The Alveolate Perkinsus marinus: Biological Insights from EST Gene Discovery

Abstract

BackgroundPerkinsus marinus, a protozoan parasite of the eastern oyster Crassostrea virginica, has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of the United States. It is classified as a member of the Perkinsozoa, a recently established phylum considered close to the ancestor of ciliates, dinoflagellates, and apicomplexans, and a key taxon for understanding unique adaptations (e.g. parasitism) within the Alveolata. Despite intense parasite pressure, no disease-resistant oysters have been identified and no effective therapies have been developed to date.ResultsTo gain insight into the biological basis of the parasite's virulence and pathogenesis mechanisms, and to identify genes encoding potential targets for intervention, we generated >31,000 5' expressed sequence tags (ESTs) derived from four trophozoite libraries generated from two P. marinus strains. Trimming and clustering of the sequence tags yielded 7,863 unique sequences, some of which carry a spliced leader. Similarity searches revealed that 55% of these had hits in protein sequence databases, of which 1,729 had their best hit with proteins from the chromalveolates (E-value ≤ 1e-5). Some sequences are similar to those proven to be targets for effective intervention in other protozoan parasites, and include not only proteases, antioxidant enzymes, and heat shock proteins, but also those associated with relict plastids, such as acetyl-CoA carboxylase and methyl erythrithol phosphate pathway components, and those involved in glycan assembly, protein folding/secretion, and parasite-host interactions.ConclusionsOur transcriptome analysis of P. marinus, the first for any member of the Perkinsozoa, contributes new insight into its biology and taxonomic position. It provides a very informative, albeit preliminary, glimpse into the expression of genes encoding functionally relevant proteins as potential targets for chemotherapy, and evidence for the presence of a relict plastid. Further, although P. marinus sequences display significant similarity to those from both apicomplexans and dinoflagellates, the presence of trans-spliced transcripts confirms the previously established affinities with the latter. The EST analysis reported herein, together with the recently completed sequence of the P. marinus genome and the development of transfection methodology, should result in improved intervention strategies against dermo disease.

Shotgun Label-Free Proteomic Analyses of the Oyster Parasite Perkinsusmarinus

“…Antioxidant proteins including peroxiredoxin-2 and thioredoxin were detected in all triplicates, as well as proteins that are known to have immunosuppressive action, such as cyclophilins [17]. Proteins that are involved in the regulation of apoptosis including TP53-regulated inhibitor of apoptosis, liver stage antigen 3, pterin-4a-carbinolamine dehydratase, ubiquitin, polyubiquitin and adenylate kinase B were identified in at least two replicates [15,17].…”

“…Several heat shock proteins were detected in all replicates mostly from the 70 kDa family that are the most common expressed heat shock proteins in response to stress [14,15]. Heat shock proteins are necessary for P. marinus adaptive survival repertoire [16,17].…”

“…Proteases are known to play important roles in P. marinus disease pathogenesis causing cellular and tissue damage [17,18]. Other identified proteases of great importance in virulence included: serine/threonine kinases, cathepsin b, c, L and z, preprocathepsin c precursor, Thiolproteinases, aspartyl aminopeptidase, 26S proteases regulatory subunit, proteasomes, m1 zinc metalloprotease, intracellular alkaline proteases and cysteine protease [15,17].…”

“…Additional proteins that are important for the pathogenesis of P. marinus and a variety of bacterial and parasitic organisms were detected in at least two replicates, including: aldose reductase [20], betaine aldehyde dehydrogenase [21], helicases [15,22], malate dehydrogenase [6], and aspartyl aminopeptidase [23].…”

“…It would be interesting to examine P. marinus proteasome as potential drug targets for mollusc diseases. Abundant interactions for ribosomal proteins might be suggestive of the rapid and extensive protein translation that accompanies parasite differentiation and multiplication following host-cell invasion [15]. The ironsulfur cluster fumarase activity has been shown to be essential for protozoan parasites such as Trypanossoma cruzi since its mitochondrial isoform is part of the tricarboxylic acid cycle and as such being central to aerobic respiration [28].…”