Characterisation of the secretome of the clam parasite, QPX
Graphical abstract
Introduction
Quahog parasite unknown (QPX) is a thraustochytrid protist that infects hard clams, Mercenaria mercenaria, along the northwestern coasts of the Atlantic Ocean from Prince Edward Islands, Canada, to Virginia, United States of America (USA) (Whyte et al., 1994, Ragone Calvo et al., 1998). The histopathological examination of tissue lesions from diseased clams typically shows QPX cells surrounded by a thick layer of muco-polysaccharide secretions characterised by positive Alcian blue staining (Ragone Calvo et al., 1998, Smolowitz et al., 1998). In addition, parasite cells, when cultivated in vitro, produce a very dense mucus secretion containing QPX extracellular products (QPX ECPs) (Perrigault and Allam, 2009). It has been suggested that the mucus layer surrounding QPX may function as a virulence factor by facilitating the infection of clam tissue and providing protection against the clam immune response by limiting parasite phagocytosis and encapsulation by clam hemocytes (Smolowitz et al., 1998). The protective role of QPX secretions has also been supported by in vitro experiments showing that the mucoid layer surrounding the parasite provides protection against the antimicrobial activity of clam plasma (Anderson et al., 2003). A preliminary study also showed that QPX ECPs contain unidentified peptidases which are capable of breaking down proteins and possibly used by the parasite to destroy clam connective tissue (Anderson, R.S., Luskey, T.M., Strauss, M.A., 2006. In vitro protease production by QPX, Abstracts of the 98th Annual Meeting of the National Shellfisheries Association, Monterey, California, USA). Further, exposure to QPX ECPs causes the death of clam hemocytes (Perrigault and Allam, 2009), suggesting that these secretions may also contain cytotoxic molecules. The specific biochemical composition of QPX ECPs and the potential role of their specific components in QPX virulence remain unknown and represent the focus of this study.
It is well documented that pathogenic organisms secrete molecules that enable host invasion, help establish infection foci and cause damage to host tissues (Kamoun, 2006, Kale and Tyler, 2011). These extracellular and cell surface molecules include hydrolysing enzymes (peptidases, glycosidases and lipases), receptors, adhesins and other recognition and attachment proteins. In silico predictions of pathogenic secretomes are often used to evaluate the pathogenic capabilities of microorganisms (Raffaele et al., 2010, Denoeud et al., 2011, Jiang et al., 2013). Proteomic approaches such as mass spectrometry (MS) represent powerful tools for large-scale identification of proteins and are also being used successfully to investigate extracellular proteins of pathogenic protozoans e.g. Trypanosoma brucei (Geiger et al., 2010) and Leishmania donovani (Silverman et al., 2008). In this study, we used a combination of transcriptomics-based prediction and MS to identify proteins secreted by QPX and to provide new insight into the molecular basis of QPX pathogenicity. We also investigated the transcription levels of QPX putative virulence factors within the clam infection sites (also known as clam nodules) to reveal their possible importance during the infection process.
Section snippets
In silico prediction of the QPX secretome
The QPX transcriptome sequence libraries were generated as part of the Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP) under the sample ID numbers: MMETSP0098 and MMETSP0100 (Keeling et al., 2014). The two transcriptome libraries were generated for QPX cells cultivated in (i) minimal essential medium (MEM) supplemented with 10% (v/v) FBS (MMETSP0098) and (ii) MEM supplemented with 10% FBS and 2% clam adductor muscle homogenates, adjusted to a concentration of 7 mg/ml of
Predicted secretome of QPX
After scanning predicted QPX peptide sequences using the SignalP bioinformatics tool, a total of 806 proteins and 641 proteins with an N-terminal secretory signal peptide were identified in MMETSP0098 and MMETSP0100 libraries, respectively (Supplementary Tables S1, S2). Among these, 192 sequences were found to have 95% or higher amino acid similarity between the two transcriptome libraries, representing the same proteins or highly similar isoforms. The 806 signal peptide-containing proteins
Discussion
QPX was predicted to secrete 1255 proteins outside its cell based on the presence of a eukaryotic signal peptide at the N-terminal of proteins. The number of proteins predicted to be secreted by QPX is within the same range as predictions for other stramenopilan pathogens such as Phytophthora infestans, Saprolegnia parasitica and Pythium ultimum which, respectively, have 1415, 970 and 747 proteins predicted to be secreted based on the presence of eukaryotic signal peptides (Levesque et al., 2010
Acknowledgments
This research was supported by projects R/XG-19 and R/FBM-34 (to BA and EPE), funded under awards NA07OAR4170010 from the National Sea Grant College Program of NOAA to the Research Foundation of State University of New York, USA on behalf of New York Sea Grant. The study was also partially supported by project IOS 1050596 (to BA and EPE) supported by the National Science Foundation, USA. The sequencing of the QPX transcriptome was made with support from the Gordon and Betty Moore Foundation’s
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