Purification of Vaccine Candidates Isolated from Streptococcus mutans UA159

1. Purification of Vaccine Candidates Isolated from Streptococcus mutans UA159 Katherine Vollmer University of Delaware Thursday July 24, 2008

2. Vaccine candidates for what?

3. Infective Endocarditis • Inflammation of the Endocardium, primarily on the heart valves • Vegetations caused by bacterial colonization on damaged valves • The fourth leading cause of life-threatening infectious disease syndromes • Fatal if left untreated • No effective defensive immune mechanisms • No preventative medicine currently available

4. Infective Endocarditis • For bacterial colonization to occur, the proteins on the surface of the cells bind to specific tissue proteins • Fibronectin • Collagen

5. How can we tackle Infective Endocarditis? A vaccine!

6. Breakthrough in genomics • In 1995, the first complete genome for a microorganism (Haemophilus influenzae) was published • In 2002 the completed sequenced genome of S. mutans UA159 was published • As of 2004, over 100 microorganisms have been sequenced with nearly 200 more being processed

7. Availability of genomes allows us to work backwards, saving time and money.

8. Predict ORFs Annotate Genes Identify Surface proteins Clone & express Purify proteins Develop Vaccines Identify antigens Immuno- assay Vaccinate animals Microbial Genomes Procedures

9. 2000, Science 2003, Science 2005, Science 2006, PNAS 2007, Nature Biotechnology 2007, Nature Biotechnology Reverse Vaccinology

10. One antigen at a time Must be done in vitro Most abundant proteins are not necessarily protective antigens Costly Multiple antigens In silico approach allows for specific antigens to be purified, including novel proteins Shorter amount of time Conventional v. Reverse

11. Why Streptococcus mutans? • Endocarditis • 31% of cases caused by Strep • 6-14% of cases caused by mutans • Underreported- difficulties in speciation • Dental Caries • Compare with other candidates from other species of Streptococcus (ex. S. pyogenes, S. sanguinis) in the hopes of finding a universal vaccine Courtesy of What is caries? http://whatiscaries.com/index.html

12. Project Goal • By using the technique of reverse vaccinology we will locate, isolate and purify the desired/predicted vaccine candidates from Streptococcus mutans • Are some of the antigens found in S. mutans conserved among other species of Strep?

13. Last summer… • Use of databases to identify all proteins • Large-scale isolation of genomic DNA • UA159 • Genomic availability • Difficulty • Surface digestion of the cells to isolate peptides • Proteinase K • Trypsin

14. Finding genes • SignalP • TMHMM • BLAST all proteins from S. mutans against the proteins of other Strep species

15. StreptoBIKE

16. Proteins identified 857 199 • 1960 total proteins in S. mutans

17. Surface Digestion Cleaved by Proteinase K Cleaved by Trypsin cytoplasm

18. Proteinase K cleaves peptide bonds adjacent to the carboxyl group of aliphatic and aromatic AAs • Trypsin also cleaves the peptide bond adjacent to the carboxyl group of lysine and arginine residues http://www.bact.wisc.edu/Microtextbook/index.php

19. Surface Digestion Cleaved by Proteinase K: 32 peptides Cleaved by Trypsin: 15 peptides 11 cleaved by both cytoplasm

20. Recap • 36 peptides identified through proteomics • Cell-wall associated proteins • Cell surface antigens • Ribosomal proteins • Substrate binding proteins • Additional gene: femA/sloC • Genomic DNA was isolated and purified • Primer design…

21. Acknowledgments • Dr. Ping Xu • Dr. Xiuchun Ge • Dr. Todd Kitten • The Kitten Lab • Joe Moeller • Jeff Elhai & BBSI participants

22. References • Capecchi, B., et al. “The Genome Revolution in Vaccine Research.” Curr. Issues Mol. Bio. 6 (2004): 17-28. • Ferretti, Joseph J., et al. “Genome Sequence of Streptococcus mutans UA159, a cariogenic dental pathogen.” The Proc of the Nat Acad of Sci. 99 (2002):14434-14439. • McGhie, D., et al. “Infective endocarditis caused by Streptococcus mutans.” British Heart Journal. 39 (1977): 456-458. • Moreillon, P., Que, Yok-Al. “Infective Endocarditis.” The Lancet. 363 (2004): 139-149. • Nemoto, H., et al. “Molecular characterization of Streptococcus mutans strains isolated from the heart of an infective endocarditis patient.” Jour of Med Microbiology. 57 (2008): 891-895. • Olsen, J.L., et al. “Trypsin Cleaves Exclusively C-terminal to Arginine and Lysine Residues.” Mol. & Cell. Proteomics 3.6. (2004): 608-614. • Rappuoli, R., et al. “Microbial genomes and vaccine design: refinements to the classical vaccinology approach.” Curr Opin in Microbiology. 9 (2006): 532-536. • Rappuoli, R., et al. “Reverse Vaccinology and Genomics.” Science. 302 (2003): 602. • Ullman, R.F., et al. “Streptococcus mutans endocarditis: report of three cases and review of the literature.” Heart Lung. 2 (1988): 209-212.

23. Topological analysis of the 37 surface-exposed proteins belonging to the membrane protein family. Each of the 37 membrane proteins belonging to the M1_SF370 surface proteome was subjected to PSORT topological prediction. (a) All proteins whose predicted topology is consistent with the experimental data are shown; the proteolytic peptides (shown in red in the figure) derived by protease treatment of whole cells are in fact located on domains protruding out of the membrane. (b,c) Shown are the 11 proteins showing discrepancies between in silico-predicted topology and experimental data (see text for details) and whose identification was deduced from the characterization of more than one (b) or one (c) peptide. ©2007 Nature Publishing Group