Identification of novel immunogens in Pasteurella multocida
© Al-Hasani et al; licensee BioMed Central Ltd. 2007
Received: 22 November 2006
Accepted: 18 January 2007
Published: 18 January 2007
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.
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.
These data show the range of bacterial immunogens recognized by the chicken immune system, including 6 novel immunoreactive proteins.
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 [1–3]. 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 , 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 , which provide only limited protection against homologous serotypes and live attenuated strains, which have been observed to revert to virulence . 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 . 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 . Outer membrane proteins also promote adherence to host cell surfaces and are therefore likely to be involved in P. multocida virulence .
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 , Streptococcus pneumoniae , Treponema pallidum , Neisseria meningitidis , and Chlamydia pneumoniae .
Results and discussion
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.
P. multocida antigens recognised by immune chicken sera. +, reaction; -, no reaction.
Database similarity (accession no.)
Outer membrane protein from P. multocida (AAK02472)
Lipoprotein from P. multocida (AAK02638)
OmpA from P. multocida (AAT57679)
Omp16 from P. multocida (CAB75338)
Outer membrane protein from Haemophilus (AAX87732)
PlpB from P. multocida (AAK03814)
SurA protein from Mannheimia (AAU38450)
Oma87 from P. multocida (CAD20126)
Outer membrane protein from P. multocida serotype D (CAA52400)
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.
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.
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 .
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.
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.
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).
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