4. Rationale
• Understand more concerning QPX
physiology
• Environmental influence
• Differences across strain
• Pathogenecity
5. 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
14. 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.
20. 21℃
Serine Protease gene expression
Media
Media
Tissue
Tissue
1
21. Summary
• Complete CDS of Subtilisin-like serine
protease
• Catalytic Triad
• Expression varies across strains
• temperature dependent
• Significantly upregulated when in contact
with host tissue
22. 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
23.
24. 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
25.
26.
27. Rationale
• Understand more concerning Clam
physiology
• Environmental Influence
• Differences in Strain
• Immune Response
28.
29.
30. 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
41. Domain indicating that protein is able to
inhibit subtilisin-like serine protease
* * * * * **
Contains cysteine residues necessary
for tertiary structure
42. Serine Protease Inhibitor
gene expression
NJ MA FL
* in other assays
have seen expressed in
all strains
43. 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?
44. 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
45. 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