Development of Immunogens to Protect Against Turkey Cellulitis. Douglas. N. Foster and Robyn Gangl. Department of Animal Science

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1 Development of Immunogens to Protect Against Turkey Cellulitis Douglas. N. Foster and Robyn Gangl Department of Animal Science University of Minnesota St. Paul, MN 55108

2 Introduction Clostridial dermatitis infections have become a problem for poultry producers in the U.S. Among the commonly implicated organisms for causing clostridial dermatitis (a.k.a., cellulitis) in turkeys is Clostridium septicum which produces symptoms that include severe necrosis of the subcutaneous tissues of the abdomen and inner thighs. While sporadic outbreaks have been reported in birds 12 weeks of age or older, outbreaks are occurring in turkeys of all ages and with increased frequency (Carver, 2007). Clostridium septicum produces a single lethal factor, α toxin which can oligomerize into a supermolecular complex that forms ion-permeable channels across cell membranes allowing release of K + ions (and eventual cell lysis). Melton et al (2004) and Melton-Witt et al., (2006), have defined the functional domains present in the α toxin to include the receptor binding domain, the oligomerization domain with subsequent membrane insertion and pore formation functions, and the cellular activation domain. To develop fusion polypeptide immunogens, wild-type portions of these domains will be generated by PCR. These peptides will by themselves not be toxic, but it is hoped that they will provide strong antigenic epitopes in order to illicit a protective immune response to Clostridium septicum upon challenge. Materials and methods To produce 7 contiguous fragments of the α toxin of Clostridium septicum, bp oligonucleotides were designed to span the entire 1227 bp exotoxin gene. Clostridium septicum α toxin gene fragments 1 through 6 were all 186 base pairs (bp) in length and each corresponded to 174bp (58 amino acids) of the α toxin gene plus 6 bp corresponding to the 5 BglII site and 6 bp for the 3 SalI site. The 7 th fragment was 204bp and corresponded to the 3 end of the α toxin (192 bp; 64 amino acids) with an additional 12 bp for the two restriction enzyme sites. The 7 fragments were generated by PCR using long oligonucleotide fragments plus sequence-specific forward and reverse primers (IDT, Inc., Coralville, IA). PCR generated fragments were digested with BglII and SalI and ligated into the pqe40 expression vector (Qiagen, Inc., Valencia, CA) that had also been digested with BglII and SalI. All 7 α toxin fragments in pqe40 were grown in mass, purified and used to transfect competent E.coli M15 bacteria for high level production of recombinant protein. Before producing any recombinant subunit proteins in the expression vector system, all fragments were verified to be in the correct open reading frames by nucleotide sequence analysis (Advanced Genetic Analysis Center, University of Minnesota, St. Paul, MN) using pqe40 vector-specific sequencing primers.

3 Results and Discussion The 7 α toxin-specific fragments were generated by PCR using long oligonucleotide fragments plus sequence-specific forward and reverse primers. Examples of the PCR fragment sizes generated for α2, α3, and α4 (each at 186bp) are shown in Figure 1. Fragments 1, 5, 6, were also 186bp in size while Fragment 7 was 204bp (data not shown). Correctly sized PCR generated α toxinspecific fragments were ligated into the pqe40 expression vector. Prior to producing recombinant subunit proteins in the expression vector system, it is essential that the open reading frame of each subunit be verified. Nucleotide sequence analysis using vector specific primers showed that all 7 α toxin-specific fragments were in frame and should correctly produce the desired recombinant peptides. As an example, the alignment of Fragment 6 to the NCBI database for the Clostridium septicum α toxin sequence is shown in Figure 2. This completes all of the proposed research aims for year one of this two year proposal. For the second year of the proposal we plan to express recombinant peptides to serve as subunit antigens to produce natural antibodies in turkeys against Clostridium septicum α toxin. The pqe40 plasmid was chosen since it has a strong promoter-operator system in E. coli M15 bacterial cells that can drive expression of the α toxin subunit DNA fragment sequences. This vector also has a fusion gene that encodes a protein (DHFR) to stabilize the recombinant α toxin subunits thus avoiding conjugation to a larger molecule for stability and immunogenicity. Finally, a 6x His tag (the amino acid, histidine repeated 6 times) will be incorporated into the final recombinant polypeptide for purification from bacterial lysates. We are currently just beginning to scale up to produce Clostridium septicum α toxin for the in-frame α Fragments 1-7 in the M15 E. coli bacteria. Once we can observe IPTG-inducible α toxin peptides on PAGE-SDS gels, we can grow liter batches of bacteria with the recombinant vector to produce milligram quantities of Clostridium septicum α toxin subunit peptide antigen. The peptides can be purified away from other host bacterial proteins using a Ni-NTA column that specifically binds only the recombinant proteins that contain a 6x His tag. We plan to produce up to 200mg of each of the purified recombinant fusion proteins which will be used in conjunction with an adjuvant to serve as an immunogen when presented to turkeys. Acknowledgements This work was supported by the Minnesota Turkey Research & Promotion Council and the Midwest Poultry Consortium/USDA Grant #

4 References Carver, D. Cellulitis/Dermatitus in Turkeys North Carolina Poultry Industry Joint Area Newsletter Vol. V, Number 1. Melton, J.A., M.W. Parker, J. Rossjohn, J.T. Buckley, and R. K. Tweten The identification and structure of the membrane-spanning domain of the Clostridium septicum alpha toxin. J. Biol. Chem 279: Melton-Witt, J.A., L.M. Bentsen, and R.K. Tweten Identification of functional domain of Clostridium septicum alpha toxin. Biochem. 45:

5 MW α2 α3 α4 200bp 100bp 186 bp PCR products of Clostridium septicum α toxin fragments 2, 3, and 4. Figure 1 100% Alignment of Clostridium septicum α toxin Fragment 6: Query 1 AGATCTCATCTTAAAGATTTATATAGTCATAAGAATATTAATGGATATTCAGAATGGGAT 60 Sbj 631 AGATCTCATCTTAAAGATTTATATAGTCATAAGAATATTAATGGATATTCAGAATGGGAT 690 Query61 TGGAAATGGGTAGATGAGAAATTTGGTTATTTATTTAAAAATTCATACGATGCTCTTACT 120 Sbj 691 TGGAAATGGGTAGATGAGAAATTTGGTTATTTATTTAAAAATTCATACGATGCTCTTACT 750 Query121 AGTAGAAAATTAGGAGGAATAATAAAAGGCTCATTTACTAACATTAATGGAACAAAAATA 180 Sbj 751 AGTAGAAAATTAGGAGGAATAATAAAAGGCTCATTTACTAACATTAATGGAACAAAAATA 810 Query 181 GTCGAC 186 Sbj 811 GTCGAC 816 Query = Fragment 6 sequence; Subject (Sbj) = National Center for Biotechnology Information (NCBI) database for Clostridium septicum α toxin Figure 2