Defence mechanisms of Pinctada fucata martensii to Vibrio parahaemolyticus infection: Insights from proteomics and metabolomics.

Survival of pearl oysters is not only challenged by coastal pollution, but also pathogen infection that may eventually incur substantial economic losses in the pearl farming industry. Yet, whether pearl oysters can defend themselves against pathogen infection through molecular mechanisms remains largely unexplored. By using iTRAQ proteomic and metabolomic analyses, we analysed the proteins and metabolites in the serum of pearl oysters (Pinctada fucata martensii) when stimulated by pathogenic bacteria (Vibrio parahaemolyticus). Proteomic results found that a total of 2,242 proteins were identified in the experimental (i.e., Vibrio-stimulated) and control groups, where 166 of them were differentially expressed (120 upregulated and 46 downregulated in the experimental group). Regarding the immune response enrichment results, the pathway of signal transduction was significantly enriched, such as cytoskeleton and calcium signalling pathways. Proteins, including cathepsin L, heat shock protein 20, myosin and astacin-like protein, also contributed to the immune response of oysters. Pathogen stimulation also altered the metabolite profile of oysters, where 49 metabolites associated with metabolism of energy, fatty acids and amino acids were found. Integrated analysis suggests that the oysters could respond to pathogen infection by coordinating multiple cellular processes. Thus, the proteins and metabolites identified herein not only represent valuable genetic resources for developing molecular biomarkers and genetic breeding research, but also open new avenues for studies on the molecular defence mechanisms of pearl oysters to pathogen infection.

The succinylome of Pinctada fucata martensii implicates lysine succinylation in the allograft-induced stress response.

Lysine succinylation is a novel protein post-translational modification associated with the regulation of a variety of cellular processes. Post-translational modifications may regulate the immune response of Pinctada fucata martensii, a marine bivalve used to produce cultured pearls, in response to the surgical implantation of the seed pearl. This allograft-induced stress response may lead to transplant rejection or host death. However, the regulatory effects of post-translational modifications following nucleus insertion surgery in P.f. martensii remain largely unknown. Here, we used 4D label-free quantitative proteomics (4D-LFQ) with LC-MS/MS to explore the effects of nucleus implantation on lysine succinylation in P.f. martensii. We identified 4430 succinylated sites on 964 succinylated proteins in P.f. martensii after nucleus insertion surgery, and seven conserved motifs were identified upstream and downstream of these sites. In total, 269 succinylation sites were differentially expressed in response to implantation (|fold-change| > 1.5 and FDR <1%; 211 upregulation and 58 downregulation), corresponding to 163 differentially expressed succinylated proteins (DESPs; 124 upregulated and 39 downregulated). The terms over-enriched in the DESPs included "cellular processes", "metabolic pathways", and "binding activity", while the significantly enriched pathways included "ECM-receptor interaction", "PI3K-Akt signaling", and "focal adhesion". "EGF-like structural domains", "platelet-responsive protein type 1 structural domains", and "laminin EGF-like (domains III and V) domains" were overrepresented in the DESPs. Parallel reaction-monitoring (PRM) analysis validated 13 DESPs from the proteomics data. The succinylome of P.f. martensii (generated here for the first time) helps to clarify the biological role of large-scale succinylation in this bivalve after nucleus insertion surgery, providing a theoretical basis for further investigations of stress-induced post-translational modifications in other mollusks and extending our knowledge of the molluscan succinylated proteome.

Comprehensive analysis of the lncRNAs, mRNAs, and miRNAs implicated in the immune response of Pinctada fucata martensii to Vibrio parahaemolyticus.

Non-coding RNAs (ncRNAs) have been implicated in a variety of biological processes. However, most ncRNAs are of unknown function and are as-yet unannotated. The immune-related functions of ncRNAs in the pearl oyster Pinctada fucata martensii were explored based on transcriptomic differences in the expression levels of long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) in the hemocytes of P.f. martensii after challenge by the pathogenic bacterium Vibrio parahaemolyticus. Across the challenged and control pearl oysters, 144 miRNAs and 14,571 lncRNAs were identified. In total, 13,375 ncRNAs were differentially expressed between the challenged and control pearl oysters; in the challenged pearl oysters as compared to the controls, 15 miRNAs and 5147 lncRNAs were upregulated, while 51 miRNAs and 8162 lncRNAs were downregulated. The sequencing results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) analysis. GO and KEGG pathway analysis showed that genes targeted by the differentially expressed ncRNAs were associated with the vascular endothelial growth factor (VEGF) signaling pathway and the nuclear factor kappa-B (NF-κB) signaling pathway. An lncRNA-mRNA-miRNA network that was developed based on the transcriptomic results of this study suggested that lncRNAs may compete with miRNAs for mRNA binding sites. This study may provide a useful framework for the detection of additional novel ncRNAs, as well as new insights into the pathogenic mechanisms underlying the response of P.f. martensii to V. parahaemolyticus.

Lysine acetylation plays a role in the allograft-induced stress response of the pearl oyster (Pinctada fucata martensii).

Implanting a spherical nucleus into a recipient oyster is a critical step in artificial pearl production using the pearl oyster Pinctada fucata martensii. However, little is known about the role of post-translational modifications (PTMs) in the response of the pearl oyster to this operation. Lysine acetylation, a highly conserved PTM, may be an essential adaptive strategy to manage multiple biotic or abiotic stresses. We conducted the first lysine acetylome analysis of the P. f. martensii gill 12 h after nucleus implantation, using tandem mass tags (TMT) labeling and Kac affinity enrichment. We identified 2443 acetylated sites in 1301 proteins, and 1511 sites on 895 proteins were quantitatively informative. We found 25 conserved motifs from all of the identified lysine sites, particularly motifs Kac H, Kac S, and Kac Y were strikingly conserved, of which Kac Y, Kac H, Y Kac, Kac K, Kac *K, Kac R, and Kac F which have been observed in other species and are therefore highly conserved. We identified 58 sites that were significantly differently acetylated in P. f. martensii in response to allograft (|fold change|>1.2, P-value ≤ 0.05); 38 newly acetylated and 20 deacetylated. According to GO functional analysis, subcellar location, and KOG classIfication, these proteins were divided into four categories: cytoskeleton, response to stimulus, metabolism, and other. The differentially acetylated proteins (DAPs) enriched pathways include aminoacyl-tRNA biosynthesis, salmonella infection, and longevity regulating pathway-worm-Caenorhabditis elegans (nematode). Parallel reaction-monitoring (PRM) validation of the differential acetylation of 10 randomly selected differentially acetylated sites from the acetylome analysis. These results indicated that our acetylome analysis results were sufficiently reliable and reproducible. These results provide an essential resource for in-depth exploration of the stress responses and adaptation mechanisms associated with lysine acetylation in marine invertebrates and P. f. martensii.