DISEASES OF AQUATIC ORGANISMSDis Aquat Org

Vol. 122: 2133, 2016doi: 10.3354/dao03058

Published November 22

INTRODUCTION

Peptidases or proteases are enzymes that hydro -lyze proteins. In pathogenic organisms peptidasesserve as virulence factors, enabling or facilitating in -fection processes (McKerrow et al. 1993, 2006, Klem -ba & Goldberg 2002). Specifically, secreted pepti-dases digest host proteins providing nutrients andallowing for infection spread, thus playing a key rolein pathogenicity of various microbial agents includ-ing bacteria (Miyoshi & Shinoda 2000), fungi (Monod

et al. 2002), and protozoa (Carruthers 2006, Alvarezet al. 2012). For example, peptidases of patho genicorganisms break down the components of the hostextracellular matrix, such as collagen or fibrin, andcomponents of blood and the immune system, suchas hemoglobin, proteins of the complement system,and immunoglobulins (McKerrow et al. 1993,Klemba & Goldberg 2002, Lecaille et al. 2002).

Quahog parasite unknown (QPX) is a protistan par-asite capable of causing deadly infections in the hardclam Mercenaria mercenaria, one of the most valu-

Inter-Research 2016 www.int-res.com*Corresponding author: bassen.allam@stonybrook.edu

Identification and characterization of peptidasessecreted by quahog parasite unknown (QPX),

the protistan parasite of hard clams

Ewelina Rubin1,4, Glenn T. Werneburg2,3, Emmanuelle Pales Espinosa1,David G. Thanassi2,3, Bassem Allam1,*

1School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USA2Center for Infectious Diseases, Stony Brook University, Stony Brook, NY 11794-5000, USA

3Department of Molecular Genetics & Microbiology, Stony Brook University, Stony Brook, NY 11794-5000, USA4Present address: Coastal Institute, University of Rhode Island, 215 South Ferry Road, Narragansett, RI 028821197, USA

ABSTRACT: Quahog parasite unknown (QPX) is a protistan parasite capable of causing deadlyinfections in the hard clam Mercenaria mercenaria, one of the most valuable shellfish species inthe USA. QPX is an extracellular parasite found mostly in the connective tissue of clam mantleand, in more severe cases of infection, other clam organs. Histopathologic examinations revealedthat QPX cells within clam tissues are typically surrounded by hollow areas that have beenhypothesized to be, at least in part, a result of extracellular digestion of clam proteins by the para -site. We investigated peptidase activity in QPX extracellular secretions using sodium dodecyl sulfate-polyacrylamide gels containing gelatin as a co-polymerized substrate. Multiple peptidaseactivity bands of molecular weights ranging from 20 to 100 kDa were detected in QPX secretionsderived from a variety of culture media. One major band of approximately 35 kDa was composedof subtilisin-like peptidases that were released by QPX cells in all studied media, suggesting thatthese are the most common peptidases used by QPX for nutrient acquisition. PCR quantificationof 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 serinepeptidase was able to digest clam plasma proteins, suggesting that this peptidase might beinvolved in the disease process, and making it a good candidate for further investigation as a possible virulence factor of the parasite.

KEY WORDS: Quahog parasite unknown Virulence factors Peptidases Zymography

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Dis Aquat Org 122: 2133, 2016

able shellfish species in the USA. QPX infection out-breaks occur in wild and aquacultured hard clamstocks along the north-eastern coast of the USA andmaritime Canada, leading to significant economiclosses to the industry in those regions. QPX belongsto the thraustochytrids, a group of protists mostly con-sisting of osmo-heterotrophic and saprophytic organ-isms (Raghukumar 2002). It has been speculated thatQPX mainly uses extracellular digestion to acquirenutrients, and possibly uses secreted peptidases to di-gest clam tissues during infection (Anderson et al.2006). The QPX transcriptome contains over 200 se-quences encoding for a variety of peptidases of allmajor types: serine (SPs), cysteine (CPs), aspartate(APs), threonine (TPs), and metallopeptidases (MPs)(Rubin et al. 2014). Over 70 of these pepti dases pos-sess an N-terminal signal peptide, suggesting thatthey are potentially directed into the secretory path-way (Rubin et al. 2015). In addition, 8 of these pepti-dases were detected in QPX extra cellular products(ECPs) using liquid chromato graphy coupled withmass spectrometry (LC/MS) (Rubin et al. 2015).

The aim of this study was to investigate the types ofpeptidases secreted by QPX cells cultivated undervarious culture conditions and among 3 QPX isolatesfrom different geographic locations. We hypothe -sized that QPX peptidase secretion will be variableand dependent on the protein supplement added tothe growth medium. We also hypothesized that diffe -rent QPX isolates will show similar peptidase se cre -tion profiles. In addition, mRNA levels of candidateextracellular peptidases were measured to determineif transcriptional changes of peptidase expressionalso occur in response to culture conditions. Ultra -filtration and ion exchange chromatography wereused to purify a major QPX peptidase before assess-ing its activity using clam plasma substrate poly-acrylamide gels.

MATERIALS AND METHODS

In silico identification of QPX extracellular peptidases

The identification of peptidases predicted to be se -creted by QPX cells was accomplished as describedpreviously (Rubin et al. 2015). Briefly, translated openreading frames (ORFs) from 2 QPX transcriptomelibraries (11 005 and 12 579 ORFs) were screened forthe presence of an N-terminal signal peptide, whichis known to direct the protein into the secretory path-way (Petersen et al. 2011). In total, 1255 unique pro-

teins were predicted to constitute the QPX secre-tome, and these included 74 putative ex tracellularpeptidases (Rubin et al. 2015). In the current study,the translated amino acid sequences of these 74 pep-tidases were further grouped into peptidase familiesbased on classification by the peptidase data base,MEROPS (Rawlings et al. 2012). Amino acidsequence alignments of peptidases belonging to thesame family were generated using the ClustalWalgorithm plug-in within Geneious v. 7.1.4 software(www. geneious. com; Kearse et al. 2012). Percentamino acid se quence identity between peptidasesbelonging to the same families and their expectedmolecular weights (MWs) were calculated using thesame software.

QPX cultures and media

To compare transcriptional expression of QPXpepti dases in response to different protein supple-ments, the following 4 media were prepared: mini-mal essential medium supplemented with fetal bo -vine serum (MEM FBS; Kleinschuster et al. 1998),MEM supplemented with yeastolate (MEM YSTO),MEM supplemented with gelatin (MEM GEL), andMEM supplemented with clam adductor musclehomogenate (MEM CAMH; Perrigault et al. 2009).The protein concentrations in all protein supple-ments were estimated using the Pierce BCA ProteinAssay Kit (Thermo Scientific) and adjusted to a con-centration of 2 mg ml1 with sterilized artificial sea-water (SASW; Instant OceanSea Salt, 28 ppt). Forthe ex perimental cultures, 1 volume of MEM wasmixed with 1 volume of 1 of the 4 protein supple-ments, resulting in a final protein concentration of1 mg ml1. The cultures were prepared in 12-wellplates filled with 4 ml of media in triplicate for eachtreatment. The QPX cell inoculum (NY1, isolateNY0313808 BC7; Qian et al. 2007) was prepared froman exponentially growing culture in MEM and FBS.The cells were collected by centrifugation (3000 g,15 min at room temperature), washed twice withSASW, and re-suspended in un-supplemented MEM.Cell concentration of QPX inocula was determinedwith a hemocytometer, and the starting concentra-tion of QPX was 8 103 cells ml1 in all cultures. QPXgrowth was monitored by cell counts using a hemo-cytometer every other day, and QPX cells were col-lected when they reached similar cell concentrations:MEM FBS (2.3 106 cells ml1), MEM GEL (2.1 106 cells ml1), MEM YSTO (2.0 106 cells ml1), andMEM CAMH (1.7 106 cells ml1). To collect the cells

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Rubin et al.: Peptidases secreted by QPX

and QPX secretions (media supernatant), 1 volume ofQPX culture was diluted with an equal volume ofSASW and passed several times through a syringe tofacilitate liquefaction of QPX extracellular mucus.The mixtures were transferred into 15 ml conicaltubes and centrifuged at 3000 g (20 min at 20C).The supernatant was collected into 1.5 ml tubes as40 l ali quots. To each aliquot, 10 l of 5 Laemmliloading dye, containing SDS but no beta-mercapto-ethanol, were added and the aliquots were stored ina 80C freezer until zymogram analyses. The cellswere used for RNA extractions (see below), whichwere conducted on the day of collection. As thezymogram analysis of QPX secretions from the abovedescribed cultures revealed peptidase activities onlyfor MEM FBS medium (see results below, Fig. 2A), anew culture based on MEM with 10% (v/v) of FBS(about 4 mg protein ml1) was used to check forpepti dase activity. Since preliminary investigationsshowed that increased protein concentrations im -prove the sensitivity of the zymograms, additionalcultures of the same QPX isolate were preparedusing MEM supplemented with FBS and CAMH,both adjusted to 3 mg protein ml1. A lower concen-tration was selected (3 instead of 4 mg ml1) becauseof technical limitations for preparing CAMH st