Open Access

Identification of novel immunogens in Pasteurella multocida

  • Keith Al-Hasani1,
  • John Boyce1,
  • Victoria P McCarl1,
  • Stephen Bottomley1,
  • Ian Wilkie2 and
  • Ben Adler1Email author
Microbial Cell Factories20076:3

https://doi.org/10.1186/1475-2859-6-3

Received: 22 November 2006

Accepted: 18 January 2007

Published: 18 January 2007

Abstract

P. multocida is a Gram-negative pathogen responsible for causing diseases in animals of economic significance to livestock industries throughout the world. Current vaccines include bacterins, which provide only limited protection against homologous serotypes. Therefore there is a need for more effective vaccines to control diseases caused by P. multocida. As a step towards developing vaccines against fowl cholera, a genomics based approach was applied for the identification of novel immunogens.

Results

Bioinformatics analysis of the P. multocida genome predicted 129 proteins as secreted, located in the outer membrane, or lipoproteins. 105 of the genes encoding these proteins were cloned and recombinant protein expressed in Escherichia coli. Polyclonal serum from P. multocida-infected chickens reacted with a subset of these proteins.

Conclusion

These data show the range of bacterial immunogens recognized by the chicken immune system, including 6 novel immunoreactive proteins.

Background

Pasteurella multocida is a Gram-negative bacterial pathogen which is the causative agent of a range of diseases in animals, including fowl cholera in avian species, hemorrhagic septicemia in ungulates, shipping fever and pneumonia in cattle, atrophic rhinitis in swine, and snuffles in rabbits [13]. The bacterium also causes infection in humans, primarily through dog and cat bites. Fowl cholera, which is generally caused by serotypes A:1, A:3 or A:4 [4], is a severe systemic disease which occurs in domestic poultry and wild birds and results in significant economic losses to poultry industries worldwide. Current vaccines against fowl cholera include bacterins [5], which provide only limited protection against homologous serotypes and live attenuated strains, which have been observed to revert to virulence [6]. Therefore, there is a need for more effective vaccines to control diseases caused by P. multocida.

The surface of Gram-negative bacteria is critical for interaction of the bacterium with the host cell environment as it mediates nutrient uptake, secretion of toxins and other products and is involved in avoidance of the host immune system [7]. Furthermore, it is the bacterial surface molecules that are the targets for host immunity. Indeed, bacterial surface proteins have been shown to be important for conferring protective immunity in a range of infection models [8, 9]. Recently, the P. multocida PlpB protein was identified as a cross-protective antigen [10, 11] and this protein is located in the P. multocida outer membrane [12]. Outer membrane proteins also promote adherence to host cell surfaces and are therefore likely to be involved in P. multocida virulence [13].

In an effort to identify novel immunogens to which chickens respond during natural infection, and as a first step towards developing protective recombinant vaccines against fowl cholera, a detailed antigen profiling analysis was undertaken. The effectiveness of this approach, which has been termed 'reverse vaccinology' or 'reverse immunology', has been reported by several authors with Plasmodium falciparum [14], Streptococcus pneumoniae [15], Treponema pallidum [16], Neisseria meningitidis [17], and Chlamydia pneumoniae [18].

Results and discussion

We utilised a range of bioinformatics analyses of the annotated P. multocida Pm70 genome sequence and previously published experimental data [12] to select genes likely to have vaccine potential. The central premise of this work was that protective antigens are likely to be surface exposed or secreted by the bacteria and therefore accessible to the host immune response. We used PSORTB [19] and ProteomeAnalyst [20] to predict all outer membrane and secreted proteins and LipoP [21] to predict all lipoproteins. We included all lipoproteins as a large number of lipoproteins was observed in the proteomics analysis of the P. multocida outer membrane [12] and we believe that precise sub-cellular prediction of lipoprotein location (inner or outer membrane) is presently unreliable. These bioinformatics analyses identified a combined list of 129 proteins (Figure 1, see Additional file 1). The prediction programs PSORTB and ProteomeAnalyst can be used to predict protein localisation in Gram-negative organisms, while LipoP specifically predicts Gram-negative lipoproteins. PSORTB and ProteomeAnalyst use different methods for prediction of localisations. PSORTB uses primary sequence analysis algorithms and gives a high precision, but low sensitivity, prediction. Proteome analyst on the other hand utilises analysis of text annotations for the closest homologue of any given query sequence and is medium-high precision and sensitivity; it thus makes a higher number of predictions. These analyses were complemented by the previously published proteomic analysis of the P. multocida outer membrane [12] which identified three proteins (PM0612, PM1357 and PM1746) not predicted by bioinformatics.
Figure 1

Venn diagram showing the different bioinformatics prediction of the outer membrane and secreted proteins from the P. multocida Pm70 genome using the algorithms, PSORTB (19) (outer membrane or extracellular), ProteomeAnalyst (20) (outer membrane or secreted) and LipoP (21) (SpII cleavage site predicted).

The large number of candidate genes necessitated the adoption of a high-throughput cloning strategy, which utilised by the Gateway™trogen Inc., Carlsbad, CA) cloning and expression system to clone PCR-amplified P. multocida ORFs. An attempt was made to clone all 129 ORFs; however, only 105 were successfully cloned and expressed in E. coli. Each of the recombinant expression clones was assessed for levels of protein expression and solubility. Because of the large inventory of proteins that were required to be expressed and purified in a time and cost effective way, protein expression was analysed using a novel inclusion body assay developed on a TECAN liquid handling robot.

To evaluate the range of P. multocida proteins recognised by the chicken antibody response, serum samples from chickens that had been repeatedly infected with P. multocida and then treated with antibiotics were tested for reactivity against 105 recombinant proteins by Western blot. Pooled serum samples from P. multocida infected chickens (8 from X-73, 3 from VP161 – a highly virulent A:1 strain [22]) and four serum samples from control chickens were reacted in immunoblot assays with comparable amounts (3 μg/lane) of recombinant proteins. The immune chicken sera generated during P. multocida strain X-73 or VP161 infection recognised a total of 12 (Table 1) recombinant proteins, ten of which were not recognised by naive sera. To our knowledge, six of these antigens have not been previously identified as capable of eliciting immune responses from chickens, demonstrating the utility of this approach for the identification of novel immunoreactive bacterial antigens (Figure 2). Although immunological reactivity to unique antigens alone does not necessarily indicate a role in protective immunity against pasteurellosis, these novel antigens constitute prime candidates as potential vaccine targets for pasteurellosis, and warrant inclusion in studies to elucidate their potential role in pathogensis. The results have important implications in understanding avian immune responses to P. multocida and in elucidating putative elements of a protective immune response.
Table 1

P. multocida antigens recognised by immune chicken sera. +, reaction; -, no reaction.

Antigen

Database similarity (accession no.)

X-73 sera

VP161 sera

Control sera

PM0388

Outer membrane protein from P. multocida (AAK02472)

+

+

-

PM0442

Hypothetical protein

+

+

-

PM0554

Lipoprotein from P. multocida (AAK02638)

-

+

-

PM0659

Hypothetical protein

+

+

-

PM0786

OmpA from P. multocida (AAT57679)

+

+

+

PM0966

Omp16 from P. multocida (CAB75338)

+

+

+

PM0979

Hypothetical protein

+

+

-

PM1614

Outer membrane protein from Haemophilus (AAX87732)

+

+

-

PM1730

PlpB from P. multocida (AAK03814)

-

+

-

PM1979

SurA protein from Mannheimia (AAU38450)

+

+

-

PM1992

Oma87 from P. multocida (CAD20126)

+

+

-

PM1993

Outer membrane protein from P. multocida serotype D (CAA52400)

+

+

-

Figure 2

Western immunoblot demonstrating recognition of six novel P. multocida antigens by pooled sera from experimentally infected chickens. Sizes of markers in kilodaltons are indicated on the left. Lanes: 1, protein molecular standards (kDa) (SeeBlue Plus2 Prestained Sandard, Novagen); 2 and 9 are blank; 3, PM0442 (pLIC-Nus); 4, PM0979 (pDEST-17); 5, PM0659 (pDEST-17); 6, PM1993 (pDEST-17); 7, PM1614 (pLIC-Nus); 8, PM1979 (pLIC-Nus); 10, P. multocida X73 whole cell lysate. All the soluble fusion proteins cloned with a NusA tag were cleaved and detected after incubation with Tev protease at room temperature for 2 h. The top arrows in the corresponding lanes show the expected size of the fusion proteins and the bottom arrows mark the recombinant proteins without the NusA tag. Lanes with single arrows correspond to the recombinant proteins expressed in the absence of a NusA tag.

One drawback of this immunogen discovery assay is that it is limited to protein coding regions that can be expressed in E. coli. In addition, antibodies that recognise only conformational epitopes that are denatured in SDS-PAGE will not be detected. Furthermore, a lack of antibody recognition to recombinant proteins may be the result of a poor immune response to these antigens during infection or alternatively, a reflection of differential expression of antigens by the VP161 and X-73 strains within the host. Four of the six novel proteins (PM0442, PM0659, PM0979, PM1614) were identified as putative lipoproteins characterised by a cysteine residue after a signal peptide. PM1993 was predicted to contain a signal peptide processed by signal peptidase 1. These results do not preclude the existence of additional immunogens present in other serotypes.

Conclusion

In summary, this work represents a systematic and comprehensive approach for the characterisation of the immune response associated with pasteurellosis by targeted computational screening of genomic sequences with predictive antigenic properties, followed by an automated high throughput expression and purification strategy using a robotic platform. It has resulted in the identification of 12 immunogenic determinants which are clearly expressed in vivo during infection, six of which were unique to this study, and thus constitute candidates for the development of a protective vaccine against fowl cholera. We are currently evaluating the protective effects of these P. multocida proteins against bacterial challenge in chickens.

Methods

Cloning of P.multocida genes

The Pm70 genome sequence was used when designing primers for X-73, which is virulent for chickens, while Pm70 does not cause disease in chickens. PCR primers were designed to amplify the gene region encoding the mature length protein (excluding the signal sequence) except where a signal sequence could not be predicted and then the primers were designed to encompass the entire gene. All 5' PCR primers included a 5'-CACC tail to facilitate directional topoisomerase cloning and where the primers had been designed to amplify only the mature length portion (without the signal sequence) the 5'-CACCATG tail was added so as to include a start codon. As the genes were to be expressed in-frame with either a C-terminal or an N-terminal tag the native stop codon was not included in the reverse primers. All PCR products were amplified from P. multocida strain X-73 (serotype A:1), which is highly virulent for chickens, and cloned into the Gateway entry vector pENTR/SD/D-TOPO® (Invitrogen Inc. Carlsbad, CA). After the cloned genes were verified by sequencing and restriction digestion analysis, they were transferred by recombination (LR Clonase kit – Invitrogen Inc., Carlsbad, CA) from the entry clone to the Invitrogen destination vectors pBAD-DEST49™, pDEST-17™ and a Gateway adapted expression vector containing a NusA solubility tag, pLIC-Nus [23].

Protein expression

Briefly, 1 ml of each culture (Overnight Express™, Novagen, Madison, WI) was chemically lysed with PopCulture (Novagen, Madison, WI), and Lysonase™ (Novagen, Madison, WI). The cell lysate was then added to a 96-well filter plate (AcroPrep™, EastHills, NY) and the solution was drawn through under vacuum. The inclusion bodies were retained while soluble proteins passed through the filter. The retained inclusion bodies were washed once with Triton X-100 to remove any remaining soluble proteins, followed by two washes with phosphate buffer. The washed proteins were then denatured by the addition of 200 μl of 8 M urea to each corresponding well, incubated for 2 hr at room temperature and collected under vacuum. Both soluble and insoluble fractions were then analysed by SDS-PAGE to assess the solubility of each protein.

Immunoblotting

The membranes were incubated with sera (diluted 1:500) from either infected or uninfected birds as the primary antibody and then with a peroxidase-conjugated anti-chicken antibody (diluted 1:1000) as the secondary antibody (Chemicon International Inc., Temecula, CA). 4-chloro-1-napthol was used as the chromogenic detection reagent.

Declarations

Acknowledgements

The authors thank Ian McPherson for his excellent technical contribution to this work. Funding for this research was provided by the Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics. Part of the results presented here have been communicated at the 4th Recombinant Protein Production Meeting (Barcelona, 2006).

Authors’ Affiliations

(1)
Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University
(2)
School of Veterinary Science, University of Queensland

References

  1. De Alwis MCL: Haemorrhagic septicaemia in cattle and buffaloes. Rev Sci Tech. 1984, 3: 707-730.Google Scholar
  2. White MP, Mukkur TKS, Cameron RD, Love RJ: Epidemiology of Pasteurella pneumonia in pigs. Pasteurellosis of production animals – ACIAR proceedings, Canberra: ACIAR. Edited by: Patten BE, Spencer TL, Johnson RB, Hoffman D, Lehane L. 1993, , 92-97.Google Scholar
  3. Manning PJ: Naturally occurring pasteurellosis in laboratory rabbits: chemical and serological studies of whole cells and lipopolysaccharides of Pasteurella multocida. Infect Immun. 1984, 44: 502-507.Google Scholar
  4. Carter GR: Pasteurellosis: Pasteurella multocida and Pasteurella hemolytica. Adv Vet Sci. 1067, 11: 321-379.Google Scholar
  5. Radostits OM, Blood DC, Gay CC: Diseases caused by bacteria. Veterinary medicine: A textbook of the diseases of cattle, sheep, pigs, goats and horses. Edited by: Radostits OM, Blood DC, Gay CC. 1994, Sydney: Bailliere Tindall, 748-750.Google Scholar
  6. Hopkins BA, Olson LD: Comparison of live avirulent PM-1 and CU fowl cholera vaccines in turkeys. Av Dis. 1997, 41: 317-325. 10.2307/1592184.View ArticleGoogle Scholar
  7. Niemann HH, Schubert WD, Heinz DW: Adhesins and invasins of pathogenic bacteria: a structural view. Microbes Infect. 2004, 6: 101-112. 10.1016/j.micinf.2003.11.001.View ArticleGoogle Scholar
  8. Brown JS, Ogunniyi AD, Woodrow MC, Holden DW, Paton JC: Immunization with components of two iron uptake ABC transporters protects mice against systemic Streptococcus pneumoniae infection. Infect Immun. 2001, 69: 6702-6706. 10.1128/IAI.69.11.6702-6706.2001.View ArticleGoogle Scholar
  9. Frazer LT, O'Brien-Simpson NM, Slakeski N, Walsh KA, Veith PD, Chen CG, Barr IG, Reynolds EC: Vaccination with recombinant adhesins from the RgpA-Kgp proteinase-adhesin complex protects against Porphyromonas gingivalisinfection. Vaccine. 2006, Google Scholar
  10. Tabatabai LB, Zehr ES: Identification of five outer membrane-associated proteins among cross-protective factor proteins of Pasteurella multocida. Infect Immun. 2004, 72: 1195-1198. 10.1128/IAI.72.2.1195-1198.2004.View ArticleGoogle Scholar
  11. Rimler RB: Purification of a cross-protective antigen from Pasteurella multocida grown in vitro and in vivo. Avian Dis. 2001, 45: 572-580. 10.2307/1592897.View ArticleGoogle Scholar
  12. Boyce JD, Cullen PA, Nguyen V, Wilkie I, Adler B: Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host. Proteomics. 2006, 6: 870-880. 10.1002/pmic.200401342.View ArticleGoogle Scholar
  13. Boyle RC, Finlay B: Bacterial pathogenesis: exploiting cellular adherence. Curr Opin Cell Biol. 2003, 15: 633-639. 10.1016/S0955-0674(03)00099-1.View ArticleGoogle Scholar
  14. Haddad D, Bilcikova E, Witney AA, Carlton JM, White CE, Blair PL, Chattopadhyay R, Russell J, Abot E, Charoenvit Y, Aguiar JC, Carucci DJ, Weiss WR: Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome. Infect Immun. 2004, 72: 1594-1602. 10.1128/IAI.72.3.1594-1602.2004.View ArticleGoogle Scholar
  15. Wizemann TM, Heinrichs JH, Adamou JE, Erwin AL, Kunsch C, Choi GH, Barash SC, Rosen CA, Masure HR, Tuomanen E, Gayle A, Brewah YA, Walsh W, Barren P, Lathigra R, Hanson M, Langermann S, Johnson S, Koenig S: Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniae infection. Infect Immun. 2001, 69: 1593-1598. 10.1128/IAI.69.3.1593-1598.2001.View ArticleGoogle Scholar
  16. McKevitt M, Patel K, Smajs D, Marsh M, McLoughlin M, Norris SJ, Weinstock GM, Palzkill T: Systematic cloning of Treponema pallidum open reading frames for protein expression and antigen discovery. Genome Res. 2003, 13: 1665-1674. 10.1101/gr.288103.View ArticleGoogle Scholar
  17. Pizza M, Scarlato V, Masignani V, Giuliani MM, Arico B, Comanducci M, Jennings GT, Baldi L, Bartolini E, Capecchi B, Galeotti CL, Luzzi E, Manetti R, Marchetti E, Mora M, Nuti S, Ratti G, Santini L, Savino S, Scarselli M, Storni E, Zuo P, Broeker M, Hundt E, Knapp B, Blair E, Mason T, Tettelin H, Hood DW, Jeffries AC, Saunders NJ, Granoff DM, Venter JC, Moxon ER, Grandi G, Rappuoli R: Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science. 2000, 287: 1816-1820. 10.1126/science.287.5459.1816.View ArticleGoogle Scholar
  18. Montigiani S, Falugi F, Scarselli M, Finco O, Petracca R, Galli G, Mariani M, Manetti R, Agnusdei M, Cevenini R, Donati M, Nogarotto R, Norais N, Garaguso I, Nuti S, Saletti G, Rosa D, Ratti G, Grandi G: Genomic approach for analysis of surface proteins in Chlamydia pneumoniae. Infect Immun. 2002, 70: 368-379. 10.1128/IAI.70.1.368-379.2002.View ArticleGoogle Scholar
  19. Gardy JL, Spencer C, Wang K, Ester M, Tusnády GE, Simon I, Hua S, deFays K, Lambert C, Nakai K, Brinkman FSL: PSORT-B: Improving protein subcellular localization prediction for Gram-negative bacteria. Nucleic Acids Res. 2003, 31: 3613-3617. 10.1093/nar/gkg602.View ArticleGoogle Scholar
  20. Lu Z, Szafron D, Greiner R, Lu P, Wishart DS, Poulin B, Anvik J, Macdonell C, R Eisner R: Predicting subcellular localization of proteins using machine-learned classifiers. Bioninformatics. 2004, 20: 547-556. 10.1093/bioinformatics/btg447.View ArticleGoogle Scholar
  21. Juncker AS, Willenbrock H, Von Heijne G, Brunak S, Nielsen H, Krogh A: Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci. 2003, 12: 1652-1662. 10.1110/ps.0303703.View ArticleGoogle Scholar
  22. Wilkie IW, Grimes SE, O'Boyle D, Frost AJ: The virulence and protective efficacy for chickens of Pasteurella multocida administered by different routes. Vet Microbiol. 2000, 72: 57-68. 10.1016/S0378-1135(99)00187-X.View ArticleGoogle Scholar
  23. Cabrita LD, Dai W, Bottomley SP: A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production. BMC Biotechnol. 2006, 6: 12-10.1186/1472-6750-6-12.View ArticleGoogle Scholar

Copyright

© Al-Hasani et al; licensee BioMed Central Ltd. 2007

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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