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  • Identification and characterization of peptidases secreted by quahog parasite unknown (QPX), the protistan parasite of hard clams.

Identification and characterization of peptidases secreted by quahog parasite unknown (QPX), the protistan parasite of hard clams.

Diseases of aquatic organisms (2016-12-03)
Ewelina Rubin, Glenn T Werneburg, Emmanuelle Pales Espinosa, David G Thanassi, Bassem Allam
ABSTRACT

Quahog parasite unknown (QPX) is a protistan parasite capable of causing deadly infections in the hard clam Mercenaria mercenaria, one of the most valuable shellfish species in the USA. QPX is an extracellular parasite found mostly in the connective tissue of clam mantle and, in more severe cases of infection, other clam organs. Histopathologic examinations revealed that QPX cells within clam tissues are typically surrounded by hollow areas that have been hypothesized to be, at least in part, a result of extracellular digestion of clam proteins by the parasite. We investigated peptidase activity in QPX extracellular secretions using sodium dodecyl sulfate-polyacrylamide gels containing gelatin as a co-polymerized substrate. Multiple peptidase activity bands of molecular weights ranging from 20 to 100 kDa were detected in QPX secretions derived from a variety of culture media. One major band of approximately 35 kDa was composed of subtilisin-like peptidases that were released by QPX cells in all studied media, suggesting that these are the most common peptidases used by QPX for nutrient acquisition. PCR quantification of mRNA encoding QPX subtilisins revealed that their expression changes with the protein substrate used in the culture media. A fast protein liquid chromatography (FPLC) was used to fractionate QPX extracellular secretions. An FPLC-fraction containing a subtilisin-type serine peptidase was able to digest clam plasma proteins, suggesting that this peptidase might be involved in the disease process, and making it a good candidate for further investigation as a possible virulence factor of the parasite.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Gelatin from porcine skin, Type A, lyophilized powder, γ-irradiated, BioXtra, suitable for cell culture
Sigma-Aldrich
Chymostatin, microbial
Sigma-Aldrich
Aprotinin from bovine lung, lyophilized powder, 3-8 TIU/mg solid