformnt expressed protein with the sme electrophoretic mobility s n A. pleuropneumonie polypeptide present only under iron-limiting growth conditions. Plsmid ws prepred from this trnsformnt nd designted ptf205/e1 (Fig. l). Three deletion derivtives were constructed nd designted s ptf205/e2, ptf205/e9, nd ptf205/e11 (Fig. l). E. coli HB101 trnsformed with ny of these plsmids, in the bsence of IPTG s well s fter induction with low concentrtions of IPTG (1 to 10,uM), bound both Congo red nd hemin. Upon induction with 1 mm IPTG, the E. coli trnsformnts produced inclusion bodies consisting of ggregted 60K protein (Fig. lb), nd the protein isolted from ptf205/e9 trnsformnts ppered to be smller by pproximtely 5 kd. The ptf205/e1 trnsformnts, in ddition, seemed to produce 30K protein bsent in the other trnsformnts. Iron-regulted expression ofa. pleuropneumonie 60K protein nd its bility to bind trnsferrin nd hemin. In Western blot, the serum rised ginst the recombinnt 60K protein detected single polypeptide in whole-cell lystes of A. pleuropneumonie grown under iron-restricted conditions. The protein ws not expressed under conditions of het, ethnol, or oxidtive stress (Fig. 2). After trnsfer onto nitrocellulose, 60K protein in whole-cell lystes from A. pleuropneumonie grown under iron-restricted conditions s well s the recombinnt protein rected with biotinylted porcine trnsferrin (Fig. 2b). In ddition, 30K, 75K, nd 100K protein were stined by biotinylted trnsferrin in A. pleuropneumonie grown under iron-restricted conditions. The 75K protein ws lso present in A. pleuropneumonie grown under stndrd conditions, nd 30K protein ws present both in A. pleuropneumonie grown under stndrd conditions nd in E. coli (Fig. 2b). The hemin-grose column retined 60K protein from A. pleuropneumonie grown under iron-restricted conditions. The protein ws eluted with 2 M gunidine hydrochloride, nd it rected with serum rised ginst the recombinnt 60K protein (Fig. 2c). Locliztion of 60K protein in A. pleuropneumonie cultures. Whole-cell lystes, totl membrnes, outer membrnes s prepred by sucrose grdient centrifugtion, nd low-speed culture superntnts contined the 60K protein but no detectble,-glctosidse ctivity. Outer membrnes prepred by srcosyl solubiliztion did not contin the 60K protein (Fig. 3). Pellets from the high-speed centrifugtion

4 VOL. 60, 1992 TRANSFERRIN-BINDING PROTEIN <-.ss. :.-f6 *::... ' ''.. : -91.+p t>is b ^_ =~~~-97 _ ~ ~~~~~~~~~ 7 FIG. 2. A. pleuropneumonie 60K protein is induced by iron restriction nd binds porcine trnsferrin nd hemin. () Expression of the 60K protein by A. pleuropneumonie AP205 upon induction with different stimuli. Whole-cell lystes stined with Coomssie blue fter SDS-PAGE (top) nd corresponding Western blot using serum rised ginst recombinnt 60K protein (bottom); lnes: 1, common strt culture; 2, control culture; 3, iron-limiting growth conditions; 4, ethnol stress; 5, peroxide stress; 6, het shock. (b) Trnsferrin binding of the A. pleuropneumonie 60K protein. Coomssie blue-stined gel (top) nd the sme preprtions on nitrocellulose membrne incubted with lkline phosphtse-lbeled porcine trnsferrin (bottom); lnes: 1, whole-cell lyste of A. pleuropneumonie AP205 control cells; 2, cells grown under iron-limiting growth conditions; 3, ggregte protein prepred from ptf205/e1 trnsformnts; 4, E. coli HB101 whole-cell lyste. (c) Hemin binding of thea. pleuropneumonie 60K protein. Coomssie blue-stined gel (top) nd the corresponding Western blot using the serum rised ginst the recombinnt 60K protein (bottom); lnes: 1, sucrose grdient-purified outer membrnes; 2, Triton X-100-treted membrnes; 3, outer membrnes prepred by Srkosyl solubiliztion; 4, hemin-grose eluent t 2 M gunidine hydrochloride; 5, ggregte protein from ptf205/e1 trnsformnts. The positions nd moleculr weights (x 103) of mrker proteins (phosphorylse b, bovine serum lbumin, ovlbumin, crbonic nhydrse) re indicted to the right of ech gel. C 2 3 ~ m i _ - 66 go_ - 97 *:r.:- XiU'AI-.~~~~~3 : of culture superntnts contined the 60K protein, wheres it ws not detected in the superntnts (Fig. 3b). Functionl chrcteriztion of the recombinnt 60K protein. Aggregte protein resuspended in nonreducing smple buffer nd subjected to SDS-PAGE ws found to bind lkline phosphtse-lbeled porcine trnsferrin (Fig. 2b). The species specificity of this binding ws demonstrted in competitive ELISA. Thus, the binding of dissolved ggregte protein to iron-sturted porcine trnsferrin could be inhibited completely by iron-sturted porcine trnsferrin nd ws 50% inhibited by 0.2,uM porcine trnsferrin (Fig. 4). In contrst, pproximtely 3.0,uM of porcine potrnsferrin ws necessry to produce 50% inhibition (Fig. 4). Using humn iron-sturted trnsferrin, only 40% inhibition could be obtined even with concentrtion 60 times higher thn the 50% inhibitory concentrtion of porcine trnsferrin, nd no inhibition ws seen with bovine trnsferrin. In ddition, 6,uM hemin could inhibit trnsferrin binding of the 60K b t _.."o so_i" -q I':do: _ ::.:: s : - 66 FIG. 3. Locliztion of the 60K protein in A. pleuropneumonie AP205 showing gel stined with Coomssie blue (top) nd the corresponding Western blot using serum rised ginst the recombinnt 60K protein (bottom). () Whole-cell lystes from A. pleuropneumonie grown under stndrd (lne 1) nd iron-restricted (lne 2) conditions, the corresponding totl membrnes (lnes 3 nd 4), the outer membrnes prepred by sucrose grdient centrifugtion (lnes 5 nd 6) or Srkosyl solubiliztion (lnes 7 nd 8), culture superntnts prepred by low-speed centrifugtion (lnes 9 nd 10), nd ggregte protein prepred from ptf205/e1 trnsformnts (lne 11). (b) Superntnts fter low-speed centrifugtion from n A. pleuropneumonie culture grown under stndrd (lne 1) nd iron-restricted (lne 2) conditions, corresponding pellets (lnes 3 nd 4) nd superntnts (lnes 5 nd 6) obtined by ultrcentrifugtion, nd ggregte protein prepred from ptf205/el trnsformnts (lne 7). The positions nd moleculr weights (x 103) of mrker proteins (phosphorylse b, bovine serum lbumin, ovlbumin, crbonic nhydrse) re indicted to the right of ech gel.

5 896 GERLACH ET AL. 0 0 M c L Inhibitor Concentrtion (pm) FIG. 4. Inhibition of trnsferrin binding by the recombinnt 60K protein in the competitive ELISA. The inhibitory compounds depicted re iron-sturted porcine trnsferrin (U), porcine potrnsferrin (E), nd hemin (-). The points re the weighted mens from two to five experiments done in duplicte, with the verticl brs depicting the stndrd devitions. Different ggregte preprtions, different ser, nd different trnsferrin preprtions were used in the individul experiments. protein by 50%, nd the inhibitory effect ws unltered in the presence of 1 mg of bovine trnsferrin per ml (Fig. 4). Congo red lso ws inhibitory, wheres porcine hemoglobin, ethylenedimine-diorthohydroxyphenyl-cette (EDDA), 2,2'- dipyridyl, nd ferric citrte did not hve n inhibitory effect in this ssy (Tble 1). Distribution of the 60K protein-encoding gene mong different serotypes ofa. pkuropneumonie. Chromosoml DNA ws prepred from 27 isoltes of A. pleuropneumonie belonging to six different serotypes, digested with the restriction endonucleses BglII nd EcoRV, nd seprted on n grose gel. A Southern blot nlysis with the EcoRV- BglII frgment of ptf205/e1 s probe showed tht frgment identicl in size to the one in ptf205/e1 ws present in ll A. pleuropneumonie serotype 2, 4, nd 7 strins s well s in one serotype 3 strin. In contrst, none of the serotype 1 nd 5 strins rected with the probe under TABLE 1. Ability of vrious substnces to inhibit binding of recombinnt 60K protein to iron-sturted porcine trnsferrin in competitive ELISA Competitive substnces.. INFECT. IMMUN. T s 1~~~~~~e S2 S3 S4 s5 S7 T",...,.!, '' 45kb -- o** * -2. FIG. 5. Southern blot nlysis of different A. pleuropneumonie isoltes. The origin of the probe used is indicted in Fig. 1; S1, S2, S3, S4, S5, nd S7 indicte the positions of chromosoml DNA from the A. pleuropneumonie serotypes 1, 2, 3, 4, 5, nd 7, respectively. The following lnes contin control DNA from E. coli HB101, E. coli JM105, nd E. coli HB101 trnsformed with ptf205/e1. The DNA in ll cses is cut with EcoRV nd BglII. The rrowheds to the right indicte the positions nd sizes of the hybridizing restric tion enzyme frgments. The 2.1-kb frgments hybridized with the 50% inhibition vluesb,ug/ml Porcine trnsferrin 15c 0.2 Porcine trnsferrin, NH2 terminus Porcine potrnsferrin Humn trnsferrin > 1,i000 >12.5 Bovine hemin Congo red Also tested nd completely noninhibitory were bovine trnsferrin (12.5 P.M), porcine hemoglobin (14 PM), EDDA (100 AM, iron sturted), 2,2'- dipyridyl (100 PM, iron sturted), nd ferric citrte (10 mm). b Inhibition vlues stte the concentrtion of competitor in the preincubtion step necessry to inhibit by 50% the rection between recombinnt 60K protein nd coting trnsferrin. c The vlue vried mong experiments between 12.5 nd 100 p.g/ml (Fig. 4); however, the reltive difference in inhibitory ctivity between the vrious substnces ws constnt. d This concentrtion inhibited binding by pproximtely 40%. P,M probe from ptf205/e1. The 3.5-kb frgments hybridized to n A. pleuropneumonie cytolysin probe (1) used s control to show the integrity of the restricted DNA. high-stringency wshing conditions (Fig. 5). To show the integrity of the DNA from the nonrective strins, we dded 3.5-kb BglII frgment from n A. pleuropneumonie cytolysin-contining bcteriophge clone (1) s control probe. Also, under low-stringency wshing conditions (3 x SSC, 55 C), wek hybridiztion of ptf205/e1-derived probe to DNA from A. pleuropneumonie serotype 1 nd 5 isoltes ws observed (dt not shown). DISCUSSION In the present report, we described the cloning nd expression in E. coli of functionlly ctive trnsferrin- nd hemin-binding protein from A. pleuropneumonie serotype 7. E. coli trnsformnts crrying the recombinnt plsmid could be induced with IPTG to produce inclusion bodies consisting of the ggregted polypeptide. The truncted protein produced by ptf205/e9 trnsformnts (Fig. lb) indictes tht the 3' end of the encoding gene is locted pproximtely 150 nucleotides downstrem from the XbI site. Therefore, bsed on the totl size of the polypeptide, the 5' end of the encoding gene (designted tfba [for trnsferrin binding A]) ws mpped to be pproximtely t the NsiI site (Fig. l). Whether the dditionl 30K protein present in ggregte preprtions from ptf205/e1 trnsformnts (Fig. lb) is gene product encoded downstrem from the tfba gene or contminting E. coli protein remins to be investigted. The protein prepred from ptf205/e2-trnsformnts ws ntigeniclly ctive, when dissolved in gunidine hydrochloride, nd ntibodies rised ginst it detected 60K wildtype protein present in A. pleuropneumonie grown under iron-restricted conditions. Expression of the protein is specificlly induced by iron restriction since other stresses such s het, ethnol, nd peroxide did not induce production of the protein (Fig. 2). A 60K wild-type protein ws shown to bind trnsferrin in Western blot-like ssy (Fig. 2b). Three other proteins of pproximtely 30, 75, nd 100 kd moleculr mss lso bound trnsferrin in this ssy (Fig. 2b). The 30K protein ws not locted in the membrne (dt not shown). The 75K protein ws present in the bcteril lystes independent of the growth conditions, nd it ws found to bind streptvidinphosphtse lone (dt not shown). The 100K protein ws found only in cells grown under iron-restricted conditions, nd it ppered to be present in substntil mounts in the

6 VOL. 60, 1992 outer membrne (Fig. 3). Gonzlez et l. (7) recently described the trnsferrin-binding bility of 56K nd 105K outer membrne proteins of A. pleuropneumonie. We suggest tht the cloned 60K protein is relted to the 56K protein identified by these reserchers. Our results using hemingrose ffinity chromtogrphy nd subsequent Western blotting indicte tht the 60K protein dditionlly hs the bility to bind hemin (Fig. 2c). Our finding tht the protein could not be eluted from the column by using 2 M NCl s described for the hemin-binding protein from Hemophilus influenze (10) suggests reltively high ffinity binding. In A. pleuropneumonie, the 60K protein ws found to be present in outer membrnes prepred by sucrose grdient centrifugtion but bsent in outer membrnes prepred by Srkosyl solubiliztion (Fig. 3). This indictes tht it is different from the iron-regulted outer membrne proteins previously described by Deneer nd Potter (5). However, this finding is consistent with the results from Bnerjee- Bhtngr nd Frsch (4), who hve shown tht 70K trnsferrin-binding protein from N. meningitidis cn be seprted from outer membrnes by detergent tretment. Our results re lso consistent with the findings of Hnson nd Hnsen (10), who showed tht the hemin-binding protein from H. influenze cn be removed from outer membrnes by tretment with Triton X-100. The presence of the 60K protein in the superntnt ofa. pleuropneumonie serotype 7 cultures in the mid-log phse could be due to either cell lysis or secretion or the formtion of membrne vesicles which hve been observed to be produced bya. pleuropneumonie isoltes (unpublished dt) s well s by other grm-negtive pthogens (6). To investigte this question, we ssyed cell cultures nd superntnts for P-glctosidse ctivity. The bsence of detectble mounts of enzyme ctivity in culture superntnts suggested tht cell lysis hd not occurred to significnt degree. Also, low-speed superntnts were further seprted by ultrcentrifugtion. The presence of detectble mounts of the 60K protein in the ultrcentrifugtion pellet nd its bsence in the superntnt (Fig. 3b) indicte tht the presence of the 60K protein in culture supemtnts is due to the formtion of membrne vesicles. After SDS-PAGE under nonreducing conditions, the recombinnt protein ws found to bind biotinylted trnsferrin in Western blot-like ssy (Fig. 2). To rule out unspecific trnsferrin binding by the recombinnt protein, we developed competitive ELISA. This ssy showed tht 15-fold-higher mount of porcine potrnsferrin thn ironsturted porcine trnsferrin ws necessry to inhibit binding to the coting iron-sturted porcine trnsferrin by 50% (Fig. 4). Fifty percent inhibition could not be chieved with humn or bovine trnsferrin with concentrtions 60 times greter thn those effective with iron-sturted porcine trnsferrin. The inbility to bind humn nd bovine trnsferrin is consistent with the previously reported inbility of A. pleuropneumonie wild-type strins to use these trnsferrins s sources of iron (7). Therefore, this result indictes tht the trnsferrin binding of the recombinnt protein is due to the sme mechnism used by the ntive polypeptide. In ddition, the stronger inhibition of 60K protein trnsferrin binding by iron-sturted trnsferrin demonstrtes tht the conformtionl chnge of trnsferrin due to iron sturtion influences its ffinity for the 60K protein. This finding suggests tht in vivo, A. pleuropneumonie is ble to efficiently distinguish between iron-sturted nd iron-depleted trnsferrin. This is in contrst to N. meningitidis, which hs TRANSFERRIN-BINDING PROTEIN 897 been found to be unble to distinguish between iron-sturted nd iron-depleted trnsferrin (28). Of further interest is the bility of the 60K protein to bind the mino terminus of the trnsferrin molecule. This, s well s the preference for iron-sturted trnsferrin, indictes tht binding does not occur in the hinge region of the molecule but in n re of the mino-terminl loop which is exposed or conformtionlly ltered in the iron-sturted stte. The inhibitory ctivity of hemin in this ssy ws consistent with the binding of the 60K wild-type protein to hemingrose s well s the Congo red nd hemin binding phenotype of E. coli(ptf205/e1) trnsformnts. Also, to exclude nonspecific interction between the coting trnsferrin nd the hemin, the competitive ELISA with hemin ws done both with nd without 1 mg of bovine trnsferrin per ml. No difference in the inhibitory effect of hemin ws noticed, thus suggesting tht the inhibition is cused by hemin binding of the 60K protein. However, whether this inhibition is due to competitive or llosteric inhibition remins to be investigted. Since A. pleuropneumonie is ble to grow on both hemin nd trnsferrin s the only iron source, the possibility of one common mechnism used to obtin iron from both substnces poses n interesting re for future reserch. Bsed on the mpped loction of the tfba gene, the EcoRV-BglII frgment ws chosen s the DNA probe to confirm the identity of the cloned DNA nd its distribution in other A. pleuropneumonie isoltes. The Southern blot nlysis demonstrtes tht the tfba gene is conserved mong the serotype 2, 4, nd 7 strins investigted. The bsence of hybridiztion under high-stringency conditions to DNA from A. pleuropneumonie serotype 1 nd 5 isoltes suggests divergence of the tfba gene mong different serotypes. This hs subsequently been confirmed by Western blot nlysis nd by cloning of the tfba gene from n A. pleuropneumonie serotype 1 isolte (6). ACKNOWLEDGMENTS This work ws supported by grnt from the Albert Agriculturl Reserch Institute, by n operting grnt from the Nturl Sciences nd Engineering Reserch Council of Cnd, by n Ontrio Ministry of Agriculture nd Food reserch contrct (OP1006), nd by the Cnd-Mnitob Economic nd Regionl Development Agreement ( ). We thnk Sndr Clver for editoril ssistnce. REFERENCES 1. Anderson, C., A. A. Potter, nd G.-F. Gerlch Isoltion nd moleculr chrcteriztion of spontneously occurring cytolysin-negtive mutnts of Actinobcillus pleuropneumonie serotype 7. Infect. Immun. 59: Archibld, F. S., nd I. W. DeVoe Removl of iron from humn trnsferrin by Neisseri meningitidis. FEMS Microbiol. Lett. 6: Archibld, F. S., nd I. W. DeVoe Iron cquisition by Neisseri meningitidis in vitro. Infect. Immun. 27: Bnerjee-Bhtngr, N., nd C. E. Frsch Expression of Neisseri meningitidis iron-regulted outer membrne proteins, including 70-kilodlton trnsferrin receptor, nd their potentil for use s vccines. Infect. 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