INFECTION

AND

IMMUNITY, OCt. 1991, p. 3398-3406

Vol. 59, No. 10

0019-9567/91/103398-09$02.00/0 Copyright © 1991, American Society for Microbiology

Molecular Studies of Ssal, a Serotype-Specific Antigen of Pasteurella haemolytica Al REGGIE Y. C.

LO,'*

CRAIG A. STRATHDEE,1t PATRICIA E. SHEWEN,2 AND BRAD J. COONEY'

Department of Microbiology1 and Department of Veterinary Microbiology and Immunology,2 University of Guelph, Guelph, Ontario, Canada NIG 2WJ Received 8 March 1991/Accepted 1 July 1991

A serotype-specific antigen of Pasteurella haemolytica Al encoded on the recombinant plasmid pSSAl is characterized. Nucleotide sequence analysis of the insert DNA in pSSAl identified the gene ssal, which codes for a protein of approximately 100 kDa. In vivo labeling of pSSAI-encoded protein in Escherichia coli maxicells showed the expression of a 100-kDa protein from the insert DNA on the recombinant plasmid. Northern blot and primer extension analyses were used to identify the mRNA transcript in P. haemolytica Al and the putative promoter of ssal. The antigen (designated Ssal) could be localized to the outer membrane of P. haemolytica Al and E. coli clones carrying pSSAl. A rabbit serum against Ssal was produced by using whole cells of E. coli expressing Ssal on the surface as the immunogen, demonstrating that Ssal is immunogenic in rabbits. The results from colony immunoblot analysis with calf serum from animals that were resistant to P. haemolytica Al-induced pneumonia suggest indirectly that Ssal is also immunogenic in the animals.

Bovine pneumonic pasteurellosis, also known as shipping fever, is the major cause of economic loss in the feedlot cattle industry in North America (25, 39). The principal microorganism associated with the disease is the bacterium Pasteurella haemolytica (11, 25). Although 16 serotypes of P. haemolytica have been recognized (3, 10, 14), only serotype 1 (Al) and rarely serotype 2 are associated with this disease (38). The serotypes are distinguished by differences in soluble or extractable surface antigens in an indirect hemagglutination procedure or a rapid plate agglutination test (13). Serotype specificity is believed to reside in the polysaccharide capsular antigens; however, it is likely that outer membrane components also contribute to this specificity. Vaccination of calves with live P. haemolytica serotype 1 organisms or with bacteria-free culture supernatants from logarithmic-phase cultures induces resistance to experimental challenge (33). These culture supernatants contain a number of soluble antigens, including a leukotoxin specific for ruminant cells (19, 32), glycoprotease (1, 2, 28), neuraminidase (12, 28), and other soluble materials shed during bacterial growth. Vaccination of calves with the culture supernatant stimulates the production of leukotoxin-neutralizing antibodies as well as serotype-specific agglutinating antibodies (33), both of which are apparently necessary for protection. A vaccine based on a P. haemolytica Al culture supernatant has been developed for commercial production (Presponse) (31). As an aid to understanding the relative role of each of the soluble antigens in protection, we are attempting to characterize each antigen at the molecular level. We previously reported cloning and characterization of the genetic determinant encoding the leukotoxin of P. haemolytica Al (23, 24, 35). Recently, we showed that recombinant leukotoxin expressed from the cloned genes enhanced the efficacy of Presponse in a vaccine trial and challenge experiment in

calves (7). We have also reported the identification of recombinant plasmids encoding for a serotype-specific antigen of P. haemolytica Al (15). We report here the molecular studies on this serotype-specific antigen. This antigen is another potential component for use in the development of a serotype-specific subunit vaccine against P. haemolytica Al.

MATERIALS AND METHODS

Bacteria, plasmids, and culture conditions. Escherichia coli HB101 and P. haemolytica Al were described previously (23). The plasmid pSSAl is a subclone of the recombinant plasmid pPH32, which codes for the serotype 1-specific antigen (15). The M13 phage vectors mpl8 and mpl9 were purchased from Pharmacia Chemicals Inc. (Dorval, Quebec, Canada). E. coli TG-1 was used for the propagation of the M13 and recombinant phages, and its genotype was described previously (24). The E. coli HB101 cultures were grown in LT medium supplemented with ampicillin (100 mg/l) where appropriate (22), whereas the P. haemolytica Al cultures were grown in brain heart infusion broth (Difco Laboratories, Detroit, Mich.). The E. coli maxicell-producing strain CSR603 [thr-I ara-14 leuB6 A(gpt-proA)62 lac YJ tsx-33 supE44 galK2 gyrA98 recAl rpsL31 kdgK51 xyl-5 mtl-l argE3 thi-I uvrA6 phr-l rac] was obtained from B. Bachman (E. coli Genetic Stock Center, Yale University) and maintained on LT medium. E. coli TG-1 was maintained on Davis minimal medium (26). Enzymes and chemicals. All restriction endonucleases and DNA-modifying enzymes were purchased from Bethesda Research Laboratories (Burlington, Ontario, Canada) or Pharmacia Chemicals Inc. and were used as suggested by the suppliers. The RNA size standard is from Bethesda Research Laboratories. The goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate, the protein A-horseradish peroxidase conjugate, and the color development reagents were purchased from Bio-Rad Laboratories (Mississauga, Ontario, Canada) or Sigma Chemicals (St. Louis, Mo.). [cx-32P]dATP (3,000 Ci/mmol) and Tran35S-Label (1,130 Ci! mmol) were purchased from ICN Biochemicals (St. Laurent,

* Corresponding author. t Present address: Department of Genetics, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.

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VOL. 59, 1991

Ssal, A SEROTYPE-SPECIFIC ANTIGEN OF P. HAEMOLYTICA Al

Quebec, Canada). The reagents for silver staining of the polyacrylamide gels were purchased from Bio-Rad. Isolation of outer membranes and Western immunoblot analysis. Outer membranes were prepared from E. coli and P. haemolytica Al by the procedure of Hancock and Carey (17), with modifications. Briefly, cells from stationary-phase cultures were washed twice in 0.01 M Tris hydrochloride (pH 6.8) and suspended in 1/50 volume of a cold Tris-sucrose solution that contained 0.01 M Tris hydrochloride (pH 6.8), 20% sucrose, lysozyme (1 mg/ml), RNase (100 ,ug/ml), and DNase (50 ,ug/ml). The cell suspensions were passed through a French press three times at a pressure of 17,000 lb/in2 at 4°C. The samples were centrifuged at 1,085 x g for 5 min to remove cellular debris, and the lysates were layered onto sucrose gradients consisting of 14 ml of 70% sucrose overlaid with 14 ml of 52% sucrose. Approximately 2 ml of the protein preparation was applied to each sucrose gradient, which was then centrifuged in a swinging-bucket rotor at 80,000 x g for 18 h at 4°C. The outer membrane fraction could be seen as the lower band at the 70% sucrose region and was recovered by aspiration. About 4 to 5 ml of the outer membrane fraction was recovered, brought up to a final volume of 12 ml with distilled water, and centrifuged in a fixed-angle rotor at 225,000 x g for 1 h to pellet the outer membranes. The pellets were then resuspended in a small volume (approximately 100 RI) of 0.01 M Tris hydrochloride (pH 6.8)-0.001 M dithiothreitol and stored at -20°C for further analysis. The samples were assayed for succinate dehydrogenase activity and 2-keto-3-deoxyoctonate as described by Squire et al. (34). The protein concentration was determined by the method of Bradford (4). The outer membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) as described by Laemmli (21). After separation by electrophoresis, the proteins were visualized either by staining with Coomassie blue R250 or silver staining reagents. For Western immunoblot analysis, the proteins were transferred to nitrocellulose paper as described previously (5) and incubated with the rabbit antiserum against the soluble antigens of P. haemolytica Al (23) or a rabbit antiserum raised against the serotype-specific antigen Ssal (see below). Both antisera were purified by affinity chromatography with total E. coli proteins as antigens to remove antibodies that cross-reacted with E. coli proteins. The goat anti-rabbit immunoglobulin G-alkaline phosphatase conjugate and the color development reagents were used for the detection of the bound first antibodies. Preimmune serum was used as the negative control in Western immunoblot analyses. Antiserum preparation and agglutination tests. E. coli HB101 carrying the plasmid pSSAl was grown to the stationary phase, and the cells were collected by centrifugation. The cell pellet was resuspended in 1/10 volume of 3% formalinized saline and incubated overnight at 4°C. The fixed bacteria were washed once in phosphate-buffered saline (0.1 M potassium phosphate [pH 7.4]) and resuspended in phosphate-buffered saline to an optical density of 1.0 at 525 nm (equivalent to approximately 109 cells per ml). The suspension was mixed with the appropriate volume of a 0.15% saline solution of the adjuvant saponin (Quil-A; Superfos a/s, Vedbek, Denmark) to yield a final concentration of 50 ,ug of Quil-A per rabbit. Two milliliters of the adjuvanted preparation was injected subcutaneously into each of five Pasteurella multocida-free New Zealand White rabbits (Hazelton Research Products Inc., Denver, Colo.) on two occasions 2 weeks apart. Serum was collected 2 weeks after the last injection and adsorbed by overnight incubation at

3399

4°C with formalinized E. coli HB101; the titers of agglutinating antibody to P. haemolytica Al were determined with a direct microagglutination assay (33). The specificity of the antiserum for P. haemolytica Al was confirmed in a series of slide agglutination tests (13) with each of the P. haemolytica serotypes 1 through 12 and E. coli HB101 as antigens. The serum used in Western immunoblot analysis had an agglutinating titer for P. haemolytica Al of 1/8 after adsorption with E. coli HB101. Identification of Ssal in E. coli maxicells. To analyze the plasmid-encoded proteins on pSSA1, E. coli CSR603 was transformed with pSSAl and maxicells were prepared by the method of Sancar et al. (30). Briefly, E. coli CSR603 carrying pSSAl was grown to the midlogarithmic phase at 37°C in Davis minimal medium (26) supplemented with ampicillin (100 ,ug/ml) and 0.5% Casamino Acids. Ten milliliters of the culture was irradiated for 15 s at 400 puW/cm2 with a germicidal lamp (General Electric). After the cells were cultured for 2 h, 100 pJ of D-cycloserine (2 mg/ml) was added, and the culture was grown overnight. Three milliliters of the culture was centrifuged, washed, and resuspended in 0.75 ml of Davis minimal medium supplemented with threonine (100 ,ug/ml), arginine (150 ,ug/ml), leucine (150 ,ug/ml), and proline (100 ,ug/ml). The cell suspension was incubated for 1 h at 37°C, after which 25 ,uCi of Tran35SLabel was added. After the cells were labeled for 1 h, they were harvested in a microfuge and lysed by resuspension in 150 ,ul of SDS sample buffer (21). The labeled proteins were separated by SDS-PAGE with the discontinuous system of Laemmli (21) and identified by direct autoradiography of the dried gel with Cronex 4 X-ray film (E. I. Du Pont de Nemours & Co., Wilmington, Del.). Molecular cloning and DNA sequence analysis. The nucleotide sequence of the insert DNA encoding pSSAl was determined by the dideoxy-chain termination method as described previously (24). The method of Dale et al. (8) was adopted and modified as follows to generate overlapping deletions of the insert DNA in either M13 mpl8 or mpl9 vectors for sequencing of the whole insert DNA. The RD22 primer (International Biotechnologies Inc., Toronto, Ontario, Canada) was used in conjunction with the mpl9 vector, and the RD29 primer (International Biotechnologies) was used in conjunction with the mpl8 vector. The restriction endonucleases EcoRI and HindlIl were used for cleavage of the annealed DNAs, whereas dGTP and dATP were used for tailing of the 3'-OH ends for the mpl9 and mpl8 recombinants, respectively. The -DNA sequence was analyzed by using Pustell Sequence Analysis programs (International Biotechnologies) on an IBM PC-XT 286 microcomputer (IBM Corp., Armonk, N.Y.). The nucleotide sequence was compared with the GenBank data base to search for homology with reported nucleic acid sequences. In addition, the deduced amino acid sequence of Ssal was analyzed for a potential secretion signal peptide, transmembrane regions, and hydrophobicity by using the SOAP program of PC Gene (Intelligenetics Inc., Mountain View, Calif.). N-terminal amino acid analysis. Outer membranes were prepared from the E. coli clones carrying Ssal by sucrose gradient centrifugation as described above. After SDSPAGE, the proteins were transferred onto polyvinylidene difluoride membranes (Millipore) by Western immunoblotting as described by Walsh et al. (37) and stained with Coomassie blue R250. The part of the polyvinylidene difluoride membrane that contained the Ssal protein band was sliced off, and the materials were subjected to N-terminal

3400

LO ET AL.

INFECT. IMMUN.

amino acid analysis by the automated Edman procedure with a gas-phase peptide microsequencer.

Mapping of ssal transcript and primer extension analysis. Total cellular RNA from P. haemolytica Al was extracted by the method of Hagblom et al. (16) with modjfications. The procedures for Northern blot transfer and hybridization with the ssal-specific probe were as described previously (36). A 2.4-kbp HindIII-PstI fragment from pSSAl was purified from low-melting-point agarose with GeneClean (Bio/Can Scientific, Mississauga, Ontario, Canada) and labeled with [aU-32P]dATP (3,000 Ci/mmol) by nick translation (29). For primer extension analysis, 15 Fg of RNA was annealed with the oligomer SSA, which has the sequence 5'-TGTTTAACC ATGAAGC-3' (Regional DNA Synthesis Laboratory, Calgary, Alberta, Canada), and reverse transcription was carried out with avian myeloblastosis virus reverse transcriptase as described previously (36). The oligomer SSA was also used as a primer in a standard dideoxy sequencing reaction with a DNA fragment that contained the promoter region of ssaI as the substrate to provide a reference for this analysis. The primer extension and reference samples were boiled and separated by PAGE followed by autoradiography as described previously (36). Colony immunoblot analysis with immune calf serum. E. coli clones carrying pSSA1 were analyzed with calf sera by a colony immunoblot method as described previously (22). The calf sera were obtained from animals on entry to a feedlot; the calves were naturally resistant to a subsequent outbreak of P. haemolytica Al pneumonia in the herd. Sera collected from animals that eventually developed pneumonia (sick animals) were also tested. Briefly, the E. coli colonies were grown on a nitrocellulose filter overlaid on agar plates, and the cells were lysed with chloroform vapor. The filter was then washed in a solution containing 50 mM Tris hydrochloride (pH 7.5), 150 mM NaCl, 5 mM MgCI2, 30% gelatin, 1 ,ug of DNase per ml, and 40 ,ug of lysozyme per ml for 30 min. After the filter was washed twice in TBS (20 mM Tris hydrochloride [pH 7.5], 500 mM NaCI), it was incubated in an antibody solution (1% gelatin in TBS) containing calf immune serum at a 1/100 dilution. After overnight incubation, the filter was washed twice with TBS and incubated in protein A-horseradish peroxidase conjugate (1/200 dilution in antibody solution) for 1 to 2 h. After two washes with TBS, the bound protein A conjugate was detected by reaction with 4-chloro-1-napthol and hydrogen peroxide until the desired color intensity appeared. Nucleotide sequence accession number. The nucleotide sequence reported herein has been submitted to GenBank and assigned the accession number M62363. RESULTS Identification of Ssal as an outer membrane protein. Plasmid pPH32 was initially isolated from a P. haemolytica Al genomic library by screening for the expression of P. haemolytica Al soluble antigens in E. coli. It is possible that the serotype 1-specific antigen (Ssal) expressed from pPH32 is a component of the outer membrane of P. haemolytica Al that was sloughed off during growth; alternatively, it may be an exported protein of P. haemolytica Al. We observed previously the expression and surface localization of Ssal in E. coli carrying pPH32 (15), and therefore it may be possible to identify Ssal directly in the outer membrane of the E. coli clones. Outer membrane proteins from E. coli carrying pSSAl were prepared and analyzed by SDS-PAGE to look for additional proteins. The plasmid pSSAl is a subclone of

ees, H

H

-v- AS

A

A

B

I

-

I KbP

SSA1

C

FIG. 1. Restriction maps of plasmids pPH32 (A) and pSSA1 (B). Open boxes represent pBR322 sequences linearized at 3 kbp, and thin lines represent P. haemolytica Al sequences. The dotted lines indicate the religation of pPH32 after partial digestion with HindIll. The arrows indicate the direction and extent of DNA sequencing. (C) The direction of the open reading frame encoding Ssal is indicated by the solid bar. Abbreviations: P, PstI; E, EcoRI; H, HindlIl; B, BamHI; S, SphI; Bg, BglII; BS, BamHI-Sau3A junction. Only one of the BamHI-Sau3A junctions regenerated a BamHI site on pPH32.

pPH32 in which a 1.8-kbp HindlIl fragment was removed after partial digestion and religation of pPH32 (Fig. 1). This plasmid still expresses the serotype-specific antigen as determined by agglutinatic n of E. coli clones with typing serum (results not shown). E. coli carrying pSSAl expresses an outer membrane protein of approximately 100 kDa in addition to the proteins expressed by E. coli carrying the vector pBR322 (Fig. 2). A protein of similar size was also observed in P. haemolytica Al outer membrane protein preparations (Fig. 2). Since the bands were visualized by silver staining, various minor bands also appear on the gel due to the extreme sensitivity of the staining method. Staining of the gel with Coomassie blue results in poor resolution of the bands, probably as a result of lipopolysaccharide material that appears to be complexed and migrating with Ssal in the

a

b

c

:

200

..... f.

116

92.5

66

45

FIG. 2. A 10% SDS-PAGE profile of the outer membrane proteins from the E. coli clones and from P. haemolytica Al visualized by silver staining. The left lane shows the molecular size standards in kilodaltons. Lanes: a, E. coli carrying pBR322; b, E. coli carrying pSSAl; c, P. haemolytica Al. The location of Ssal is indicated by the arrow.

VOL. 59, 1991

Ssal, A SEROTYPE-SPECIFIC ANTIGEN OF P. HAEMOLYTICA Al

FIG. 3. Autoradiogram showing E. coli maxicell analysis of plasmid-encoded proteins expressed from pBR322 or pSSA1. The arrow indicates the 100-kDa protein expressed from pSSA1. Bla indicates the P-lactamase encoded on pBR322. The location of the molecular size standards in kilodaltons are indicated on the left.

P. haemolytica Al outer membrane preparations (data not

shown). Enzymatic analysis of the succinate dehydrogenase and 2-keto-3-deoxyoctonate of the outer membrane fractions

showed that the outer membrane fractions had less than 10% contaminating inner membrane material (data not shown). These results suggest that Ssal could be the 100-kDa outer membrane protein in P. haemolytica Al. Maxicell labeling of pSSA1-encoded proteins. The plasmid pSSAl was transformed into E. coli CSR603, which is used for in vivo labeling of plasmid-encoded proteins. A protein of approximately 100 kDa was expressed from the insert DNA in pSSAl (Fig. 3). The size of this plasmid-encoded protein is very similar to that of the 100-kDa protein observed in the outer membrane of the E. coli clones and in P. haemolytica Al (Fig. 2). This result agrees with the above observation that Ssal is a protein of 100 kDa. In related experiments, attempts to prepare minicells from E. coli carrying pSSAl for the in vivo labeling of pSSAl-encoded proteins failed repeatedly. This may be attributed to the failure of the minicells to separate from the mother cells due to the presence of Ssal in the outer membrane of the E. coli clones. Nucleotide sequence of ssal. The strategy used to determine the nucleotide sequence of the insert DNA in pSSAl is shown in Fig. 1, and the DNA sequence is presented in Fig. 4. Eighty percent of both DNA strands was sequenced either directly from the subclones or by using overlapping deletions in the cloned DNA. In all cases, each nucleotide was sequenced at least twice independently by using overlapping deletions of the insert DNA fragments in the M13 mpl8 and mpl9 vectors. This sequence covers a continuous region of 3,628 bp that contains a single large open reading frame encoding a polypeptide of 932 amino acids with a predicted molecular mass of 103.6 kDa. This estimation is very close to the deduced size of Ssal determined by SDS-PAGE of the outer membrane proteins in the E. coli clones and by maxicell analysis of pSSAl-encoded proteins. This open reading frame is designated ssaL. The predicted amino acid sequence of Ssal was analyzed for its hydrophobicity and for any potential membranespanning regions. Figure 5 shows a hydropathy plot of Ssal as analyzed with the SOAP program (20). This is based on

3401

discriminant analysis of the characteristics of a protein sequence with parameters such as maximum local hydrophobicity (20). The analysis identified one potential transmembrane region on Ssal between amino acids 11 and 27. Further, a potential cleavage site for the removal of a secretory leader peptide was located between amino acids 24 and 25 (Fig. 4). These analyses support the interpretation that Ssal is transported through the inner membrane into the outer membrane; however, the actual amino acid position used for the removal of a signal peptide remains to be determined. Finally, the nucleotide sequence of ssaI was used to search the GenBank data base for homology, and no significant homology was detected. N-terminal analysis of Ssal expressed from E. coli. Results from the N-terminal amino acid analysis of the heterologously expressed Ssal showed that the first seven amino acids of the peptide correspond to position 58 onwards (Fig. 4). From this position, the predicted molecular mass of the protein would be 97.5 kDa, which is very close to the molecular mass of 103.6 kDa predicted from the nucleotide sequence data. These two proteins would be difficult to resolve in the SDS-PAGE system used. It is possible that the initial translated product from ssaI is a protein of 103.6 kDa, which was processed into the smaller protein. This discrepancy in the molecular mass of Ssal is not understood at this moment and will be addressed in the Discussion section. Mapping of the mRNA transcript and the promoter of ssal. To further characterize the expression of ssaI, Northern blot analysis was carried out with a restriction fragment of ssal as a probe against total RNA from P. haemolytica Al. The autoradiogram in Fig. 6 shows ,a unique transcript of approximately 3 kb. The size of this transcript is in agreement with the predicted coding length of ssaI as deduced from the nucleotide sequence. The primary transcript expressed from ssaI was characterized by primer extension with avian myeloblastosis virus reverse transcriptase together with an oligomer that is complementary to positions 179 to 194 according to the numbering system in Fig. 4. Figure 7 shows a sequencing gel that contains sequences reading down to the ribosome-binding site and initiation codon of ssaL. In the primer-extended RNA sample, the longest transcript had a 5' end corresponding to position -51 with respect to the ssaI initiation codon and initiating at a guanosine residue (Fig. 4). Three other possible initiation sites corresponding to positions -49, -42, and -41 can also be seen; however, they may represent degradation products from the primary transcript. Examination of the nucleotide sequence of this region shows sequences homologous to the E. coli consensus -10 and -35 promoter sequences (Fig. 4). In fact, a perfect match to the consensus TATAAT sequence can be seen. Starting from the guanosine at position -51, the transcript expressed will be approximately 3 kb long and will terminate at a putative rho-independent transcriptional termination signal identified downstream of ssaI (Fig. 4). This is in good agreement with the actual length of the transcript detected by Northern blot analysis. In addition, a potential ribosomal binding sequence that closely resembles the E. coli consensus sequence can also be seen upon transcription of the mRNA at a proper position preceding the AUG initiation codon (Fig. 4). These features are probably involved in the expression of ssal. Immunogenicity of Ssal. E. coli expressing Ssal on the cell surface was used to immunize rabbits as described in Materials and Methods. Figure 8A shows a Western immunoblot of outer membrane proteins from E. coli carrying pSSAl and from P. haemolytica Al blotted with this rabbit serum. A

3402

INFECT. IMMUN.

LO ET AL.

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1510

CMA GIG TOG GGC AMA TCT Gtu Vet Lou Gly Lye .Ser

GAl AMA TTA ACA GMA AMA ACC GMA GCA GOT GAT 011 TCA GCG ACC GTA GTA CAG ITOG Asp Lye Lou Thr Gtu Lye Thr Gtu Ate Gty Asp Vet Ser Ate Thr Vet Vei Gin Lou

0630

GAG CTT TAT OTT GCA ACG MAG ICA TAT MAA COA ACT 106 GMA MAA GOT AlT CMA OAT Gisu Lou Tyr Vet Ate Thr Lye Ser Tyr Lys Ars Thr Lou Gtu Lye Vet lito Gin Asp

1500

1550

1540

1590

1580

480

470

1450

000 CIA AlT 0CC MIT OCT CMA 0CC MIT AlA CAT TIA GCC GGC TCG TTA AMA All OAT Gty Lou It* Ate Asn Ate GLn Ate Ass Ito His Lou Ate GLy Sot Lou Lye IL., Asp

430

OCT CMA GCA OAT MAA ACT CII GOT OAT GGC TAT AGO GOY GOT All GCA MAG GGA OCT Ate GLn Ate Asp Lye Thr Lou Gty Asp Gty Tyr Sor Gty Gty Ieo Ate Lye Gty Ate 440

1530

1520

All OAT CTT All CCT OCT CII GMA ACA CAC GGT GCA GOT GIA OCT GOT All AOl GCC Ito Asp Lou ILo Pro Sor Lou Gtu Thr His Gty ALa Oty Vet Ate Gty ite Iie Ate 380

110

1440

GOT GAG CMA MIT TAT TCT TCG OCT GOA CMA AGC CMA TTA VoL Gtu Gin Asn Tyr Ser Set Ser Gty Gin Sot Gin Lou

370

360

350

GCC ITA MAC GOT Gty Arg Leu AL. Lou Aen Gty

GOT AGA

1490

1480

1470

1460

ATC TAC GAT GCA AGT TAC CCI CMA TIC GAG GTA MAC CCC GTA GMA MAA GMA GAT GGA Ite Tyr Asp Ate Scr Tyr Pro Gin Ph. GLu Vet Asn Pro Vet Gtu Lye GLu Asp Gty

320

1400

1390

1380

1430

1420

CTG CAT

AAAACTIMGGTG AAAACA ATCAGAAI C CAT GCAMAAITAGCTGTCMAT GGA ACCGMA Lye Thr Lye Vet Lye Thr Ie* Sot Asn His Ate Asn Leu Ate Vet Asn GLy Thr GiLu

310

300

290

TCA

1370

1410

AGO ICA CATCTT CAT CCT CTT ATTCATICMAATAACA ACA CCC GAAGGCGMOAAOGAAA Set Ser His Lou His Pro Lou Ite His Gtn Lou Thr Thr Pro GLu Gty Gtu Vat Ars

270

1340

TTA OAC TOT AMA AlT AMA AMA 000 OAC AMA Ph. Lys ILo Ihr Lye Lys Gty Asp Lys Sot Loss Nis Loss Asp

1360

1350

TOG TCG MAT Trp Sot Asn

1330

ACA

GGC GMA MIT CAT TTA OAT ACG GIA GCA GOT GMA GMA Gty Gtu Asn His Leu Asp Thr Vat Ate Vet Gtu Gtu

CAC CCI COC CAT His Pro Arg His

Lye GLy Vat Lye Lou Gly Vet Het Asp GLu Gty Pho Net Vet_Lye

ACT COT GAT OAC CAT Thr Arg Asp Asp His

1320

1310

1300

CCA CTC GCC AGC CMA Pro Leu ALe Sct Gtn

1280

1270

1260

CMA TIC CIA MIT OAT GMA ACG All ACC GTA GLn Ph. Lou Asn Asp GLu Thr Ite Thr Vat

1290

GAG MAT CCA CMA CCI ATC AlT CMA TTA TCT GAG AGC CTC AGC TCA AMA TAT AGT GGC Gtu Asn Pro GLn Pro IL. ILo Gin Lou Ser GLu Ser Lou Sor Sot Lys Tyr Sot Gty

TGG

1250

1240 ACT hr

140

130

120

110

100

GMA TCA AlA Gtus Ser lito

GCA TAC OCT ACA Yhr

CIT AlT GCA All AGC All TCT AOl OTT TTA TCT All Leu Lie Ate Ito Ser lite Sr Ser Ph. Less Ser Ite

1230

1220

1210

G00 CTC All AAC COO AMA AMA OCT GTT MAT GGC CCA Gly Trp Gly Leus Ito Asn Leu Lys Lys ALe Vet Asn Gty Pro

TAT GGC

Val Asp Asn Val Tyr

90

80

70

60

MAT OTT

GTC GAO

1200

1190

1180

ITT AAC AAA ACA Ph. Asn Lys Thr

CAT TCA Lys lie Lys His Ser

AMA

NHot Tyr

s0

40

30

20

10

GGT AAA ATT ATG TAT

SD

666

1170

1160

1150

1140

1130

1120

-30

-40

-50

GCG ACT CMA ATT CGC GAT ACT TTA TTG ACT ACC GCC ACT OAT T16 GGT GMA AMA GGG ATA MAA MAT TAA CIA CGT AMA AlA `TAA TTA TTT G*TA.P.AL& Arg Asp Thr Leu Leu Thr Thr Ate Thr Asp Less Gly Gtu Lys GLY Thr Gtn -to

GA TCT ITA TCA ITTA ACT AGA TTA

His

2290

CCA Pro

2190 TTT

GCA

ACC 116

Phe Ate Gin Ate Thr

2240

116

CMA OCT

Lou

2250

OAT MAC OCT 100 Pho 601 SHr TTT

Lou Asp Asn Ate Trp

2300

2310

GMA OCT ACA TOG C66 OCT CGOCAG6 MAA lAO CM CMAAAC COOT COA OCT AGC Gtus Ate Ohr Leu Gtn Phe Ate Arg Gin Lye Tyr Gin Gtn Sot AtH Arg Pho Ate Sot TTT

TTT

Ssal, A SEROTYPE-SPECIFIC ANTIGEN OF P. HAEMOLYTICA Al

VOL. 59, 1991

2370

2360

2350

2340

2330

2320

CAT CAA CTA GGT ACT GCA GAA ACC AGA GOT TCA ACG CTC GOT GT GAA ATG COG ATC His GLn Leu Gly Thr ALa Glu Thr Arg Gly Ser Thr Lou Gly Gly Glu Net Arg lIe

2420

2410

2400

2390

2380

GGT TAT CM TTT ATG CCA AAC CAA TOO ATA ATT GAA CCA AMC CTT GGC GTA CAA TOG Gly Tyr Gln Phe Met Pro Asn Gln Trp lIe lIe Giu Pro Ser L*u Gly V.l Gin Trp

248

2470

2460

2450

240

2430

ATT CAA ACC AA ATG AAT GOT TTA AAT GAA AMT GGC GAA CTT CCG ACT CM ACG GCT lIe Gin Thr Lys Net Asn Gly Lou Asn Glu Ser Gly Glu Lou Ata Thr Gln Thr Alt

2500

2490

2540

2530

2520

2510

GCA ATG COT TAT COT AT

AAT ATT GTT CCA AC GTG AAA TTA CG CGA ACT TTC met Arg Tyr Arg Asp Val Asn Ito Val Pro Ser Vat Lys Lou Gin Arg Thr Pht

Al.

GTG

2s5o

2570

2560

2550

CAA CTT GAG CAA GGC TCT ATT TCC CCT TAT ATC Ser Pro Tyr lIe Gln Leu Giu Gin Gly Ser

2630

2620

2610

2670

2680

GCA ACG ACG AAA CGT AAT CGC CAA TTA AAC 00C

2720 AAA AAT

TGG

0C0 ATOAMT

ACT

TTT

Gly

Gly

TTA

2780

AMT CGA

TAC

2800

2790

GTG AAA CTG CAT TAT

Gly

Vat Lys Lou Nis

2830

2850

2840 TGA AAA

TCA

Vat Ly

Ser Cys Lw Pro

AM

AAA CCT TGA ACA ACT OTT CAA

AAT TTA

2890

2900

3000

3020

3010

TGC TAC

CTT ATC

3130

ACC

TTT

AM

3110

3100

MGC TCC TA CAT

TTG

3150

3180

3190

3200

3210

3220

CTG ATA TTT CCA CCA AAT AM ACT AA AAT MT CGC AAT TAT COC AMA TAA CC AAT

3240

3230

3250

3260

3300

3310

3320

MG

CGA 0C0

3330

AAA TCC CAC CAC ATA ATA AMG ACA CCC CTA ATA CTA ATT CTA ATc CMA GTA TA CMA 3340

GC

3350

CTA ATA

TGC

3400

3360

TCC

AGC CAA

3410

CTC AAT TMA TCC

GOT

3460

CTA

3380

AM

CC AGT

3430

TGA

3390

CCA TAC TCC CTC CAT ATA

3450

3440

OCT TOT TTT ATT OCT TTT TM

3490

3480

ACC AAT CAA ATT TCC AMC TTC TAA COG CAT

3530

3520

AAT TAM

3120 TTT A80

3470

AAT ACC 8GC MC A80

3370

TCC

3S40

*

ACT GTT ATC T1A AGA ACC TAT TTC TTT CMA

AGC

TT

DISCUSSION Outer membrane preparations from E. coli carrying pSSAl contained a 100-kDa protein encoded by the plasmid pSSAl. A protein of similar size was also detected in the

3280

3270

TOC TAA CGC TTG CCC TAA TOT AAA AAM TCC AMC OAT GMA AGC AMG MT

3290

band at approximately 100 kDa can be detected in the outer membrane of E. coli carrying pSSAl. This band is not present in E. coli carrying pBR322. In the outer membrane preparation from P. haemolytica Al, two closely migrating bands can be seen. It is possible that the two bands represent the primary translated and processed forms of Ssal in P. haemolytica Al. A number of other bands are also detected with this antiserum in the E. coli samples. This could represent degradation materials of Ssal or antigens that cross-react with E. coli antigens, since E. coli whole cells were used as the immunogen. By using the rabbit serum against the soluble antigens of P. haemolytica Al in Western immunoblots, the Ssal protein was detected in the outer membrane preparations from E. coli carrying pSSAl and from P. haemolytica Al (Fig. 8B). This is not unexpected, since this serum was used in the initial isolation of pPH32. Since no bands were detected in the preparation from E. coli carrying pBR322, the other bands could be due to degradation materials from Ssal. Preimmune sera do not detect the 100-kDa protein in all of the outer membrane preparations (data not shown). Calf serum obtained from healthy animals resistant to shipping fever was used in colony immunoblot analysis against E. coli carrying pSSAl to determine whether the serum contains antibodies that recognize this P. haemolytica Al antigen. In addition to pSSAl, E. coli carrying pLKT52 (35), which encodes the leukotoxin, was included in the immunoblots as a positive control, since it is known that leukotoxin is one of the protective antigens (33). E. coli expressing Ssal or the leukotoxin is recognized by the calf serum (Fig. 9), suggesting that animals that are healthy and resistant to pneumonia have serum antibodies that recognize Ssal and the leukotoxin. E. coli carrying pBR322 does not show any positive response with the sera. Further, sera from sick animals do not have antibodies that recognize Ssal or the leukotoxin (Fig. 9), suggesting that an immune response to these two antigens may contribute to resistance. These data should be interpreted with caution, since the actual titers in serum of the antibodies against these antigens are not known. However, the data are consistent with the notion that both the leukotoxin and Ssal are produced in P. haemolytica Al in calves and are immunogenic.

3160

AG G0A ACT OCT CTG ATC CTC TTT CTT GTC TTT TCC ATG

0C0 ATG CTC TCG A8A GTT

3170

3050

CT ATC

3090

3140

A8

CTG GAC TTC MAT

TTT

3040

Trc AC OAT 0CC ATT AMG TAA AT TTC AMC CAC AMA GCC

3120

ATA

2990

AC ACC CCA ACT

308

3070

3060 TCC

2940

TMA

GA

2m0

3030

TTG TA ATT ATT 8CC TAT

GAG

2930

AAT TCC CTC CAC

GT

288 ACC OCT TC

TCA

TTC TAT

GTG

TTA

2970

ACG GAC AM TAA

TTG CTT

ACA CMA

GGT

2870

2920 TOT

TTT

CCT

2320

CTA TTT

ACT

2910

2960

2950 AAA AAC TTA

AMT TTC

GOT

TGC

2810

2860

TTTGq

GTT

AMA

GTG A

ATT TOG TTG GA AGT AAA TOT TGG CTT TAM TtT CTA ACT TAT TAT lie Trp Leu Glu Ser Lys Cys Trp Lou ---

0C0

Tyr

2760

Lys Asn Trp Phe Thr Ala Met Mn Lou Asp Tyr Ser Arg

2770

GGG

2750

OAT

2710

2700

CTC Glu Vol GM

2740

2730

AAA ACA CTG CAT AMT GAM Lys Thr Lou Nis Ser Glu

2690

Ala Thr Thr Lys Arg Asn Arg Gin Lou Asn

2650

2640

AAT AAA ATA ACC AAA ATT ACC AMT AAC ATT GCA Asn Gly Lys lIe Thr Lys lIe Thr Ser Asn ILe Ala

2660

AMn

Gly Lou

lie

2600

2590 AAT TAC TTG CAT AGA TTA Tyr Leu Wis Arg Leu

CT

GGG

3403

MT TTC

3500

AC

3510 TAC

AAC

TTT

FIG. 4. Nucleotide sequence of the insert DNA on pSSAl. The numbers above each line refer to the nucleotide positions, which are arbitrarily numbered from position +1 at the ATG initiation codon of the open reading frame of ssaL. The predicted amino acid sequence of Ssal is shown beneath the DNA sequence. The locations of the -35 and -10 consensus promoter sequences and the Shine-Dalgarno sequence (SD) on the transcribed mRNA are indicated. The arrowhead (o--_) indicates the guanosine residue identified as the first nucleotide transcribed (see Fig. 6). The position of the predicted cleavage site for the removal of signal sequence is identified (A). The amino acids underlined at positions 58 to 64 were identified from Edman degradation analysis of the heterologously expressed Ssal (a valine was identified at the methionine residue underlined by the dots). The arrows between nucleotides 2837 and 2865 indicate the inverted repeats located 3' to the coding sequence that could form a stem structure and function in rho-independent termination of transcription.

3404

INFECT. IMMUN.

LO ET AL.

40

30 20

-16 -28

-30 -40

90 70 we 40 600 SW FIG. 5. Hydropathy plot of the predicted amino acids of Ssal. The method used was that of Klein et al. (20). The vertical axis represents the scale of the hydrophobic (positive) and hydrophilic (negative) values established for each window of nine amino acids. The horizontal axis shows the positions of the amino acids in the protein. 530 10 200

outer membrane of P. haemolytica Al. It is not surprising to find Ssal in the outer membrane fraction, since the recomwas originally isolated by screening of a genomic library of P. haemolytica Al DNA with an antiserum directed against the soluble antigens of the bacterium (15). During growth of the bacterium, this protein is probably shed into the culture supernatant as a result of cell division or lysis. Previous results have demonstrated the surface location of this protein in the E. coli clones (15), and the present data support that observation. There have been a number of reports in the literature on the characterization of outer membrane proteins from P. haemolytica Al and their possible role(s) as protective antigens (6, 27). These data were obtained primarily by SDS-PAGE analysis of outer membrane proteins extracted by various methods or by the use of Western immunoblot analysis of the bacterial antigens with immune serum from P. haemolytica Al-infected ani-

binant clone

9:5 7. 15;

:

4A

1.4

24

FIG. 6. Northern blot analysis of the ssaI-specific transcript. Shown is an autoradiogram of RNA extracted from P. haemolytica Al separated by a 1.0% agarose gel and hybridized with the 2.4-kbp PstI-HindIII fragment from pSSA1. The positions of the RNA size standards are indicated in kilobases on the right.

A._

.0 ,Mt

.

i

FIG. 7. Mapping of the ssaI-specific promoter. Shown is an autoradiogram of an 8% polyacrylamide-8 M urea sequencing gel showing the primer-extended transcripts together with a standard dideoxy sequencing run of a subclone of pSSAl with the synthetic oligomer SSA as the primer. The transcriptional start site is indicated at the guanosine residue. The Pribnow box at position -10, the RNA polymerase binding site at position -35, the ShineDalgarno sequence, and the ATG initiation codon of ssal are also shown.

mals (6, 27). However, the identity of these proteins or antigens is often unknown, making it difficult for follow-up and detailed characterization. This is the first incidence in which a serotype-specific outer membrane protein of P. haemolytica Al has been identified and its gene has been cloned and sequenced. This characterization will make future experimentation on Ssal more amenable. The results from the nucleotide sequence analysis of the insert DNA on pSSAl identified a large open reading frame that could be encoding Ssal. This is supported by the following evidence. The size of the open reading frame and the predicted molecular mass of the encoded polypeptide are in good agreement with the apparent molecular mass of Ssal extrapolated from SDS-PAGE analysis of the outer membrane of the E. coli clones and from P. haemolytica Al. In vivo labeling of E. coli maxicells carrying pSSAl showed a protein expressed from the insert DNA with a molecular mass similar to that estimated from SDS-PAGE analysis of outer membrane proteins. Northern blot analysis of RNA transcripts from P. haemolytica Al with the cloned DNA identified an RNA transcript of the expected size. Finally, primer extension data identified the promoter involved in the expression of the open reading frame. The regions of the DNA immediately upstream from the predicted open reading frame show close similarity to the promoter sequences identified in E. coli (18). All of these results indicate that the insert DNA in pSSAl did encode the Ssal protein, and the identified gene has been designated ssaL. The localization of Ssal to the outer membrane of the E. coli clones indicates that Ssal must be transported across the inner membrane by the E. coli protein export mecha-

VOL. 59, 1991

Ssal, A SEROTYPE-SPECIFIC ANTIGEN OF P. HAEMOLYTICA Al

A 1

B

2 3

1

2

3

iiri

FIG. 8. Western immunoblot analysis of outer membrane proteins from the following (lanes): 1, E. coli carrying pBR322; 2, E. coli carrying pSSAl; 3, P. haemolytica Al. Rabbit serum raised against whole cells of E. coli expressing Ssal on the surface was used to probe the blot in panel A, whereas a rabbit serum raised against the soluble antigens of P. haemolytica Al was used to probe the blot in panel B. The arrows indicate Ssal.

nism. This suggests that Ssal must contain the necessary signal(s) for its recognition and translocation in E. coli. Analysis of the deduced amino acid sequence of Ssal predicted the presence of a transmembrane region and a potential signal sequence cleavage site (Fig. 4). Interestingly, N-terminal analysis of the heterologously expressed Ssal from E. coli shows that this protein begins at amino acid 58 on the predicted open reading frame. It is unlikely that the methionine at position 57 was used as the initiation codon, since there is no ribosome binding site immediately upstream of this position. Further, it would be difficult to explain the secretion of this protein without a signal seIn addition, the data from mapping of the mRNA transcript clearly identified the promoter of ssaI at the position shown in Fig. 4, suggesting that a 103-kDa protein is probably expressed from the gene. It is possible that this discrepancy in size is due to processing of Ssal and may not be directly associated with its translocation through the quence.

3405

inner membrane. Alternatively, the site for the cleavage of the signal peptide may not be that predicted by the computer analysis, which was based primarily on data from E. coli proteins. In Western blot analysis of the P. haemolytica Al outer membrane, two closely migrating bands could be detected (Fig. 8A, lane 3). This supports the hypothesis that in P. haemolytica Al the primary translated product is 103 kDa in mass and is processed to the smaller 97-kDa protein. On the other hand, silver staining of the outer membrane proteins after SDS-PAGE did not resolve two proteins corresponding to Ssal (Fig. 2). This could be due to the extreme sensitivity of the staining procedure and to the presence of contaminating lipopolysaccharide material, resulting in less defined bands on the gel. Analysis of the N-terminal amino acid sequence of Ssal prepared from P. haemolytica Al will clarify the actual size of Ssal in P. haemolytica Al. However, because of the difficulties in recovering quantities of sufficiently pure Ssal from P. haemolytica Al, this is not possible at this moment. Quantitative preparation of Ssal expressed from the cloned gene in E. coli is currently in progress for the production of monoclonal antibodies. Characterization of the surface location and distribution of Ssal in P. haemolytica Al and in the E. coli clones with the monoclonal antibodies may shed some light on this dilemma. Since Ssal is an outer membrane protein of P. haemolytica Al, it is possible that it is recognized by the host immune system during an infection by P. haemolytica Al. Indeed, antibodies to Ssal were detected in immune calf sera by colony immunoblot analysis of E. coli expressing Ssal, suggesting that Ssal is immunogenic in calves. It is possible that this outer membrane protein plays a role in the attachment of the bacterium to the host tissue or in the evasion of host defenses. Alternatively, Ssal may be involved in other aspects of virulence, such as iron acquisition by P. haemolytica in vivo. It has been reported that P. haemolytica Al produces a number of iron-regulated outer membrane proteins, one of which is approximately the same size as Ssal (9). Further characterization of the role(s) of Ssal in P. haemolytica and in protective immunity will be greatly facilitated by the molecular data presented here. We have shown that Ssal is an outer membrane protein of P. haemolytica Al. Previous investigations had demonstrated the serotype specificity of this antigen (15), and the molecular data presented here provide a foundation for further study of this antigen with respect to its surface location on P. haemolytica Al as well as its possible role(s) in pathogenesis. This P. haemolytica Al antigen is a promising candidate for further investigation in the formulation of an efficacious, antigenically defined vaccine against bovine pneumonic pasteurellosis.

11

FIG. the

9.

Colony immunoblot analysis

following

(rows):

1,

pBR322;

separate colonies of each clone paper and

probed with

typical

signs

calves that

against the

of

were

sick

were

patched

c were

animals.

resistant to

soluble

pSSA1;

the different

and Methods. Sera a, b, and

antigens

sera as

3,

d,

e,

clones

carrying

pLKT52.

onto

Four

nitrocellulose

described in Materials

from calves with

Sera

and

pneumonia. Serum of P.

coli

of E.

2,

f

pneumonia and

were

from

healthy

g is the rabbit

haemolytica Al.

serum

ACKNOWLEDGMENTS We thank Carla Wilkie for assistance in DNA sequencing and Lori Merner for helpful suggestions to the manuscript. We also thank Khalid Abdullah for assistance with the preparation of Ssal for N-terminal amino acid analysis and the Biotechnology Center of the Hospital for Sick Children, Toronto, for conducting the analysis. This work is supported by research grants from the Natural Sciences and Engineering Research Council of Canada and from the Ontario Ministry of Agriculture and Foods to R.Y.C.L. and P.E.S. REFERENCES 1. Abdullah, K. M., R. Y. C. Lo, and A. Mellors. 1991. Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica Al glycoprotease gene. J. Bacteriol. 173:5597-5603.

3406

LO ET AL.

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