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Characterization of a fourth lipoprotein from Pasteurella haemolytica A1 and its homology to the OmpA family of outer membrane proteins
FEMS Microbiology Letters 165 (1998) 71^77
Characterization of a fourth lipoprotein from Pasteurella haemolytica A1 and its homology to the OmpA family of outer membrane proteins Patrick M. Nardini a , Alan Mellors a , Reggie Y.C. Lo b; * a
Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ont. N1G 2W1, Canada b Department of Microbiology, University of Guelph, Guelph, Ont. N1G 2W1, Canada Received 23 April 1998; revised 19 May 1998 ; accepted 20 May 1998
Abstract A fourth lipoprotein gene from Pasteurella haemolytica A1 was cloned and characterized. The plpD gene encodes a 31-kDa lipoprotein (Plp4) which could be recognized in Western immunoblot by sera from calves immunized with the culture supernatant vaccine Presponse. This suggests that Plp4 is one of the immunogenic molecules in the P. haemolytica A1 culture supernatant. The lipoprotein nature of Plp4 was confirmed by labelling with [3 H]palmitate and inhibition of leader peptide cleavage with globomycin. A homology search with databanks showed extensive homology between Plp4 and a 31-kDa antigen from Haemophilus somnus and a 19.2-kDa antigen from Neisseria meningitidis. Additional homology of the distal half of Plp4 was identified with a number of bacterial outer membrane proteins belonging to the OmpA family. Plp4 appears to be a novel type of outer membrane protein that contains motifs typical of OmpA but which is also lipid modified. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Bovine pneumonia; Lipoprotein; Pasteurella haemolytica A1; OmpA
1. Introduction Pasteurella haemolytica A1 is the microorganism associated with bovine pneumonic pasteurellosis, also called shipping fever. Shipping fever is the major cause of sickness and death as well as economic loss in the feedlot cattle industry [1]. A commercially available vaccine (Presponse) has been developed which showed greater than 70% e¤cacy in the ¢eld [2]. This culture supernatant vaccine contains anti* Corresponding author. Tel.: +1 (519) 824-4120, ext. 3363; Fax: +1 (519) 837-1802; E-mail: [email protected]
gens which were secreted by the bacteria as well as surface molecules sloughed o¡ during growth. In our continuing e¡orts to examine and characterize the protective antigens of P. haemolytica A1, we have isolated a collection of recombinant plasmids each expressing a P. haemolytica A1 antigen in Escherichia coli [3]. These clones were identi¢ed by the screening of an E. coli clone bank of P. haemolytica A1 DNA utilizing sera from Presponse vaccinated calves that were protected from P. haemolytica A1 infections. In this paper, we report the characterization of two recombinant plasmids which encode a 31-kDa lipoprotein of P. haemolytica A1. This is
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 2 2 2 - 5
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the fourth lipoprotein gene we have characterized from this bacterium. We also compared the amino acid sequence of this 31-kDa lipoprotein with the databanks and identi¢ed signi¢cant homologies with a number of bacterial outer membrane proteins.
2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions P. haemolytica A1 and E. coli strains were from our laboratory collection and have been described previously [3]. Haemophilus somnus was a pneumonic strain isolated from a calf at the Veterinary Diagnostic Laboratory, Ontario Veterinary College, University of Guelph. The isolation of the recombinant plasmids pGS3-42 and pSA2-9 has been described [3]. E. coli strains were cultured in LT broth supplemented with ampicillin (100 Wg ml31 ) when required. P. haemolytica A1 was cultured in brain heart infusion broth (BHIB), H. somnus was cultured in BHIB supplemented with 0.01% thiamine monophosphate and 0.1% Tris. All cultures were grown at 37³C. 2.2. Enzymes, chemicals and antibodies Restriction endonucleases, T4 DNA ligase, protein and DNA molecular mass standards were purchased from Pharmacia Chemicals, Gibco/Bethesda Research Laboratories or Bio-Rad Laboratories and used according to the manufacturers' instructions. The goat anti-bovine IgG alkaline phosphatase conjugate and the rabbit anti-bovine Fab were purchased from Jackson Immunological Laboratories. 2.3. DNA methods Plasmid DNA was isolated from E. coli cells using Qiagen columns (Chatsworth, CA). Standard techniques were used for restriction analysis, subcloning, ligation and recovery of DNA fragments [4]. The nucleotide sequence of the insert DNA was determined by the dideoxy sequencing method according to our laboratory procedure [5] using a combination of manual and automated sequencing approaches. Automated sequencing was performed at the GenA-
lyTiC facility at the University of Guelph with an Applied Biosystems 377 Prism automated sequencer. 2.4. In vivo labelling in E. coli maxi-cells The recombinant plasmids were transformed into E. coli CSR603 and plasmid encoded proteins were labelled with 35 S according to our laboratory procedure [6]. The labelled proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane and immunostained with the calf anti-Presponse serum as described [3]. Afterwards, autoradiography on an X-ray ¢lm revealed the location of 35 S-labelled protein bands. 2.5. [3H]Palmitate labelling Lipoproteins were labelled with [3 H]palmitate according to a published procedure [7]. Brie£y, an overnight culture of E. coli/pGS3-42 grown in LT supplemented with ampicillin, glycerol (0.5% w/v) and casamino acids (2% w/v) was subcultured 1/50 in fresh medium. At an OD550 of 0.4, [3 H]palmitate (51 Ci mmol31 , Amersham) was added to a ¢nal speci¢c activity of 50 WCi ml31 and the culture grown for another 2 h. The cells were pelleted, washed twice with methanol, air dried and resuspended in 100 Wl of SDS-PAGE sample bu¡er. A 20^30-Wl aliquot of the sample was analyzed by SDS-PAGE and autoradiography. To inhibit the activity of signal peptidase II, globomycin (10 mg ml31 in dimethyl sulfoxide) was added to a ¢nal concentration of 100 Wg ml31 , 5 min prior to the addition of the [3 H]palmitate. 2.6. Western immunoblot analysis and antibody puri¢cation Western immunoblot analysis of P. haemolytica A1 antigens expressed from the E. coli clones, using bovine serum from Presponse vaccinated calves, was carried out as described [3]. Antibodies speci¢c against the 31-kDa protein were immunopuri¢ed according to Huang and Forsberg [8]. Brie£y, after SDS-PAGE and transfer of the proteins from E. coli/pGS3-42 onto a piece of nitrocellulose membrane, a vertical strip was excised and immunostained with the calf anti-Presponse serum. After de-
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velopment with conjugated antibodies to reveal the location of the 31-kDa antigen, the strip was aligned with the untreated nitrocellulose membrane, a horizontal strip corresponding to the 31-kDa antigen was excised and cut into small pieces. After blocking with 3% gelatin in TTBS, the nitrocellulose pieces were incubated with the calf anti-Presponse serum overnight. The nitrocellulose pieces were then washed extensively with TTBS and the bound antibodies eluted with 1 ml of 0.2 M glycine-HCl (pH 2.7), 0.5 M NaCl at room temperature for 5 min. The eluate was immediately neutralized with 0.2 ml of 1.5 M Tris-HCl (pH 8.8) and stored at 4³C for future use. Outer and inner membranes were prepared by sucrose gradient separation as previously described [7]. Succinate dehydrogenase and 2-keto-3-deoxyoctonate assays were performed to determine the amount of cross-contamination in the membrane fractions. The results showed that the inner membrane fraction is contaminated with up to 40% of outer membrane proteins. On the other hand, the outer membrane fraction contains less than 10% of inner membrane proteins.
Fig. 1. Western immunoblot analysis of total proteins from E. coli clones. Total proteins from E. coli carrying pGS3-42 (lane 2), pGSv26 (lane 3) and pBR322 (lane 4) were separated on a 15% SDS-PAGE and immunostained with bovine antibodies from calves vaccinated with Presponse and resistant to subsequent challenge with P. haemolytica A1. Molecular mass size standards in kDa are as shown in lane 1. The arrow points to the 31-kDa antigen expressed from E. coli/pGS3-42.
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Fig. 2. Western immunoblot and autoradiogram of 35 S-labelled proteins. A: Western immunoblot using immunopuri¢ed antibodies from Presponse-vaccinated calves. B: The corresponding autoradiogram obtained from the nitrocellulose membrane. 35 S-labelled proteins from E. coli carrying pBR322 (lane 1), pGSv26 (lane 2) and pGS3-42 (lane 3) were applied on the lanes. The arrows indicate the location of Plp4 at 31 kDa.
3. Results 3.1. Characterization of recombinant plasmid pGS3-42 From the collection of the E. coli clones expressing P. haemolytica A1 antigens, two clones GS3-42 and SA2-9 were found to express a P. haemolytica A1 antigen of 31 kDa (Fig. 1). Restriction analysis of the corresponding recombinant plasmids showed that the plasmids pGS3-42 and pSA2-9 are identical, each carrying an insert DNA of 6.6 kb. Therefore, all subsequent experiments were performed with plasmid pGS3-42. A subclone of pGS3-42 (pGSv26) was made in which a 2.6-kb ClaI fragment was removed. Analysis of the proteins expressed from E. coli carrying pGSv26 by Western immunoblot showed that the 31-kDa antigen is no longer expressed, indicating that the gene (or parts of it) is encoded on the 2.6-kb ClaI fragment deleted in the subclone. Without any further subcloning, the nucleotide sequence of the 2.6-kb ClaI fragment was determined and analyzed for the presence of a corresponding ORF. The nucleotide sequence of the 2.6-kb ClaI fragment has been deposited in GenBank under the accession number AF58703. 3.2. Nucleotide sequence analysis Analysis of the nucleotide sequence of the 2.6-kb ClaI fragment showed the presence of one complete ORF. The ORF encodes a polypeptide of 284 amino acids with a predicted molecular mass of 31.4 kDa,
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Fig. 3. Amino acid alignment of Plp4 with homologous proteins. The predicted amino acid sequence of Plp4 (Ph) was used as the reference and aligned with the sequences from H. somnus (Hs) and N. meningitidis (Nm). indicates identical amino acids ; - indicates gaps introduced to maximize aligment ; + indicates the lipoprotein cleavage sequence ; # the amino acids GIDPENTSGVEGITT; % the amino acids VFPKDAACPPPAPKAEPQVIIREIVPAKPKRIRQ at the C-terminal end which do not ¢t into the alignment and are placed here for clarity. In addition, the amino acid sequences of a number of outer membrane proteins from H. in£uenzae (Hi), A. actinomycetemcomitans (Aa), S. dysenteriae (Sd) and K. pneumoniae (Kp) are shown with Plp4 to illustrate the conserved regions of these outer membrane proteins. In each case, only the pertinent conserved regions are shown. It can also be seen that these four outer membrane proteins have additional conservations between them. Space regions are amino acids that do not ¢t into alignment and are left out for clarity. The highly conserved amino acids are dotted : these have been suggested to form an K-helical motif that interacts with the peptidoglycan. The numbers refer to the amino acid residue positions of the respective proteins.
which corresponds to that observed for the antigen in the Western immunoblot data. Maxi-cell labelling experiments further support the nucleotide sequence evidence that this ORF encodes the 31-kDa antigen of interest. 3.3. In vivo labelling of plasmid encoded proteins Plasmids pGS3-42 and pGSv26 were transformed
into E. coli maxi-cell strain CSR603 and plasmid encoded proteins labelled with 35 S as described. Proteins from the maxi-cells were separated by SDSPAGE, immunostained by Western immunoblot and the blot exposed to X-ray ¢lm to obtain a corresponding autoradiogram. The results showed that a 31-kDa protein was expressed from pGS3-42 which could be detected by Western immunoblot using the anti-Presponse sera (Fig. 2A). This protein corre-
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Fig. 4. [3 H]Palmitate labelling of lipoprotein. Autoradiogram of labelled proteins from E. coli/pGS3-42. The proteins were labelled in the absence (lanes 1) and in the presence (lanes 2) of globomycin. `p' refers to the prolipoprotein at 31 kDa, `m' refers to the mature Plp4 at 29 kDa.
sponded exactly to one of the 35 S-labelled proteins detected in the autoradiogram (Fig. 2B). This protein was not produced in the subclone pGSv26. This con¢rms that the 31-kDa protein is encoded on the 2.6kb ClaI fragment and corresponds to the ORF from the nucleotide sequence data. 3.4. Properties of the 31-kDa antigen The N-terminal amino acid sequence of the 31kDa antigen shows properties characteristic of a lipoprotein. The ¢rst 24 amino acids contain features that conform to the S1-I2-S2-I2 model of Inouye et al. [9] for bacterial lipoproteins. Further, a putative prolipoprotein cleavage motif of V21 -A-A-C24 can be identi¢ed (Fig. 3). This suggest that the 31-kDa antigen is lipid modi¢ed and in accordance with previous practice, we named it Plp4 (the gene plpD). Removal of the leader peptide will result in a mature protein of 28.9 kDa based on the predicted amino acid sequence. The lipid modi¢cation of Plp4 was con¢rmed by labelling the protein with [3 H]palmitate as shown in Fig. 4. In the presence of globomycin, cleavage by peptidase II was inhibited and the prePlp4 could be observed at a higher molecular mass. 3.5. Homology analysis with databank A homology search comparing Plp4 with sequences in GenBank showed signi¢cant homologies with several prokaryotic outer membrane proteins and lipoproteins. The greatest similarity was detected with a 31-kDa `hemolytic' antigen of H. somnus [10]. The amino acids of Plp4 and the H. somnus antigen are 63% identical and 13.9% similar for a total of 76.9% homology over 273 amino acids. In addition, signi¢cant homology was also detected with a 19.2-kDa antigen of Neisseria meningitidis
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[11]. The amino acids in the N. meningitidis protein and Plp4 are 33.9% identical and 15.8% similar for a total of 49.7% homology over the shorter length (171 amino acids) of the N. meningitidis protein. All three proteins contain typical lipoprotein cleavage and modi¢cation motifs. It is of particular interest that the C-terminal halves of Plp4 and the H. somnus 31-kDa antigen contain motifs typical of the OmpA family of outer membrane proteins characterized from a number of bacteria. Examples of these include the Omp P5 of Haemophilus in£uenzae [12], Omp34 of Actinobacillus actinomycetemcomitans [13], OmpA of Shigella dysenteriae [14] and OmpA of Klebsiella pneumoniae [15]. Thus, it is likely that Plp4 and its homologues are located in the outer membranes. Further, Plp4 contains a region which is thought to be involved in interaction with peptidoglycan [16,17], suggesting another property for this protein. An alignment showing some of the conserved regions in these proteins is shown in Fig. 3. 3.6. Detection of Plp4 in membrane fractions Antibodies speci¢c for Plp4 were immunopuri¢ed from the calf serum using the recombinant protein from E. coli/pGS3-42 as the antigen. The speci¢c antibodies were used to screen for the presence of Plp4 in the outer membrane and inner membrane fractions of P. haemolytica and H. somnus. The results show that most of Plp4 is located in the outer membrane in both P. haemolytica and H. somnus as expected (data not shown).
4. Discussion A number of P. haemolytica A1 antigens in the 28^31-kDa range have been suggested to be protective antigens by their correlation with resistance to pneumonic pasteurellosis [18]. Previously we have reported the characterization of three 30-kDa lipoproteins Plp1, 2 and 3 from P. haemolytica A1. The present 31-kDa lipoprotein is distinct from these three and is named Plp4. Therefore, it appears that there are multiple lipoproteins in this size range which are capable of inducing an immune response in cattle. It is likely the lipid moieties of these lip-
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oproteins that are responsible for their highly immunogenic properties. It is not surprising to observe a signi¢cant homology between Plp4 and the H. somnus 31-kDa antigen since both microorganisms belong to the Pasteurellaceae family. We have identi¢ed at least four other homologous antigen genes within the two bacteria and are in the process of characterizing them. A previous report of the failure to detect a homologous gene in P. haemolytica A1 by Southern hybridization using the H. somnus 31-kDa antigen gene as a probe [10] could be due to the conditions used in the hybridization. Using plpD as the probe, we have detected hybridization signals in P. haemolytica A1 and H. somnus (data not shown) in agreement with the present sequence comparison. The results of the homology analysis of Plp4 with sequences in the database suggest some interesting properties of Plp4. The signi¢cant homology with the H. somnus and N. meningitidis lipoproteins suggests that these proteins carry out some common function in these species. They are also highly antigenic molecules for the respective bacteria. The signi¢cant homology with members of the OmpA family suggests that Plp4 may have some properties similar to the OmpA proteins. However, OmpA proteins have not been shown to be lipid modi¢ed as is Plp4. In addition, the homology with the N. meningitidis lipoprotein stopped at the same position where homology with OmpA proteins begins. It is possible that plpD is the result of a gene fusion between a precursor gene similar to the N. meningitidis lipoprotein gene and a gene encoding the C-terminal half of an OmpA-like protein. This fusion gene product is a hybrid molecule, a novel outer membrane protein which may combine the functions normally attributed to the lipoprotein and the OmpA proteins. We are currently examining other P. haemolytica serotypes for the presence of plpD and investigate a possible gene fusion event as well as attempting to ascertain some function for Plp4.
Acknowledgments This work is supported by research grants from the Natural Sciences and Research Council of Can-
ada. We thank Dr. Andy Potter for the supply of globomycin.
References [1] Yates, W.D.G. (1982) A review of infectious bovine rhinotracheitis, shipping fever pneumonia and viral-bacterial synergism in respiratory disease of cattle. Can. J. Comp. Med. 46, 225^263. [2] Shewen, P.E., Sharp, A. and Wilkie, B.N. (1988) E¤cacy testing of a Pasteurella haemolytica extract vaccine. Vet. Med. 10, 1078^1083. [3] Lo, R.Y.C. and Mellors, A. (1996) The isolation of recombinant plasmids expressing secreted antigens of Pasteurella haemolytica A1 and the characterization of an immunogenic 60 kDa antigen. Vet. Microbiol. 51, 381^391. [4] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [5] Lo, R.Y.C., Strathdee, C.A. and Shewen, P.E. (1987) Nucleotide sequence of the leukotoxin genes of Pasteurella haemolytica A1. Infect. Immun. 55, 1987^1996. [6] Abdullah, K.M., Lo, R.Y.C. and Mellors, A. (1991) Cloning, nucleotide sequence, and expression of the Pasteurella haemolytica A1 glycoprotease gene. J. Bacteriol. 173, 5597^5603. [7] Cooney, B.J. and Lo, R.Y.C. (1993) Three contiguous lipoprotein genes in Pasteurella haemolytica A1 which are homologous to a lipoprotein gene in Haemophilus in£uenzae type b. Infect. Immun. 61, 4682^4688. [8] Huang, L. and Forsberg, C.W. (1988) Puri¢cation and comparison of the periplasmic and extracellular forms of the cellodextrinase from Bacteroides succinogenes. Appl. Environ. Microbiol. 54, 1488^1493. [9] Inouye, S., Wang, S., Sekizawa, J., Halegoua, S. and Inouye, M. (1977) Amino acid sequence for the peptide extension on the prolipoprotein of the Escherichia coli outer membrane. Proc. Natl. Acad. Sci. USA 74, 1004^1008. [10] Won, J. and Gri¤th, R.W. (1993) Cloning and sequencing of the gene encoding a 31-kilodalton antigen of Haemophilus somnus. Infect. Immun. 61, 2813^2821. [11] Yang, Q.-L., Tinsley, C.R. and Gotschlich, E.C. (1995) Novel lipoprotein expressed by Neisseria meningitidis but not by Neisseria gonorrhoeae. Infect. Immun. 63, 1631^1636. [12] Munson, R.S., Grass, S. and West, R. (1993) Molecular cloning and sequence of the gene for outer membrane P5 of Haemophilus in£uenzae. Infect. Immun. 61, 4017^4020. [13] White, P.A., Nair, S.P., Kim, M.-J., Wilson, M. and Henderson, B. (1998) Molecular characterization of an outer membrane protein of Actinobacillus actinomycetemcomitans belonging to the OmpA family. Infect. Immun. 66, 369^372. [14] Braun, G. and Cole, S.T. (1982) The nucleotide sequence coding for major outer membrane proteins OmpA of Shigella dysenteriae. Nucleic Acids Res. 10. 2367^2378. [15] Lawrence, J.G., Ochman, H. and Hartl, D.L. (1991) Molec-
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P.M. Nardini et al. / FEMS Microbiology Letters 165 (1998) 71^77 ular and evolutionary relationship among enteric bacteria. J. Gen. Microb. 137, 1911^1921. [16] De Mot, R. and Vanderleydon, J. (1994) The C-terminal sequence conservation between OmpA-related outer membrane proteins and MotB suggests a common function in both grampositive and gram-negative bacteria, possibly in the interaction of the domains with peptidoglycan. Mol. Microbiol. 12, 333^334.
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[17] Koebnik, R. (1995) Proposal for a peptidoglycan-associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269^1270. [18] Mosier, D.A., Simons, K.R., Confer, A.W., Panciera, R.J. and Clinkenbeard, K.D. (1989) Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis. Infect. Immun. 57, 711^716.
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Characterization of a fourth lipoprotein from Pasteurella haemolytica A1 and its homology to the OmpA family of outer membrane proteins Patrick M. Nardini a , Alan Mellors a , Reggie Y.C. Lo b; * a
Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ont. N1G 2W1, Canada b Department of Microbiology, University of Guelph, Guelph, Ont. N1G 2W1, Canada Received 23 April 1998; revised 19 May 1998 ; accepted 20 May 1998
Abstract A fourth lipoprotein gene from Pasteurella haemolytica A1 was cloned and characterized. The plpD gene encodes a 31-kDa lipoprotein (Plp4) which could be recognized in Western immunoblot by sera from calves immunized with the culture supernatant vaccine Presponse. This suggests that Plp4 is one of the immunogenic molecules in the P. haemolytica A1 culture supernatant. The lipoprotein nature of Plp4 was confirmed by labelling with [3 H]palmitate and inhibition of leader peptide cleavage with globomycin. A homology search with databanks showed extensive homology between Plp4 and a 31-kDa antigen from Haemophilus somnus and a 19.2-kDa antigen from Neisseria meningitidis. Additional homology of the distal half of Plp4 was identified with a number of bacterial outer membrane proteins belonging to the OmpA family. Plp4 appears to be a novel type of outer membrane protein that contains motifs typical of OmpA but which is also lipid modified. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Bovine pneumonia; Lipoprotein; Pasteurella haemolytica A1; OmpA
1. Introduction Pasteurella haemolytica A1 is the microorganism associated with bovine pneumonic pasteurellosis, also called shipping fever. Shipping fever is the major cause of sickness and death as well as economic loss in the feedlot cattle industry [1]. A commercially available vaccine (Presponse) has been developed which showed greater than 70% e¤cacy in the ¢eld [2]. This culture supernatant vaccine contains anti* Corresponding author. Tel.: +1 (519) 824-4120, ext. 3363; Fax: +1 (519) 837-1802; E-mail: [email protected]
gens which were secreted by the bacteria as well as surface molecules sloughed o¡ during growth. In our continuing e¡orts to examine and characterize the protective antigens of P. haemolytica A1, we have isolated a collection of recombinant plasmids each expressing a P. haemolytica A1 antigen in Escherichia coli [3]. These clones were identi¢ed by the screening of an E. coli clone bank of P. haemolytica A1 DNA utilizing sera from Presponse vaccinated calves that were protected from P. haemolytica A1 infections. In this paper, we report the characterization of two recombinant plasmids which encode a 31-kDa lipoprotein of P. haemolytica A1. This is
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 2 2 2 - 5
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the fourth lipoprotein gene we have characterized from this bacterium. We also compared the amino acid sequence of this 31-kDa lipoprotein with the databanks and identi¢ed signi¢cant homologies with a number of bacterial outer membrane proteins.
2. Materials and methods 2.1. Bacterial strains, plasmids and culture conditions P. haemolytica A1 and E. coli strains were from our laboratory collection and have been described previously [3]. Haemophilus somnus was a pneumonic strain isolated from a calf at the Veterinary Diagnostic Laboratory, Ontario Veterinary College, University of Guelph. The isolation of the recombinant plasmids pGS3-42 and pSA2-9 has been described [3]. E. coli strains were cultured in LT broth supplemented with ampicillin (100 Wg ml31 ) when required. P. haemolytica A1 was cultured in brain heart infusion broth (BHIB), H. somnus was cultured in BHIB supplemented with 0.01% thiamine monophosphate and 0.1% Tris. All cultures were grown at 37³C. 2.2. Enzymes, chemicals and antibodies Restriction endonucleases, T4 DNA ligase, protein and DNA molecular mass standards were purchased from Pharmacia Chemicals, Gibco/Bethesda Research Laboratories or Bio-Rad Laboratories and used according to the manufacturers' instructions. The goat anti-bovine IgG alkaline phosphatase conjugate and the rabbit anti-bovine Fab were purchased from Jackson Immunological Laboratories. 2.3. DNA methods Plasmid DNA was isolated from E. coli cells using Qiagen columns (Chatsworth, CA). Standard techniques were used for restriction analysis, subcloning, ligation and recovery of DNA fragments [4]. The nucleotide sequence of the insert DNA was determined by the dideoxy sequencing method according to our laboratory procedure [5] using a combination of manual and automated sequencing approaches. Automated sequencing was performed at the GenA-
lyTiC facility at the University of Guelph with an Applied Biosystems 377 Prism automated sequencer. 2.4. In vivo labelling in E. coli maxi-cells The recombinant plasmids were transformed into E. coli CSR603 and plasmid encoded proteins were labelled with 35 S according to our laboratory procedure [6]. The labelled proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane and immunostained with the calf anti-Presponse serum as described [3]. Afterwards, autoradiography on an X-ray ¢lm revealed the location of 35 S-labelled protein bands. 2.5. [3H]Palmitate labelling Lipoproteins were labelled with [3 H]palmitate according to a published procedure [7]. Brie£y, an overnight culture of E. coli/pGS3-42 grown in LT supplemented with ampicillin, glycerol (0.5% w/v) and casamino acids (2% w/v) was subcultured 1/50 in fresh medium. At an OD550 of 0.4, [3 H]palmitate (51 Ci mmol31 , Amersham) was added to a ¢nal speci¢c activity of 50 WCi ml31 and the culture grown for another 2 h. The cells were pelleted, washed twice with methanol, air dried and resuspended in 100 Wl of SDS-PAGE sample bu¡er. A 20^30-Wl aliquot of the sample was analyzed by SDS-PAGE and autoradiography. To inhibit the activity of signal peptidase II, globomycin (10 mg ml31 in dimethyl sulfoxide) was added to a ¢nal concentration of 100 Wg ml31 , 5 min prior to the addition of the [3 H]palmitate. 2.6. Western immunoblot analysis and antibody puri¢cation Western immunoblot analysis of P. haemolytica A1 antigens expressed from the E. coli clones, using bovine serum from Presponse vaccinated calves, was carried out as described [3]. Antibodies speci¢c against the 31-kDa protein were immunopuri¢ed according to Huang and Forsberg [8]. Brie£y, after SDS-PAGE and transfer of the proteins from E. coli/pGS3-42 onto a piece of nitrocellulose membrane, a vertical strip was excised and immunostained with the calf anti-Presponse serum. After de-
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velopment with conjugated antibodies to reveal the location of the 31-kDa antigen, the strip was aligned with the untreated nitrocellulose membrane, a horizontal strip corresponding to the 31-kDa antigen was excised and cut into small pieces. After blocking with 3% gelatin in TTBS, the nitrocellulose pieces were incubated with the calf anti-Presponse serum overnight. The nitrocellulose pieces were then washed extensively with TTBS and the bound antibodies eluted with 1 ml of 0.2 M glycine-HCl (pH 2.7), 0.5 M NaCl at room temperature for 5 min. The eluate was immediately neutralized with 0.2 ml of 1.5 M Tris-HCl (pH 8.8) and stored at 4³C for future use. Outer and inner membranes were prepared by sucrose gradient separation as previously described [7]. Succinate dehydrogenase and 2-keto-3-deoxyoctonate assays were performed to determine the amount of cross-contamination in the membrane fractions. The results showed that the inner membrane fraction is contaminated with up to 40% of outer membrane proteins. On the other hand, the outer membrane fraction contains less than 10% of inner membrane proteins.
Fig. 1. Western immunoblot analysis of total proteins from E. coli clones. Total proteins from E. coli carrying pGS3-42 (lane 2), pGSv26 (lane 3) and pBR322 (lane 4) were separated on a 15% SDS-PAGE and immunostained with bovine antibodies from calves vaccinated with Presponse and resistant to subsequent challenge with P. haemolytica A1. Molecular mass size standards in kDa are as shown in lane 1. The arrow points to the 31-kDa antigen expressed from E. coli/pGS3-42.
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Fig. 2. Western immunoblot and autoradiogram of 35 S-labelled proteins. A: Western immunoblot using immunopuri¢ed antibodies from Presponse-vaccinated calves. B: The corresponding autoradiogram obtained from the nitrocellulose membrane. 35 S-labelled proteins from E. coli carrying pBR322 (lane 1), pGSv26 (lane 2) and pGS3-42 (lane 3) were applied on the lanes. The arrows indicate the location of Plp4 at 31 kDa.
3. Results 3.1. Characterization of recombinant plasmid pGS3-42 From the collection of the E. coli clones expressing P. haemolytica A1 antigens, two clones GS3-42 and SA2-9 were found to express a P. haemolytica A1 antigen of 31 kDa (Fig. 1). Restriction analysis of the corresponding recombinant plasmids showed that the plasmids pGS3-42 and pSA2-9 are identical, each carrying an insert DNA of 6.6 kb. Therefore, all subsequent experiments were performed with plasmid pGS3-42. A subclone of pGS3-42 (pGSv26) was made in which a 2.6-kb ClaI fragment was removed. Analysis of the proteins expressed from E. coli carrying pGSv26 by Western immunoblot showed that the 31-kDa antigen is no longer expressed, indicating that the gene (or parts of it) is encoded on the 2.6-kb ClaI fragment deleted in the subclone. Without any further subcloning, the nucleotide sequence of the 2.6-kb ClaI fragment was determined and analyzed for the presence of a corresponding ORF. The nucleotide sequence of the 2.6-kb ClaI fragment has been deposited in GenBank under the accession number AF58703. 3.2. Nucleotide sequence analysis Analysis of the nucleotide sequence of the 2.6-kb ClaI fragment showed the presence of one complete ORF. The ORF encodes a polypeptide of 284 amino acids with a predicted molecular mass of 31.4 kDa,
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Fig. 3. Amino acid alignment of Plp4 with homologous proteins. The predicted amino acid sequence of Plp4 (Ph) was used as the reference and aligned with the sequences from H. somnus (Hs) and N. meningitidis (Nm). indicates identical amino acids ; - indicates gaps introduced to maximize aligment ; + indicates the lipoprotein cleavage sequence ; # the amino acids GIDPENTSGVEGITT; % the amino acids VFPKDAACPPPAPKAEPQVIIREIVPAKPKRIRQ at the C-terminal end which do not ¢t into the alignment and are placed here for clarity. In addition, the amino acid sequences of a number of outer membrane proteins from H. in£uenzae (Hi), A. actinomycetemcomitans (Aa), S. dysenteriae (Sd) and K. pneumoniae (Kp) are shown with Plp4 to illustrate the conserved regions of these outer membrane proteins. In each case, only the pertinent conserved regions are shown. It can also be seen that these four outer membrane proteins have additional conservations between them. Space regions are amino acids that do not ¢t into alignment and are left out for clarity. The highly conserved amino acids are dotted : these have been suggested to form an K-helical motif that interacts with the peptidoglycan. The numbers refer to the amino acid residue positions of the respective proteins.
which corresponds to that observed for the antigen in the Western immunoblot data. Maxi-cell labelling experiments further support the nucleotide sequence evidence that this ORF encodes the 31-kDa antigen of interest. 3.3. In vivo labelling of plasmid encoded proteins Plasmids pGS3-42 and pGSv26 were transformed
into E. coli maxi-cell strain CSR603 and plasmid encoded proteins labelled with 35 S as described. Proteins from the maxi-cells were separated by SDSPAGE, immunostained by Western immunoblot and the blot exposed to X-ray ¢lm to obtain a corresponding autoradiogram. The results showed that a 31-kDa protein was expressed from pGS3-42 which could be detected by Western immunoblot using the anti-Presponse sera (Fig. 2A). This protein corre-
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Fig. 4. [3 H]Palmitate labelling of lipoprotein. Autoradiogram of labelled proteins from E. coli/pGS3-42. The proteins were labelled in the absence (lanes 1) and in the presence (lanes 2) of globomycin. `p' refers to the prolipoprotein at 31 kDa, `m' refers to the mature Plp4 at 29 kDa.
sponded exactly to one of the 35 S-labelled proteins detected in the autoradiogram (Fig. 2B). This protein was not produced in the subclone pGSv26. This con¢rms that the 31-kDa protein is encoded on the 2.6kb ClaI fragment and corresponds to the ORF from the nucleotide sequence data. 3.4. Properties of the 31-kDa antigen The N-terminal amino acid sequence of the 31kDa antigen shows properties characteristic of a lipoprotein. The ¢rst 24 amino acids contain features that conform to the S1-I2-S2-I2 model of Inouye et al. [9] for bacterial lipoproteins. Further, a putative prolipoprotein cleavage motif of V21 -A-A-C24 can be identi¢ed (Fig. 3). This suggest that the 31-kDa antigen is lipid modi¢ed and in accordance with previous practice, we named it Plp4 (the gene plpD). Removal of the leader peptide will result in a mature protein of 28.9 kDa based on the predicted amino acid sequence. The lipid modi¢cation of Plp4 was con¢rmed by labelling the protein with [3 H]palmitate as shown in Fig. 4. In the presence of globomycin, cleavage by peptidase II was inhibited and the prePlp4 could be observed at a higher molecular mass. 3.5. Homology analysis with databank A homology search comparing Plp4 with sequences in GenBank showed signi¢cant homologies with several prokaryotic outer membrane proteins and lipoproteins. The greatest similarity was detected with a 31-kDa `hemolytic' antigen of H. somnus [10]. The amino acids of Plp4 and the H. somnus antigen are 63% identical and 13.9% similar for a total of 76.9% homology over 273 amino acids. In addition, signi¢cant homology was also detected with a 19.2-kDa antigen of Neisseria meningitidis
75
[11]. The amino acids in the N. meningitidis protein and Plp4 are 33.9% identical and 15.8% similar for a total of 49.7% homology over the shorter length (171 amino acids) of the N. meningitidis protein. All three proteins contain typical lipoprotein cleavage and modi¢cation motifs. It is of particular interest that the C-terminal halves of Plp4 and the H. somnus 31-kDa antigen contain motifs typical of the OmpA family of outer membrane proteins characterized from a number of bacteria. Examples of these include the Omp P5 of Haemophilus in£uenzae [12], Omp34 of Actinobacillus actinomycetemcomitans [13], OmpA of Shigella dysenteriae [14] and OmpA of Klebsiella pneumoniae [15]. Thus, it is likely that Plp4 and its homologues are located in the outer membranes. Further, Plp4 contains a region which is thought to be involved in interaction with peptidoglycan [16,17], suggesting another property for this protein. An alignment showing some of the conserved regions in these proteins is shown in Fig. 3. 3.6. Detection of Plp4 in membrane fractions Antibodies speci¢c for Plp4 were immunopuri¢ed from the calf serum using the recombinant protein from E. coli/pGS3-42 as the antigen. The speci¢c antibodies were used to screen for the presence of Plp4 in the outer membrane and inner membrane fractions of P. haemolytica and H. somnus. The results show that most of Plp4 is located in the outer membrane in both P. haemolytica and H. somnus as expected (data not shown).
4. Discussion A number of P. haemolytica A1 antigens in the 28^31-kDa range have been suggested to be protective antigens by their correlation with resistance to pneumonic pasteurellosis [18]. Previously we have reported the characterization of three 30-kDa lipoproteins Plp1, 2 and 3 from P. haemolytica A1. The present 31-kDa lipoprotein is distinct from these three and is named Plp4. Therefore, it appears that there are multiple lipoproteins in this size range which are capable of inducing an immune response in cattle. It is likely the lipid moieties of these lip-
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oproteins that are responsible for their highly immunogenic properties. It is not surprising to observe a signi¢cant homology between Plp4 and the H. somnus 31-kDa antigen since both microorganisms belong to the Pasteurellaceae family. We have identi¢ed at least four other homologous antigen genes within the two bacteria and are in the process of characterizing them. A previous report of the failure to detect a homologous gene in P. haemolytica A1 by Southern hybridization using the H. somnus 31-kDa antigen gene as a probe [10] could be due to the conditions used in the hybridization. Using plpD as the probe, we have detected hybridization signals in P. haemolytica A1 and H. somnus (data not shown) in agreement with the present sequence comparison. The results of the homology analysis of Plp4 with sequences in the database suggest some interesting properties of Plp4. The signi¢cant homology with the H. somnus and N. meningitidis lipoproteins suggests that these proteins carry out some common function in these species. They are also highly antigenic molecules for the respective bacteria. The signi¢cant homology with members of the OmpA family suggests that Plp4 may have some properties similar to the OmpA proteins. However, OmpA proteins have not been shown to be lipid modi¢ed as is Plp4. In addition, the homology with the N. meningitidis lipoprotein stopped at the same position where homology with OmpA proteins begins. It is possible that plpD is the result of a gene fusion between a precursor gene similar to the N. meningitidis lipoprotein gene and a gene encoding the C-terminal half of an OmpA-like protein. This fusion gene product is a hybrid molecule, a novel outer membrane protein which may combine the functions normally attributed to the lipoprotein and the OmpA proteins. We are currently examining other P. haemolytica serotypes for the presence of plpD and investigate a possible gene fusion event as well as attempting to ascertain some function for Plp4.
Acknowledgments This work is supported by research grants from the Natural Sciences and Research Council of Can-
ada. We thank Dr. Andy Potter for the supply of globomycin.
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P.M. Nardini et al. / FEMS Microbiology Letters 165 (1998) 71^77 ular and evolutionary relationship among enteric bacteria. J. Gen. Microb. 137, 1911^1921. [16] De Mot, R. and Vanderleydon, J. (1994) The C-terminal sequence conservation between OmpA-related outer membrane proteins and MotB suggests a common function in both grampositive and gram-negative bacteria, possibly in the interaction of the domains with peptidoglycan. Mol. Microbiol. 12, 333^334.
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[17] Koebnik, R. (1995) Proposal for a peptidoglycan-associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269^1270. [18] Mosier, D.A., Simons, K.R., Confer, A.W., Panciera, R.J. and Clinkenbeard, K.D. (1989) Pasteurella haemolytica antigens associated with resistance to pneumonic pasteurellosis. Infect. Immun. 57, 711^716.
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