1. Genomic Sequencing of Mycoplasma hyopneumoniae, Virulence Factors, and Vaccines

2. Acknowledgements The High Throughput Sequencing Center University of Washington Lee Hood, Greg Mahairas, Steve Swartzell, Jim Wallace The University of Alabama in Birningham Elliot Lefkowitz Iowa State University Tina VanDyk, Barbara Cleary, Melissa Lovekamp NSW Agriculture, Australia Steve Djordjevic

3. How Does M. hyopneumoniae Cause Disease? How does Mhyo colonize?How does Mhyo colonize? What genes are expressed?What genes are expressed? What do these products do for the pathogen?What do these products do for the pathogen? What do these products do to the host?What do these products do to the host?

4. Functional Genomics Define function of every geneDefine function of every gene Establish relationship to pathogenesisEstablish relationship to pathogenesis Identify virulence factorsIdentify virulence factors Develop new therapeuticsDevelop new therapeutics

5. Genome Statistics OrganismSizeG+CC D M. hyopneumoniae 903,350 28.6 %93% M. genitalium580,07432.0 %90% M. pneumoniae816,39440.0 %89% M. pulmonis963,87926.6 %93% U. urealyticum751,71925.5 %93%

6. Mhyo Potential Protein Coding ORFs Total Protein Coding ORFs:766Total Protein Coding ORFs:766 –Functional identification:392(51%) –Hypothetical:374(49%) Conserved (Database match):64(17%)Conserved (Database match):64(17%) Unique (No database match):311(83%)Unique (No database match):311(83%)

7. Metabolic Assignments Amino acid BiosynthesisAmino acid Biosynthesis Biosynthesis of cofactors, prosthetic groups, and carrierBiosynthesis of cofactors, prosthetic groups, and carrier Cell envelopeCell envelope Cellular processesCellular processes Central intermediary metabolismCentral intermediary metabolism Energy metabolismEnergy metabolism Fatty acid and phospholipid metabolismFatty acid and phospholipid metabolism Purines, pyrimidines, nucleosides, and nucleotidesPurines, pyrimidines, nucleosides, and nucleotides Regulatory functionsRegulatory functions ReplicationReplication TranscriptionTranscription TranslationTranslation Transport and binding proteinsTransport and binding proteins Other categories, UnassignedOther categories, Unassigned HypotheticalHypothetical

9. Mycoplasma hyopneumoniae 900 kb Ribosomal and Transfer RNAs

10. PTS Gluc6P Fruc6P Fruc1,6P GA3P DHAP PyruvateAc-CoA Ac-P Acetate Lactate G3P ABC +ATP +NAD + +ATP + NADH +NAD + Glycolysis

11. Ribose5P 5Phosphoribose diphosphate Adenine Thymidine Uracil Guanine Adenosine Thymine UMP AMP dTMP CMP GMP Uridine Guanosine ADP dTDP CDP GDP NDP NTP dNDP dNTP +ATP +5PRPP dUMP +5PRPP Cytidine UDP +5PRPP ? ? ? Purine and Pyrimidine Metabolism

12. Membrane Transport F O F 1 ATPas e ADP ATP H + /Na + ~45lipoproteins P-type ATPas e ADP ATP PTS mglA MgtE Mg 2+ / Co 2+ Fe 3+ / haemin phosphat e spermidineputrescine oligopeptide s cations carbohydrate amino acids,bases, nucleosides ??

13. Chaperones DnaKDnaK DnaJDnaJ GrpEGrpE Trigger FactorsTrigger Factors No GroEL-GroESNo GroEL-GroES Secretion SecASecA SecYSecY ClpBClpB

14. Mycoplasma hyopneumoniae 900 kb Locations of Putative Lipoproteins

15. Mycoplasma hyopneumoniae Attaches only to ciliated epitheliumAttaches only to ciliated epithelium Attaches exclusively to the ciliaAttaches exclusively to the cilia

16. cilium binding F2G5  NP97 F2G5  NP97 NP97

17. Immunogold EM Image of M. hyopneumoniae reacted with F2G5 monoclonal antibody R1 R2  ∆NP97  NP97 F1B6

18. Immunogold labeling of in vitro grown M. hyopneumoniae with  -∆NP97 peptide antiserum R1 R2  ∆NP97  NP97 F1B6

19. Immunogold EM Images of M. hyopneumoniae reacted with anti-NP97.2 mouse antiserum R1 R2  ∆NP97  NP97 F1B6

20. Immunogold EM Image of M. hyopneumoniae reacted with anti-P102 mouse antiserum

21. 6 Mycoplasma hyopneumoniae cilium adhesin-related operons 1 2 3 4 5

22.       

23. P97 homolog alignment P102 homolog alignment

24. Therapeutic Targets Lipoproteins as vaccine components Lipoproteins as vaccine components Membrane transporters Membrane transporters Adherence-related proteins Adherence-related proteins P97 and P102 paralogs P97 and P102 paralogs Unique membrane activities Unique membrane activities