nsa qpx 2008
DESCRIPTION
TRANSCRIPT
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Characterization of a serine protease in QPX
Steven RobertsRoxanna Smolowitz
Jackie DeFaveri
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Rationale
• Understand more concerning QPX physiology
• Environmental influence
• Differences across strain
• Pathogenecity
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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
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QPX Biology
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Factors- Temperature- Strain
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Factors- Temperature- Strain
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Proteases
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Complete Protein
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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.
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*
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Asp/Ser/His Catalytic Triad
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Seri
ne P
rote
ase
gene
exp
ress
ion
ATCC Env_66
10℃
10℃10℃
21℃21℃
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Seri
ne P
rote
ase
gene
exp
ress
ion
ATCC Env_66
10℃
10℃10℃
21℃21℃
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Seri
ne P
rote
ase
gene
exp
ress
ion
Media
10℃21℃
MediaTissue Tissue
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Summary
• Complete CDS of Subtilisin-like serine protease
• Catalytic Triad
• Expression varies across strains
• temperature dependent
• Significantly upregulated when in contact with host tissue
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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
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Characterizing gene expression patterns in three strains of Northern Quahogs Mercenaria mercenaria in response to
infection by QPX
Jackie DeFaveri, Steven Roberts, Roxanna SmolowitzDiane Murphy, Bill Walton
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Rationale
• Understand more concerning Clam physiology
• Environmental Influence
• Differences in Strain
• Immune Response
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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
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QPX
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NJ MA FL
Percent Mortality
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What is different in relation to the immune
response across strains?
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Approach
• Differential Display Analysis
• Construction of cDNA library from hemocytes
• Gene Discovery
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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)
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Results
• Few ribosomal proteins
• Hemocyte receptors
• Important signaling cascade
• Unexpected difficulty identifying products
• ????
• Serine Protease Inhibitor
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Inhibitors of proteases
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Hard Clam Serine Protease Inhibitor
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Domain indicating that protein is able to inhibit subtilisin-like serine protease
* * **** *
Contains cysteine residues necessaryfor tertiary structure
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NJ MA FL
Serine Protease Inhibitorgene expression
* in other assayshave seen expressed in all strains
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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?
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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
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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