NSA QPX 2008

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NSA QPX 2008

  1. 1. Characterization of a serine protease in QPX Steven Roberts Roxanna Smolowitz Jackie DeFaveri
  2. 2. Rationale • Understand more concerning QPX physiology • Environmental influence • Differences across strain • Pathogenecity
  3. 3. QPX • Single-celled relative of slime mold that has both animal and fungal characteristics • QPX secretes a thick layer of mucus to ward off the clam’s immune response • Killing nine out of every ten clams in some plots when it first hit the Massachusetts coast in 1993 Hugh Powell
  4. 4. QPX Biology
  5. 5. Factors - Temperature - Strain
  6. 6. Factors - Temperature - Strain
  7. 7. Proteases
  8. 8. Complete Protein
  9. 9. Sequence Analysis •Subtilase family. •Subtilases are a family of serine proteases. •They appear to have independently and convergently evolved a catalytic triad •the three amino acid residues found inside the active site of certain protease enzymes: serine (S), aspartate (D) and histidine (H).They work together to break peptide bonds on polypeptides. •Structure is an alpha/beta fold containing a 7-stranded parallel beta sheet.
  10. 10. Asp/Ser/His Catalytic Triad * * *
  11. 11. Serine Protease gene expression 21℃ ATCC 10℃ 21℃ Env_66 10℃ 1
  12. 12. Serine Protease gene expression 21℃ ATCC 10℃ 21℃ Env_66 10℃ 1
  13. 13. 21℃ Serine Protease gene expression Media Media Tissue Tissue 1
  14. 14. Summary • Complete CDS of Subtilisin-like serine protease • Catalytic Triad • Expression varies across strains • temperature dependent • Significantly upregulated when in contact with host tissue
  15. 15. Future Work • Sequence variation in strains? • Does structure of local QPX community influence disease outbreak • Does Strain or Tissue affect SP expression • Further analysis of expression in environmental samples • incorporate into modeling
  16. 16. Characterizing gene expression patterns in three strains of Northern Quahogs Mercenaria mercenaria in response to infection by QPX Jackie DeFaveri, Steven Roberts, Roxanna Smolowitz Diane Murphy, Bill Walton
  17. 17. Rationale • Understand more concerning Clam physiology • Environmental Influence • Differences in Strain • Immune Response
  18. 18. Current Field Trial • Clams from MA, FL, NJ • First Winter • No QPX • High Mortality in FL Clams • Summer of 2007 QPX detected in all plots
  19. 19. QPX
  20. 20. Percent Mortality NJ MA FL
  21. 21. What is different in relation to the immune response across strains?
  22. 22. Approach • Differential Display Analysis • Construction of cDNA library from hemocytes • Gene Discovery
  23. 23. Differential Expression • Infected versus uninfected • 7 upregulated • 6 downregulated • Comparison across strains • 10 upregulated FL (June 7: Aug 3) • 8 upregulated NJ (June 2: Aug 6) • 1 upregulated MA (June)
  24. 24. Results • Few ribosomal proteins • Hemocyte receptors • Important signaling cascade • Unexpected difficulty identifying products • ???? • Serine Protease Inhibitor
  25. 25. Inhibitors of proteases
  26. 26. Hard Clam Serine Protease Inhibitor
  27. 27. Domain indicating that protein is able to inhibit subtilisin-like serine protease * * * * * ** Contains cysteine residues necessary for tertiary structure
  28. 28. Serine Protease Inhibitor gene expression NJ MA FL * in other assays have seen expressed in all strains
  29. 29. Inhibitor Expression • Currently do not have a complete picture • Different across strains • Is it induced upon recognition of QPX? • Upon QPX expression of protease? • Difference in sequence? • What is the role temperature?
  30. 30. Summary • Difficulty in identifying homologus sequences • Serine Protease Inhibitor is highly regulated and likely associated with proper immune function • Expression pattern could be developed as a marker
  31. 31. Future Work • Full-length sequence of clam SPI • 3-D Modeling • Protein - protein interaction • Recombinant production of clam SPI • Assay effects on SP expression in vitro

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