Shotgun Label-Free Proteomic Analyses of the Oyster Parasite Perkinsus Marinus

Perkinsus marinus is an intracellular parasitic protozoan that is responsible for serious disease epizootics in marine bivalve molluscs worldwide. Despite all available information on P. marinus genomics, more baseline data is required at the proteomic level. Our aim was to study the proteome profile of in vitro cultured P. marinus isolated from oysters Crassostrea spp. using a label-free shotgun UDMS approach. A total of 4073 non redundant proteins were identified across three biological replicates with stringent identification. Proteins specifically related to adaptive survival, cell recognition, antioxidants, regulation of apoptosis and others were detected. Important virulence factors of P. marinus were identified including serine protease and irondependent superoxide dismutase. Other proteins with involvement in several pathogens invasion strategies were rhoptries, serine-threonine kinases and protein phosphatases. Interestingly, peptides corresponding to retroviruses polyproteins were identified in all replicates. The interactomic analysis of P. marinus proteins demonstrated important cluster networks related to biological processes. In conclusion, we provide the first comprehensive proteomic profile of P. marinus that can be useful for further investigations on Perkinsus biology and virulence mechanisms. DOI : 10.14302/issn.2326-0793.jpgr-17-1571 Corresponding author: Henrique César Pereira Figueiredo, Department of Preventive Veterinary Medicine, Veterinary School, Federal University of Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, Brazil. Phone: +55-31-34092120, Fax: +55-31-34092075, E-mail address: figueiredoh@yahoo.com Running title: Proteome of Perkinsus marinus


Introduction
Marine bivalve molluscs production worldwide is being affected by disease epizootics caused by intracellular protozoan parasites of the genus Perkinsus. Numerous species have been described but only two species namely Perkinsus marinus and Perkinsus olseni require notification to the World Organization for Animal Health [1]. P. marinus has been associated with significant mortalities of the eastern oyster Crassostrea virginica but other species such as the pacific oyster Crassostrea gigas and the mangrove oyster Crassostrea rhizophorae are also susceptible [2].
The parasite belongs to a new independent phylum Perkinsozoa that is positioned between the phyla Apicomplexa and Dinoflagellata [3]. Species of Perkinsus are believed to be transmitted directly from host to host during the feeding process [4]. Different developmental stages are observed that are all believed to be infective [2]. No effective therapies have been developed to date.
A considerable amount of genomic information is available for P. marinus. In contrast, few studies available on comparisons of proteomic profiles of different Perkinsus species and on proteome variability of P. olseni resulted in low throughputs: 28 and 19 annotated proteins, respectively [5,6]. More proteomic baseline data is required for a better understanding of P. marinus biology, including virulence mechanisms.
Proteome techniques nowadays allow us to observe whole cellular events by directly visualizing the proteins being expressed. Neither genome nor transcriptome investigations would allow the analysis of such complex parasitic responses [7].
In the present study, we analyzed the proteomic profile of in vitro cultures of P. marinus by a high throughput label-free shotgun UDMS E approach using nano ultra-performance liquid chromatography mass spectrometry (nanoUPLC-MS). of PLGS data of all replicates. The data was decreasingly ordered and plotted using R software [11] (Fig. 3). A protein-protein interaction network was built using the algorithm STRING [12]. STRING data setting adjusted parameters were active interaction sources to experiments, gene fusion, databases, co-ocurrence and co-expression; and the minimum required interaction score to custom value of 0.980. Proteins present in all replicates with at most a unique peptide were included as query in STRING web server (n = 881).

Results
A total of 4073 non redundant proteins were identified across the replicates and a combined total of 2810 proteins were present in at least two of the biological replicates ( Figure 1). partner repository with the dataset identifier PXD003727.
The identified proteins were divided into 14 categories, according to Gene Ontology (GO) classification. Most of the identified proteins (82.09 %) were involved in metabolic, cellular and single-organisms processes. Other identified proteins were involved in response to stimulus, localization, biological regulations, cellular component organization or biogenesis, signaling, detoxification, developmental process, biological adhesion and less than only 1 % were involved in multicellular, organismal process and locomotion ( Figure  2). The predicted proteins were blasted against Uniprot/   (Fig 4).

Discussion
The proteome of in vitro cultured P. marinus was analyzed. In this study, we provided the first broad- For proteome dataset analyses, particular attention was given to proteins that are known to represent virulence factors in P. marinus and in other pathogens, including closely related apicomplexans.
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]. 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 TP53regulated 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].
Serine proteases and iron-containing superoxide dismutase that are suggested to be virulence determinants of P. marinus were detected in at least two replicates [18,19]. 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].
Rhoptries are club-shaped secretory organelles that discharge their contents during infection. Rhoptry proteins were detected in all replicates. P. marinus is well known to infect oyster hemocytes residing inside phagosome-like vesicles where they remain viable and multiply. Rhoptries are considered to be key mediators of virulence by enabling many parasites, including apicomplexans to invade hosts erythrocytes [24].  Rhoptries contains a number of novel proteins (ROP) including serine-threonine kinases and protein phosphatases that were detected in all replicates and are also believed to be parasitic virulence determinants [24]. Furthermore, the merozoite surface protein 3 was detected in all replicates. This protein is reported to be important parasitic surface antigens as well as virulence factors [25].
One remarkable finding was that peptides corresponding to retroviruses polyproteins were detected. An analysis of the P. marinus genome reveals that there are three putative endogenous retroviruses present and our findings shows that these sequences are being transcribed and translated. The presence of a virus and/or retrotransposon elements in the P. marinus genome based on genomic annotation together with EST data was previously reported [15]. There are several reports that have confirmed the role of proteasomes in parasite differentiation and proliferation processes, which are key steps in pathogen colonization [26]. A great variety of proteasome inhibitors have been studied as molecular targets for treatment development of many parasitic diseases by selectively killing or disrupting parasite multiplication [26,27]. 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]. The TCP1 complex belongs to the HSP60 family protein that was detected in P.
marinus from our study. This protein is involved in folding and assembly of wide range of cytosolic proteins after stress related denaturation being critical for maintaining the integrity of cellular proteins [15]. In most organisms, ATP synthase taps the energy stored in the proton gradient generated by the respiratory complexes to synthesize ATP from ADP and phosphate (P i ). However, it has been suggested that in blood-stage trypanosomes that has no mitochondrial respiratory enzymes, this enzyme might operate in reverse, as an ATPase hydrolyzing ATP into ADP and P i and pumping protons into the intermembrane space. This reverse reaction would be important to maintain the membrane potential during anoxia [29]. Since P. marinus primarily infects host hemocytes, it is interesting to further examine Perkinsus ATP synthase mechanisms. It has been reported that P. marinus possesses genes for a plant-type ferredoxin system that possibly encodes plastid-targeting signals [30]. In addition to ferredoxins, triose phosphate proteins that are also predicted to target plastids were detected in our study, however its implications are unknown. Clathrin-mediated trafficking is known to be responsible for endocytosis and post-Golgi transport in trypanosomes. It also represents an important interface with the host, in addition to play multiple roles in immune evasion and host cell invasion that are vital for effective infection and persistence [31].
In conclusion, this is the first comprehensive identification of P. marinus proteins by a label-free shotgun proteomic approach. These results might serve as a valuable resource for future investigations involving