A 24-kDa cloned zinc metalloprotease from Actinobacilluspleuropneumoniae is common to all serotypes and cleaves actin in vitro

Claudia Garcia-Cuellar, Cecilia Montafiez, Victor Tenorio, Jorge Reyes-Esparza,Maria de Jesus Duran, Erasmo Negrete, Alma Guerrero, Mireya de la Garza

AbstractActinobacillus pleuropneumoniae causes pleuropneumonia in swine. This bacterium secretes proteases that degrade porcine hemo-globin and IgA in vitro. To further characterize A. pleuropneumoniae proteases, we constructed a genomic library expressed inEscherichia coli DH5ox, and selected a clone that showed proteolytic activity. The recombinant plasmid carries an 800-base pairA. pleuropneumoniae gene sequence that codes for a 24-kDa polypeptide. A 350-base pair PstI fragment from the sequence

hybridized at high stringency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA from Actinobacillus suis,Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida A or D, or E. coli DH5cx, thus showing specificity for A. pleu-ropneumoniae. The expressed polypeptide was recognized as an antigen by convalescent-phase pig sera. Furthermore, a

polyclonal antiserum developed against the purified polypeptide recognized an A. pleuropneumoniae oligomeric protein in bothcrude-extract and cell-free culture media. This recombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of theproteolytic activity by diethylpyrocarbonate suggests that this polypeptide is a zinc metalloprotease.

ResumeActinobacillus pleuropneumoniae est l'agent etiologique de la pleuropneumonie porcine. Cette bacterie secrete des proteases quidegradent l'hemoglobine et les IgA d'origine porcine in vitro. Afin de mieux caracteriser les proteases d'A. pleuropneumoniae, une librairiegenomique capable de s'exprimer dans Escherichia coli DH5o fut constituee et un clone possedant une activite proteolytique fut selec-tionne. Le plasmide recombinant etait porteur d'une sequence genomique de 800 paires de bases provenant d'A. pleuropneumoniae quicodait pour un polypeptide de 24-kDa. Un fragment de 350 paires de bases obtenu par digestion de la sequence genomique avec PstI a re'agilors de reactions d'hybridation sous conditions rigoureuses avec l'ADN provenant des 12 serotypes d'A. pleuropneumoniae, mais ne reagis-sait pas avec l'ADN provenant d'Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida Aou D, ou E. coli DH5o-, demontrant ainsi sa specificite pour A. pleuropneumoniae. Le polypeptide exprimifut reconnu comme un antigenepar un serum de porc convalescent. De plus, un antiserum polyclonal produit contre le polypeptide purifie a reconnu une proteine oligomeriqued'A. pleuropneumoniae dans un extrait brut ainsi que dans le milieu sans bacteries. Le polypeptide recombinant a degrade l'azocoll, lagelatine et l'actine. L'inhibition de l'activite prote'olytique par le diethylpyrocarbonate suggere que ce polypeptide est une metalloproteaseliee au zinc.

(Tradiuit par docteuir Serge Messier)

We described A. pleuropneurnoniae proteolytic activity againstporcine hemoglobin and IgA (7). A >200-kDa metalloprotease com-

Actinobacillus pleuropneumoniae, the etiological agent of porcine mon to all serotypes was purified from cell-free culture medium (8).

pleuropneumonia, is a gram-negative bacterium of the Pasteurellaceae When this protease was treated with reducing agents, it showed

family, of which 14 serotypes have been described (1). Actinobacillus oligomerization, suggesting its ability to form complexes with

pleuropneumoniae often causes fatal disease in swine (2,3). Survivors proteolytic activity. Actinobacillus pleuropneumoniae proteases maygenerally show chronic lesions and weight loss, become subclinical be involved in virulence.

carriers (4,5), and originate acute outbreaks in non-affected herds. To continue studying the A. pleuropneumoniae protease activity,Serotypes 1, 3, 5, and 7 are the most frequent in Mexico (6). we constructed a genomic library from a strain of serotype 1 and

Departamento de Biologia Celular, (Garcia-Cuellar, de la Garza, DurAn, Negrete, Guerrero) and Genetica y Biologia Molecular (Montanez),Centro de Investigaci6n y de Estudios Avanzados del IPN. Ap. 14-740, Mexico D.F. 07000, Mexico; Instituto Nacional de Cancerologia. San

Fernando No. 22, Tlalpan D.F. 14000, Mexico. (Garcia-Cuellar, Reyes-Esparza); Centro Nacional de Investigaciones Microbiol6gicas, Instituto

Nacional de Investigaciones Forestales, Agricolas y Pecuarias, Mexico D.F., M6xico (Tenorio).

Address correspondence and reprint requests to Dr. Mireya de la Garza, telephone: 52-5-747-3800 ext. 5548; fax: 52-5-747-7081; e-mail:

mireya@cell.cinvestav.mx.Received July 9, 1999.

88 The CanaiIn J ral of t hnorv-,rr osoc,-s e, h

select a clone which carries an 800 bp DNA. The clone showed anantigenic and proteolytic phenotype. The recombinant polypeptideshowed metalloprotease characteristics and actin-degrading activity.This is the first report of a cloned protease from A. pleuropneumoniae.

Bacterial strains and growth conditionsActinobacillus pleuropneumoniae strain 35 is a field isolate (7); it was

classified as serotype 1 with pig antisera, donated by R. F. Ross(College of Veterinary Medicine, Iowa State University, Ames,Iowa, USA). The 12 A. pleuropneumoniae strains used, donated byE. M. Kamp (ID-DLO, Department of Bacteriology, The Netherlands),included S4047 (serotype 1), 1536 (serotype 2), 1421 (serotype 3), M62(serotype 4), K17 (serotype 5a), Fem 0 (serotype 6), WF 53 (serotype7), 405 (serotype 8), CVI 13261 (serotype 9), D13039 (serotype 10),56153 (serotype 11), and 6329 (serotype 12). Actinobacillus pleurop-neumoniae strains were grown in brain-heart infusion broth (BHI,Bioxon, Mexico) supplemented with 10 ,ug/mL P-nicotinamide ade-nine dinucleotide, incubated at 37°C in a rotatory shaker at 200 rpm,and harvested at 5 X 109 cells/mL by centrifugation (11 000 X g, 4°C,20 min). Actinobacillus suis was a field isolate obtained by B. Fenwickand donated to us by J. A. Montaraz (Universidad NacionalAutonoma de Mexico, Mexico). It was grown in BHI. Haemophilusparasuis was a field isolate obtained by V. M. Perez (Lab. BioVetSA,Mexico) and grown in BHI supplemented with 1% pig serum.Escherichia coli DH5ao (Gibco BRL) was grown in Luria-Bertani(LB) broth. Recombinant E. coli 2732 was obtained in this work, andwas grown in LB broth containing 50 jig/mL ampicillin and 0.5 mMisopropyl 3-D-thiogalactopyranoside (IPTG). Pasteurella haemolyt-ica Al and Pasteurella multocida types A and D strains were donatedby the Bacteriology Laboratory of INIFAP, Mexico and were culturedin BHI.

DNA extraction and genomic library constructionGenomic DNA was prepared according to Sambrook et al (9).

Plasmids were obtained by alkaline lysis (10). For gene libraryconstruction, DNA from A. pleuropneumoniae serotype 1 (strain 35)was digested with Sau3AI to generate fragments ranged fromapproximately 0.5-10 kb in size. Plasmid Bluescript II KS (Stratagene)was linearized with BamHI, dephosphorylated, and ligated to A. pleu-ropneumoniae DNA fragments. Competent E. coli DH5aL cells weretransformed and spread on LB agar containing ampicillin.

Gene library screeningRecombinant clones were tested in a colony immunoblot assay (9).

A pig sera pool (1:10), positive for A. pleuropneumoniae by comple-ment fixation test, was used as primary antibody and adsorbed withan extract of E. coli DH5a prior to use (9). Sera were obtained fromconvalescent pigs of a farm with a recent pleuropneumonia outbreak.The secondary antibody was rabbit anti-pig-IgG peroxidase-linked,1:5000 (Sigma Chemical Co., St. Louis, Missouri, USA). The immunereaction was detected with 3,3'-diaminobenzidine. Positive cloneswere screened for protease activity on LB agar containing 1% skimmilk and ampicillin (11). Proteolysis was detected in several clonesby a slight halo around the colonies.

PCR amplification of the 800-bp cloned genesequence from pTG2732Polymerase chain reaction (PCR) amplification reagents

(GeneAmp Kit) were from Perkin-Elmer Cetus. Primers used were

the 5' and 3' lacZ universal oligonucleotides (Amplitaq SequencingKit Perkin-Elmer). Amplification involved 30 cycles of denaturationfor 1 min at 94°C, primer annealing for 1 min at 55°C, and primerextension-polymerization for 1 min at 72°C (Perkin-Elmer 480 ther-mal cycler). The PCR product (800 bp) was incubated with EcoRI,BamHI, HindIII, and PstI (1 U/,ug DNA, 1 h). The PCR and digestionproducts were separated by electrophoresis in 1.5% agarose gels andstained with ethidium bromide.

Southern blot hybridizationChromosomal DNA (3 jig) from A. pleuropneumoniae and E. coli

DH5Sa were digested with Sau3AI (1 U/jig DNA, 10 min), separatedby electrophoresis in a 1% agarose gel, and transferred to 0.45-jimnylon filters (Schleicher & Schuell) (12). The 800-bp gene sequence

was used as a probe. This insert was labeled with 5'(a-32P)dCTP(Multiprime DNA labelling system, Amersham International).Filters were hybridized under high stringency conditions 5X SSC (OXSSC contains 0.15 M NaCl, 0.015 M sodium citrate) without for-mamide at 60°C and washed in 0.05X SSC-0.1% SDS at 60°C in a

hybridization oven. Hybridization signals were detected by autora-diography (Kodak X-OMAT film, Kodak Corporation).

Dot-blot hybridizationSamples with 3, 0.3, and 0.03 jig of chromosomal DNA from

each of the 12 A. pleuropneumoniae serotype strains, A. suis, H. para-

suis, P. haemolytica Al, P. multocida types A and D, and E. coliDH5Sa were dotted on a nylon filter using a dot-blot apparatus. Thecomplete cloned gene sequence of pTG2732 (800 bp), as well as its450 bp and 350 bp PstI fragments, were the probes. Hybridization,washing conditions, and signal detection were as for Southernblot hybridization.

SDS-PAGE and immunoblottingcrude extracts

1'of E. coli 2732

Crude extract was prepared from 1 mL E. coli 2732 culture ofA6M = 0.6 (5 X 108 cells/mL), peileted at 11 000 X g, resuspended in100 jiL of 150 mM Tris-HCl, plus an equal volume of Laemmlisample buffer, and boiled 5 min, as was described (9), with the addi-tion of protease inhibitors (13). Crude extract proteins (10 ,uL)were separated by SDS-PAGE using 15% acrylamide gels (14).Cell-free medium was obtained by centrifugation at 7000 X g andfiltration through PVDF-D 0.22 jim Durapore membrane with lowprotein-binding capacity. Proteins were precipitated with coldethanol (1:3 v/v). Protein concentration was determined byBradford's method (15). The same electrophoresis procedure wasused for crude extracts of A. pleuropneumoniae and E. coli DH5a,transformed with pBluescript in order to compare the protein pat-tern. Crude extract and cell-free culture medium were tested byimmunoblot, as described (16), using 0.45 jim nitrocellulose mem-branes (Schleicher & Schuell). To identify the fusion peptide, cloneGal 40 monoclonal anti 3-galactosidase antibody (1:1000, SigmaChemical Co.) was used; the secondary antibody was sheep

The Canadian Journal of Veterinary Research- - -. I -I IC ILI-11. .-l- rt L- n r%89

kbp

23.19.46.64.4

800 bp1 2

2.32.0-450 bp

X 350 bpB

0.5

A,

Figure 1. Actinobacillus pleuropneumonlae DNA cloned In pTG2732. TheInsert was amplified by PCR with 5',3' lacZ universal primers, and run inan agarose gel (1.5%, stained with ethidlum bromide). A. Lane 1: DNA stan-dards (100 bp ladder); lane 2: TG800; lane 3: TG800 digested with Psti(1 U/jig DNA) B. Lane 1: TG450; lane 2: TG350. Size Is Indicated in bp.

anti-mouse-Ig peroxidase-linked, 1:200 (Amersham NXA 931). Toidentify the expressed polypeptide as an antigen, a serum-poolfrom pigs positive for A. pleuropneumoniae (1:10, same conditions as

in library screening) was used. The secondary antibody was rabbitanti-pig-IgG peroxidase-linked, 1:5000 (Sigma Chemical Co.).

Purification of the recombinant polypeptideAcetone powder, obtained from an E. coli 2732 overnight culture

(17), was exposed to preparative SDS-PAGE with 15% acrylamide.To purify the recombinant polypeptide, the 24-kDa band was cut outand electroeluted. Its separation from other proteins with similarmigration was obtained by passing the sample recovered from theelution through an anion exchanger column (Waters-MilliporeProtein-Pak DEAE-5PW, 8.0mm X 7.5 cm) anion exchanger column.This step was carried out with a Waters 650E Advanced ProteinPurification System, coupled with a 486 Tunable AbsorbencyDetector (Waters-Millipore). The column was equilibrated in 50 mMTris-HCl, pH 8.5. Elution was carried out by washing with thesame buffer, followed by an 80 min linear NaCl gradient rangingfrom 0 to 0.5 M in the initial buffer. The sample recovered at 0.4MNaCl containing the purified polypeptide was subjected to molec-ular gel filtration on a Protein-Pak 125 column (7.8 mm X 30 cm)equilibrated with 50 mM Tris-HCl, pH 8.5.

Polyclonal antibodies developed against therecombinant polypeptideTwo New Zealand rabbits were injected subcutaneously with

50 jig of the purified recombinant protease, at 15-day intervals, until

1 2 3 4

Figure 2. Southern blot hybridization of TGSO0 with A. pleuropneumoniaeand E. coil DNA. Chromosomal DNA (3 jig) was digested with Sau3AI(1 U/jig DNA, 10 min). The digestlon products were electrophoresed on

1% agarose gel, transferred onto a nylon membrane, and hybridized with(32P)-TG800 probe under high stringency conditlons. Lane 1: (uP)-DNA sizestandards (DNA digested with Hindlil); lane 2: A. pleuropneumonlae DNA(arrowhead shows the hybridization signal); lane 3: E. coil DH5a DNA;lane 4: TG800 (0.03 jg as positive control).

a titer of 1:1000 was reached, as tested by ELISA assay. Freund's com-plete adjuvant was used for the first immunization, and the incom-plete adjuvant for subsequent antigen injections. Rabbits were

bled and sera adsorbed with E. coli DH5a, according to Sambrooket al (9), and tested by Western blot against crude extracts andcell-free supernatants of A. pleuropneumoniae and E. coli 2732. Thesecondary antibody was a donkey anti-rabbit Ig, peroxidase-linked,1:200 (Amersham NXA 934). Preimmune sera were used as negativecontrol.

Gelatinase zymogram and protease activityassays

Gelatinase zymogram analysis (18) was performed by exposing10 jig of the native purified protein to electrophoresis in SDS-polyacrylamide with 0.1% porcine gelatin, as described for A. pleu-ropneumoniae proteases (7). SDS-PAGE of non-boiled samples wasrun in parallel with the zymogram gels. To determine the opti-mal pH and temperature for proteolytic activity, azocoll cleavage wasmeasured, as described for bacterial proteases (19). Different pH val-ues (4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) and temperatures (18, 37, and 50°C)were tested. Coilagenase IV from Clostridium histolyticum was usedas positive control of proteolysis. Other substrates, includingchicken-gizzard actin, bovine serum albumin (Sigma ChemicalCo.), and pig fibronectin, purified in our laboratory (5 ,ug of bothnative and heat-denatured), were tested for cleavage by the purifiedprotein. These substrates were incubated with 0.5 jig of the

90 The Canadian Journal of Veterinary Research

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Figure 3. Dot blot hybridization using TG800 and Pstl fragments (TG450and TG350) as probes. Chromosomal DNA (3 ug) tested under high strin-gency conditions from: A. (Dot 1) A. pleuropneumonlae serotype 1; (dot 2)serotype 3; (dot 3) serotype 5; (dot 4) serotype 7; and controls: (dot 5)TG450; (dot 6) TG350; (dot 7) TG800. Total DNAs were hybridized with(32P)-TG450 or (32P)-TG350. B. (Dot 1) A. p/europneumonlae serotype 1;(dot 2) A. suls; (dot 3) H. parasuls; (dot 4) P. haemolytica Al; (dot 5)P. multocida type A; (dot 6) P. multocida type D; (dot 7) E. coil DH5a; andcontrols: (dot 8) TG450; (dot 9) TG350; (dot 10) TG800. Total DNAs werehybridized with (32P)-TG800, (32P)-TG450, and (32P)-TG350.

recombinant protein/mL in 0.1 M Tris-HCl, 10 mM CaCl2, 5%2-mercaptoethanol (2-ME), pH 7.0, at 37°C for 24 h. Proteolyticactivity was analyzed by SDS-PAGE.

Proteolysis inhibition assaysInhibition of proteolysis by the recombinant polypeptide was on

azocoll in the presence of 10 mM ethylenediamine-tetraacetic acid(EDTA), 1 mM 1,10-phenanthroline (OPA), 10 mM phenyl methylsulfonyl fluoride (PMSF), 2 mM diethyl pyrocarbonate (DEPC),or 10mM ZnCl2. All incubations were done in the presence of 10mMCaCl2 and 5% 2-ME at pH 7.0 and 37°C.

Specificity of the A. pleuropneumoniae clonedgene fragment

Clone 2732 was selected by its antigenic and proteolytic charac-teristics from among 10 000 recombinants. It carries the plasmidpTG2732 which has an A. pleuropneumoniae 800 bp (TG800) fragment.The fragment was amplified by PCR (Figure 1A, lane 2). OnlyTG800 was restricted by PstI, producing a fragment of 450 bp(TG450) and other one of 350 bp (TG350) (Figure 1A, lane 3). TG800hybridized with DNA from A. pleuropneumoniae, but not with thatof E. coli DH5aL, as was confirmed by Southern blot hybridization(Figure 2, lanes 2 and 3, respectively). TG450 and TG350 werepurified (Figure 1B) and used as probes in a dot-blot hybridizationwith the 12 serotypes of A. pleuropneumoniae (Figure 3A). All theA. pleuropneumoniae serotypes hybridized with both fragments;only serotypes 1, 3, 5, and 7 are shown (Figure 3A, dots 1-4). The con-

I

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Figure 4. Crude extract protein pattems of E co/i 2732 and immunomeactwith pig sera positive for A. pleuropneumoniae. Proteins of crude extractsfrom I X 106 cells were run In a 15% SDS-PAGE and stained withCoomassie brilliant blue. A. Lane 1: molecular weight markers; lane 2:A. pleuropneumonlwe lane 3: E. coli DH5a; lane 4: E. coil DH5a transfomedwith pBluescript; lane 5: E. coil DH5ax transformed with pTG2732.Arrowhead points to the major form of the recombinant protein.B. Corresponding Westem blot from panel A. Arrowheads point to the majorform of the recombinant polypeptide and Its oligomers that were recognizedby the pig sera positive to A. pleuropneumonlae. C. Westem blot recognitionof A. pleuropneumoniae proteins of crude extract tested with a poly-clonal antibody raised to the recombinant polypeptide purifled from E. coli2732. Arrowheads point the bands that show reactivity.

trols used, TG450 and TG350 (dots 5 and 6), are shown in the samefigure. Dot 7 corresponds to the complete 800 bp fragment used asthe control from which the probes were derived. TG800, TG450, andTG350 were used as probes for hybridization with A. pleuropneu-moniae serotype 1, A. suis, H. parasuis, P. haemolytica, P. multocida A,P. multocida D, and E. coli DH5at (Figure 3B, dots 1-7). The hybridiza-tion control, using purified probes TG450, TG350, and TG800, isshown in the same figure, dots 8-10. As Figure 3B shows, TG350probe hybridized only with A. pleuropneumoniae. We show thehybridization with 3 p,g of DNA, but a good signal was alsodetected with 0.3 jig DNA.

A 24kDa polypeptide is expressed by E. coilA prominent 24-kDa polypeptide was seen in crude extracts of

clone 2732 (Figure 4A, lane 5) but not in cell-free medium (notshown). It was also absent in E. coli DH5a, both with and withoutpBluescript (Figure 4A, lanes 4 and 3, respectively). When crudeextracts (Figure 4A, lanes 2-5) were tested by Western blot with ananti-,B-galactosidase antibody, there was recognition (data notshown). These extracts also were tested with the pool of pig sera pos-itive for A. pleuropneumoniae (Figure 4B). Actinobacillus pleuropneu-moniae crude extract was used to check the sera (Figure 4B, lane 2).Positive sera recognized proteins of > 200, 60, 40, and 24 kDa in E. coli2732 (Figure 4B, lane 5). There was no reactivity with untrans-formed and transformed E. coli DH5a (Figure 4B, lanes 3 and 4).When proteins from A. pleuropneumoniae crude extract and cell-free medium were tested by Western blot with a polyclonal anti-serum raised against the 24-kDa purified polypeptide, a band ofsimilar molecular weight and other oligomeric bands were recog-nized, showing reactivity in crude extract, as well as a secretionproduct (Figure 4C, for crude extract).The recombinant polypeptide was obtained by electroelution

from SDS-PAGE, passed through DEAE-cellulose for purification.

The Canadian Journal of Veterinary Research 91

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46.0

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Figure 6. Gelatin zymogram of the recombinant polypeptide from E. coil clone2732. SDS-PAGE (15%, containing 0.1% porcine gelatin) was Incubated I hIn 2.5% Triton X-100 and for 12 h in 100 mM Tris-HCI, pH 7.0, plus 10 mMCaCI2 prior to staining with Coomassie brilliant blue. Lanes I and 2:cell-free medium from A. pleuropneumoniae culture in SDS-PAGE andzymogram gel, respectively, as positive control of protease activity(arrowhead); lanes 3 and 4: the E. coll DH5at proteins recovered at 0.4 MNaCi from DEAE-cellulose chromatography, SDS-PAGE and zymograms,respectively, as negative controls; lanes 5 and 6: the purified recombinantpolypeptide purified under conditions described above, SDS-PAGE andzymogram, respectively. Arrowheads Indicate the recombinant polypeptideand its oligomers In lane 5. Arrowheads indicate protease activities of therecombinant protein in lane 6.

Table 1. Inhibition of the A.protease

pleuropneumoniae recombinant

Figure 5. Temperature and pH optimum for the activity of A. pleuropneu-monlae protease. Purified protease (0.5 pg) was incubated with azo-coil (5 mg/mL In 100 mM Tris-HCI) for 6 h at: A) Different temperaturesB) Several pH values for 6 h at 37°C. Dye release was measured spec-

trophotometrically (A4,).

This polypeptide eluted in the 0.4 M fraction of the NaCl gradient,and was filtered through a molecular filtration chromatography col-umn. After this, oligomers of different molecular weight (24, 40, 60,and 90 kDa) were found (data not shown).

Protease activity of the purified polypeptideThe oligomeric protein mentioned above was tested for proteolytic

activity on azocoll. Maximum activity was observed at 37°C, pH 7.0-8.0. At this temperature, there was no activity below pH 6.0 and itdiminished at pH 9.0 (Figure 5). Enzymatic activity was inhibited(50-60%) by EDTA, OPA, PMSF and ZnCl2, and approximately80% by DEPC (Table I). Ten micrograms of the purified protein,under conditions that preserved the oligomers, were assayed forgelatinase activity. This activity was observed in 40, 60, and 90 kDa,but not in 24 kDa (clear bands of Figure 6, lane 6). The proteinfraction eluted at 0.4 M of NaCl of E. coli DH5ax was obtainedunder the same conditions as that for clone 2732 and it showed no

proteolytic activity (Figure 6, lane 4).

Inhibitor Concentration (mM) Inhibition (%)aEDTA 10 52 ± 2.0OPA 1 56 ± 4.0PMSF 10 60 ± 4.0DEPC 2 77 ± 5.0ZnCI2 10 63 ± 5.0

EDTA - ethylenediamine-tetraacetic acid; OPA - 1,10-phenan-throline; PMSF- phenyl methyl sulfonyl fluoride; DEPC - diethyl pyro-

carbonate; ZnCI2 - zinc chloridea Inhibition of dye released from azocoll, determined at 440 nm after4 h incubation at 370C (data expressed as mean of duplicates from2 experiments ± standard deviations)

As a control of A. pleuropneumoniae protease activity, proteins fromthe cell-free culture medium were used. A >200-kDa polypeptideshowing proteolytic activity appears in Figure 6, lane 2. In additionto azocoll, other substrates like heat-denatured and native chickengizzard actin, bovine serum albumin, and pig fibronectin weretested. Only heat-denatured chicken gizzard actin was cleaved bythe recombinant protease when incubated in the absence of urea(Figure 7, lanes 4, 5 and 6). In order to confirm that we were work-ing with oligomers, the recombinant polypeptide was incubated with

92 The Canadian Journal of Veterinary Research

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*.4~~~~~~~~~~~~~~~~~~. _..._Figure 7. Proteolytic activity against actin of the recombinant purifiedpolypeptide from E. coil DH5aL clone 2732. Samples were mn in a SDS-PAGEand stained with Coomassie brilliant blue. Proteolysis time course ofheat-denatured chicken gizzard actin. Lane 1: molecular weight markers;lane 2: actin (5 ,uLg); lane 3: purified recombinant polypeptide (0.5 ,ug) Incu-bated with 3 M urea. Ten ,Lg actin were Incubated with 0.5 ,ug of recom-binant polypeptide without urea; lane 4: 0 h; lane 5: 12 h; lane 6: 24 h.Arrowheads point to the purifled polypeptide from E. co/i DH5a clone 2732.

3M urea, and only the component of 24 kDa was found under thisstrong denaturing condition (Figure 7, lane 3).

The role that bacterial proteases play in virulence has been elu-cidated in various pathogenic species (11,20,21). Studies of proteaseshave been designed to determine their influence in promotingadhesion, tissue invasion, and colonization. They are important mol-ecules that degrade structural tissues and defense molecules (22,23)and act in cytotoxicity processes (24).

Recent evidence indicates that Actinobacillus pleuropneumoniaeis able to secrete a complex protease formed by oligomers in vitroand in vivo (8). Its activity is non-specific, degrading differentsubstrates such as hemoglobin from different sources, and porcineIgA (7). This activity suggests a function in the disease. In addition,P. multocida secretes a similar protease to that of A. pleuropneumoniae;it was recognized by a polyclonal antiserum directed against themajor A. pleuropneumoniae protease (25). We also have evidence thatP. haemolytica and A. suis secrete proteases into the culture medium(data not published).

Following protease studies in A. pleuropneumoniae, we selected theclone 2732 from a chromosomal library of A. pleuropneumoniae. It wasfirst selected as an antigenic clone to determine its importance in thepig immune response. Later, this clone was positive when tested forprotease activity. It contains the recombinant plasmid pTG2732,which possesses an 800 bp insert (TG800) that has an internal

restriction site for the enzyme PstI. By Southern blot hybridizationunder high stringency conditions, this insert hybridized with A. pleu-ropneumoniae serotype 1 but not with E. coli. This confirms thatthe gene sequence belongs to A. pleuropneumoniae. TG800 hybridizedby dot blot at high stringency with A. suis and H. parasuis; thisresult suggests that these species are highly homologous to A. pleu-ropneumoniae at the gene level. Pasteurella haemolytica and P. multocidadid not hybridize with this probe. However, TG800 hybridizedwith these 2 species under low stringency conditions, and by thecross-reaction result using our A. pleuropneumoniae protease poly-clonal antibody, we think that both proteases are related.

Since TG800 has a restriction site for PstI, then the 2 restrictedproducts were tested to see if one of them was specific for A. pleu-ropneumoniae. When TG350 was tested by dot blot hybridizationunder high stringency conditions with 12 A. pleuropneumoniaeserotypes, it hybridized with all of them. However, it did nothybridize with A. suis, H. parasuis, P. haemolytica, P. multocida, orE. coli. In Mexico, all of the serotypes have been found, withserotypes 1, 3, 5, and 7 being the most frequently diagnosed. Otherpig respiratory tract residents, such as A. suis, H. parasuis, and P. mul-tocida, are commonly found on Mexican pig farms. A species-specificdiagnostic method that detects any serotype is therefore needed, inorder to diagnose subclinically infected pigs. On the other hand,A. pleuropneumoniae and A. suis are pig pathogens and thus close rel-atives, and some of their proteins are highly conserved (26); sinceTG350 can discriminate between A. pleuropneumoniae and A. suis, andthis sequence is conserved in all the A. pleuropneumoniae serotypes,it could be useful in pleuropneumonia diagnosis. Other pig respi-ratory tract pathogens and normal flora must be tested in order toperform a diagnosis test.An oligomeric recombinant protein was isolated from clone

2732. Crude extract and cell-free culture medium proteins showeda weak gelatinase activity in polyacrylamide-gelatin gels. However,the activity was more evident after the expressed polypeptide waspurified. This behavior could be explained by the presence of anendogenous inhibitor in vivo or by a non-functional conforma-tion of the protease inside the bacteria, as has been reported inother species (27), or by the different culture conditions on plate orin liquid media. The antigenic property of the cloned proteasewas confirmed in the crude extract of the clone and in the purifiedrecombinant polypeptide by using convalescent-phase pig sera.Thus, results confirm the presence of an antigen expressed in vivoby A. pleuropneumoniae. Furthermore, an A. pleuropneumoniaeoligomeric protein in cell-free culture medium, as well as in crudeextract, was recognized by antibodies obtained against the recom-binant protein. The recombinant protein was not recognized by apolyclonal antibody directed against a > 200-kDa A. pleuropneumoiaepurified metalloprotease (8), which cross-reacted with P. multo-cida (25). This could be due to the fact that we are dealing with 2 dif-ferent proteases or that the recombinant one has been modified.

Incubation of the recombinant polypeptide with DEPC or highconcentrations of zinc inhibited its proteolytic activity, as has beenreported for zinc-metalloproteases (28,29); therefore, it probablybelongs to this group of proteases, which contain histidine at thezinc-bininog site (19). These zinc-containing metalloproteases clus-ter hemagglutinins, elastases, and neutral or acid proteases from

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diverse gram-positive and gram-negative bacterial species, includ-ing P. haemolytica, a member of the same family but a non-pathogenin pigs (30). Many of them have been described as adhesins and othercolonization factors (30). Calcium and 2-mercaptoethanol werenecessary to achieve the maximal proteolytic activity at 37°C and theoptimal pH was neutral. Since the proteolytic activity was sensitiveto PMSF, serine and thiol groups could be involved at the active site.

During the purification of the 24-kDa polypeptide, 3 bands of dif-ferent molecular weight were obtained. This suggests that it is amonomer capable of forming a complex, and that in this form itcleaved the substrates tested. The same behavior was observedfor A. pleuropneumoniae proteases (8). Protease oligomerization is fre-quently found (31,32).

There are few reports of bacterial proteases which cleave actin, andthis has not been demonstrated as an in vivo mechanism of action(19,33,34,35). We have evidence that A. pleuropneumoniae, but notA. suis, cell-free culture medium has proteolytic activity against pigand chicken actin in vitro (unpublished data). Actinobacillus pleu-ropneumoniae cloned protease cleaved chicken gizzard actin, whichpossesses the 3 major isoforms of this protein (36). Since actin is anintracellular protein, it is possible that actin present in themacrophage cytoplasm could be a target for A. pleuropneumoniae pro-teases. Extracellular A. pleuropneumoniae secreted proteases could beinternalized, as has been described for the 56-kDa protease fromSerratia marcescens (37), subtilisin from Bacillus subtilis, and ther-molysin from Bacillus stearothermophilus (38). Bacteroides fragilismetalloprotease toxin cleaves actin in vitro (19) and rearrangesactin cytoskeleton without proteolysis in a cellular model (35).Thus, more studies are needed to understand the role that A. pleu-ropneumoniae proteases play in degrading host structural proteinslike actin and other cytoskeleton proteins.

We wish to express our gratitude to Claudia Torres, FernandoAvalos, Lourdes Rodriguez, Edgar Rangel, Delfino Godinez, RobertoHamer and Ricardo Valdez for technical assistance. We also thankManuel Ortega and Ruben Lopez-Revilla for their critical readingof the manuscript. This work was supported by CONACYT grantsF383-M9304 and 0038-M9106, and by CONACYT scholarship 55556.

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