p53 Ubiquitination Comediated by HUWE1 and TRAF6 Contributes to White Spot Syndrome Virus Infection in Crustacean.

p53, the guardian of the genome, is a short-lived protein that is tightly controlled at low levels by constant ubiquitination and proteasomal degradation in higher organisms. p53 stabilization and activation are early crucial events to cope with external stimuli in cells. However, the role of p53 ubiquitination and its relevant molecular mechanisms have not been addressed in invertebrates. In this study, our findings revealed that both HUWE1 (HECT, UBA, and WWE domain-containing E3 ubiquitin-protein ligase 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) could serve as E3 ubiquitin ligases for p53 in mud crabs (Scylla paramamosain). Moreover, the expression of HUWE1 and TRAF6 was significantly downregulated during white spot syndrome virus (WSSV) infection, and therefore the ubiquitination of p53 was interrupted, leading to the activation of apoptosis and reactive oxygen species (ROS) signals through p53 accumulation, which eventually suppressed viral invasion in the mud crabs. To the best of our knowledge, this is the first study to reveal the p53 ubiquitination simultaneously induced by two E3 ligases in arthropods, which provides a novel molecular mechanism of invertebrates for resistance to viral infection. IMPORTANCE p53, which is a well-known tumor suppressor that has been widely studied in higher animals, has been reported to be tightly controlled at low levels by ubiquitin-dependent proteasomal degradation. However, recent p53 ubiquitination-relevant research mainly involved an individual E3 ubiquitin ligase, but not whether there exist other mechanisms that need to be explored. The results of this study show that HUWE1 and TRAF6 could serve as p53 E3 ubiquitin ligases and synchronously mediate p53 ubiquitination in mud crabs (Scylla paramamosain), which confirmed the diversity of the p53 ubiquitination regulatory pathway. In addition, the effects of p53 ubiquitination are mainly focused on tumorigenesis, but a few are focused on the host immune defense in invertebrates. Our findings reveal that p53 ubiquitination could affect ROS and apoptosis signals to cope with WSSV infection in mud crabs, which is the first clarification of the immunologic functions and mechanisms of p53 ubiquitination in invertebrates.

KCMF1-like suppresses white spot syndrome virus infection by promoting apoptosis in mud crab (Scylla paramamosain).

Potassium channel modulatory factor 1 (KCMF1), an E3 ubiquitin ligase, plays a vital role in renal tubulogenesis, preeclampsia, and tumor development in mammals. Nevertheless, the function of KCMF1 in invertebrates remains to be investigated. Here, we identified KCMF1-like from Scylla paramamosian, encoding 242 amino acids with two zinc finger domains at the N-terminal. Real-time quantitative PCR analysis revealed that KCMF1-like was expressed in all tested tissues, including hemocytes, brain, mid-intestine, subcuticular epidermis, gills, muscle, heart, and stomach, with higher levels in muscle and mid-intestine. KCMF1-like was up-regulated in the hemocytes of mud crabs challenged with white spot syndrome virus (WSSV). RNA interference (RNAi) was performed to investigate the impact of KCMF1-like on the proliferation of WSSV in mud crabs. Knock-down of KCMF1-like resulted in an increase of the WSSV copy number and an impairment of the hemocytes apoptosis rate in vivo. In addition, KCMF1-like could also affect the mitochondrial membrane potential. Collectively, these results revealed that KCMF1-like might play a crucial role in the defense against virus infection in mud crab. This study contributes a novel insight into the role of KCMF1-like in the antiviral immune defense mechanism in crustaceans.

Exosome-mediated apoptosis pathway during WSSV infection in crustacean mud crab.

MicroRNAs are regulatory molecules that can be packaged into exosomes to modulate cellular response of recipients. While the role of exosomes during viral infection is beginning to be appreciated, the involvement of exosomal miRNAs in immunoregulation in invertebrates has not been addressed. Here, we observed that exosomes released from WSSV-injected mud crabs could suppress viral replication by inducing apoptosis of hemocytes. Besides, miR-137 and miR-7847 were found to be less packaged in mud crab exosomes during viral infection, with both miR-137 and miR-7847 shown to negatively regulate apoptosis by targeting the apoptosis-inducing factor (AIF). Our data also revealed that AIF translocated to the nucleus to induce DNA fragmentation, and could competitively bind to HSP70 to disintegrate the HSP70-Bax (Bcl-2-associated X protein) complex, thereby activating the mitochondria apoptosis pathway by freeing Bax. The present finding therefore provides a novel mechanism that underlies the crosstalk between exosomal miRNAs and apoptosis pathway in innate immune response in invertebrates.

Transcriptome and metabolome integration analysis of mud crab Scylla paramamosain challenged to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common pathogen for marine crustacean, which causes severe illnesses in aquatic animals. Therefore, it is meaningful to explore the mechanism during V. parahaemolyticus infection. In this study, to investigate the immune responses of mud crab Scylla paramamosain (S. paramamosain) to V. parahaemolyticus, we established the metabolic and transcriptional profiles of mud crab hemocytes challenged with V. parahaemolyticus. The results indicated that V. parahaemolyticus infection could induce a series of metabolism alterations at both metabolome and transcriptome levels, including biosynthesis of amino acids and Aminoacyl-tRNA, Purine and pyrimidine metabolism, TCA cycle and glutamine metabolism. In this context, through the integration of metabolomics and transcriptomics, our study provided a more comprehensive understanding of the biological process in mud crab against pathogen infection.

The miRNAs profiling revealed by high-throughput sequencing upon WSSV infection in mud crab Scylla paramamosain.

microRNAs (miRNAs) are known to regulate various immune functions by silencing the target genes in both vertebrates and invertebrates. However, in mud crab Scylla paramamosain, the role of miRNAs during the response to virus invasion remains unclear. To investigate the roles of miRNAs in S. paramamosain during virus infection, the mud crab was challenged with white spot syndrome virus (WSSV) and then subjected to the transcriptional analysis at different conditions. The results of high-throughput sequencing revealed that 940,379 and 1,306,023 high-quality mappable reads were detected in the hemocyte of normal and WSSV-infected mud crabs, respectively. Besides, the total number of 261 unique miRNAs were identified. Among them, 131 miRNAs were specifically expressed in the hemocytes of normal mud crabs, 46 miRNAs were specifically transcribed in those of WSSV-infected individuals, the other 84 miRNAs were expressed in both normal and WSSV-infected individuals. Furthermore, a number of 152 (89 down-regulated and 63 up-regulated) miRNAs were found to be differentially expressed in the WSSV-infected hemocytes, normalized to the controls. The identified miRNAs were subjected to GO analysis and target gene prediction and the results suggested that the differentially regulated miRNAs were mainly correlated with the changes of the immune responses of the hemocytes, including phagocytosis, melanism, and apoptosis as well. Taken together, the results demonstrated that the expressed miRNAs during the virus infection were mainly involved in the regulation of immunological pathways in mud crabs. Our findings not only enrich the understanding of the functions of miRNAs in the innate immune system but also provide some novel potential targets for the prevention of WSSV infection in crustaceans.

SpTGase plays an important role in the hemolymph clotting in mud crab (Scylla paramamosain).

Transglutaminase (TGase) is important in blood coagulation, a conserved immunological defense mechanism among invertebrates. This study is the first report of the TGase in mud crab (Scylla paramamosain) (SpTGase) with a 2304 bp ORF encoding 767 amino acids (molecular weight 85.88 kDa). SpTGase is acidic, hydrophilic, stable and thermostable, containing three transglutaminase domains, one TGase/protease-like homolog domain (TGc), one integrin-binding motif (Arg270, Gly271, Asp272) and three catalytic sites (Cys333, His401, Asp424) within the TGc. Neither a signal peptide nor a transmembrane domain was found, and the random coil is dominant in the secondary structure of SpTGase. Phylogenetic analysis revealed a close relation between SpTGase to its homolog EsTGase 1 from Chinese mitten crab (Eriocheir sinensis). Expression of SpTGase was investigated using qRT-PCR (1) in eight tissues from healthy mud crabs, with the highest expression in hemocytes, and (2) in response to various immune challenges (Vibrio parahaemolyticus, lipopolysaccharide (LPS) or Poly I:C infection), revealing a major up-regulation in hemocytes, skin, and hepatopancreas during the 96-h post injection. The recombinant SpTGase showed a capacity of agglutination activities on both Gram-negative bacteria and yeast. SpTGase was found to directly interact with another important blood coagulation component clip domain serine protease (SpcSP). Moreover, knockdown of SpTGase resulted in a decreased expression of both clotting protein precursor (SppreCP) and SpcSP and an increase of duration time in the blood coagulation. Taken together, the findings of this study suggest SpTGase play an important role in the hemolymph clotting in mud crab S. paramamosain.

A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain.

The clip domain serine proteinases (clip-SPs) play vital roles in embryonic development and in various innate immune functions in invertebrates such as antimicrobial activity, cell adhesion, hemolymph clotting, pattern recognition and regulation of the prophenoloxidase system. However, little is known about the role of the clip domain serine proteinase in Scylla paramamosain (designated SpcSP) immunity. In the present study, we cloned a clip-SP from S. paramamosain hemocytes using rapid amplification of cDNA end (RACE) approach. The full-length cDNA of SpcSP was 1823 bp, containing a 5' untranslated region (UTR) of 334 bp, an open reading frame of 1122 bp, and a 3' UTR of 367 bp. The open reading frame encoded a polypeptide of 373 amino acids with a calculated molecular weight of 39.7 kDa and an isoelectric point of 6.64. Structurally, SpcSP has a predicted 21-residue signal peptide and possessed the characteristic features of the clip domain family of serine proteases, namely one clip domain in the amino-terminal with six highly conserved cysteine residues and one enzyme active serine proteinase domain in the carboxyl-terminal with a highly conserved catalytic triad (His156, Asp226, Ser321). Phylogenetic analysis showed that SpcSP was clustered together with PtcSP (clip domain serine proteinase from Portunus trituberculatus). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of SpcSP was constitutively expressed at different levels in all tested tissues in untreated S. paramamosain, with hemocytes and skin expressing the most. The transcriptional level of SpcSP in hemocytes was significantly up-regulated upon challenge with V. parahaemolyticus and LPS, indicating its involvement in antibacterial immune response. Indirect immunofluorescence analysis showed that SpcSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant SpcSP protein exhibited strong binding ability and has antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. Moreover, knockdown of SpcSP resulted in increased hemolymph clotting time and decreased the mRNA expression of SpproPO mRNA in hemocytes. These findings therefore suggest that SpcSP plays an important role in the antimicrobial defense mechanism of S. paramamosain by regulating the expression of SpproPO and hemolymph clotting in S. paramamosain.

Scavenger receptor B promotes bacteria clearance by enhancing phagocytosis and attenuates white spot syndrome virus proliferation in Scylla paramamosian.

Phagocytosis and apoptosis are key cellular innate immune responses against bacteria and virus in invertebrates. Class B scavenger receptors (SRBs), which contain a CD36 domain, are critical pattern recognition receptors (PRRs) of phagocytosis for bacteria and apoptotic cells. In the present study, we identified a member of SRB subfamily in mud crab Scylla paramamosain, named Sp-SRB. The full-length cDNA of Sp-SRB is 2593 bp with a 1629 bp open reading frame (ORF) encoding a putative protein of 542 amino acids, and predicted to contain a CD36 domain with two transmembrane regions at the C- and N-terminals. Real-time qPCR analysis revealed that Sp-SRB was widely expressed in all tissues tested, and the expression of Sp-SRB was up-regulated upon challenge with Vibrio parahaemolyticus, white spot syndrome virus (WSSV), lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (PolyI:C). Moreover, in vitro experiments indicated that recombinant Sp-SRB protein (rSp-SRB) could bind to fungi, Gram-positive and Gram-negative bacteria. RNA interference of Sp-SRB resulted in significant reduction in the expression level of phagocytosis related genes, antimicrobial peptides (AMPs) and Toll-like receptors (TLRs), which consequently led to impairment in both bacterial clearance and the phagocytotic activity of hemocytes. In addition, we found that Sp-SRB had the ability to attenuate the replication of WSSV proliferation in mud crab S. paramamosain. Collectively, this study has shown that Sp-SRB contributed to bacteria clearance by enhancing phagocytosis and up-regulating the expression of AMPs possibly in a TRLs (SpToll 1 and SpToll 2)-dependent manner. Besides, Sp-SRB inhibited the replication of WSSV in S. paramamosian probably through enhancement of hemocytes phagocytosis of apoptotic cells.

Changes in Hemolymph Microbiota of Chinese Mitten Crab (Eriocheir sinensis) in Response to Aeromonas hydrophila or Staphylococcus aureus Infection.

The Chinese mitten crab (Eriocheir sinensis) has significant economic potential in both the Chinese domestic and global markets. The hemolymph microbiota is known to play a critical role in regulating physiological and biochemical functions in crustaceans. However, the study of the hemolymph microbiota of E. sinensis in response to infections has not been undertaken. In this study, changes in the composition and function of the hemolymph microbiota in E. sinensis infected with either Staphylococcus aureus (Sa) or Aeromonas hydrophila (Ah) were investigated using 16S rRNA sequencing, with a phosphate buffer saline (PBS) injection serving as the control. Results showed that the dominant hemolymph microbiota of E. sinensis were Proteobacteria, Bacteroidota, and Firmicutes. The relative abundance of the phyla Firmicutes, Bdellovibrionota, and Myxococcota was significantly reduced in both Sa and Ah groups compared to the PBS group. At the genus level, compared to the PBS group, a significant increase in the abundance of Flavobacterium and Aeromonas was found in both Ah and Sa groups. The analysis of the functional profile showed that pathways related to 'cell growth and death', 'metabolism of terpenoids and polyketides', 'cancers', 'lipid metabolism', 'neurodegenerative diseases', 'metabolism of other amino acids', 'xenobiotics biodegradation and metabolism', and 'circulatory system and endocrine system' were predominant in the Ah group. Meanwhile, pathways related to 'metabolism or genetic information progressing', such as 'translation', 'metabolic diseases', and 'cellular processes and signaling', were enriched in the Sa group. This study revealed the effects of pathogens (S. aureus or A. hydrophila) on the maintenance of the hemolymph microbiota in E. sinensis. It shed light on the mechanisms employed by the hemolymph microbiota of E. sinensis under pathogen stimulation.

Pattern recognition receptors and their roles on the innate immune system of mud crab (Scylla paramamosain).

The innate immune system is the first line of defense protecting the hosts against invading pathogens. Mud crab (Scylla paramamosain) is widely distributed in China and Indo-west Pacific countries, which develops a very complicated innate immune system against pathogen invasions. Innate immunity involves the humoral and cellular responses that are linked to the pattern recognition receptors (PRRs). PRRs initially recognize the infection and trigger the activation of signaling cascades, leading to transcriptional regulation of inflammatory mediators that function in pathogenic control and clearance. In mud crab S. paramamosain, the Toll/Toll-like receptors, lipopolysaccharide and ß-1,3-glucan binding proteins, C-type lectins, scavenger receptors, and down syndrome cell adhesion molecules have been identified as receptor families responsible for the recognition of bacteria, fungi, and viruses, and are important components in the innate immune system. In this review, we summarize the literature on the current knowledge and the roles of PRRs in the immune defenses of mud crab, which in an effort to provide much information for further researches.

Elucidation of metabolic responses in mud crab Scylla paramamosain challenged to WSSV infection by integration of metabolomics and transcriptomics.

White spot syndrome virus (WSSV) is a severe pathogen of mud crab Scylla paramamosain (S. paramamosain). Hemolymph, containing three types of hemocytes, is the key immunoregulatory tool of mud crab in response to pathogens. Herein, the metabonomics and transcriptomics analysis of hemocytes were adopted to investigate the immune response of S. paramamosain challenged to WSSV. We established the metabolic and transcriptional profiles of mud crab hemocytes with different treatments, including the control group (WT), WSSV early infected group (WSSV-6 h) and WSSV later infected group (WSSV-72 h). The results showed that 68 metabolites were dysregulated both in WSSV-infected mud crab of early stage and later stage, while 4452 genes were up-regulated and 9746 genes were down-regulated in WSSV-6 h, and 2016 genes were up-regulated and 6229 genes were down-regulated compared in WSSV-72 h. We found that several pathways were dysregulated at both metabolic and transcriptional levels, including ABC transporters, purine metabolism, taurine and hypotaurine metabolism in the WSSV early infected group, cysteine metabolism, methionine metabolism and biosynthesis of unsaturated fatty acids in the WSSV later infected group. In this context, through the integration of metabolomics and transcriptomics, our study provided a more comprehensive understanding of the biological process in mud crab against viral invasion.

Elucidation of Gut Microbiota in Mud Crab Scylla paramamosain Challenged to WSSV and Aeromonas hydrophila.

Mud crab Scylla paramamosain (S. paramamosain) is an economically important marine crab species around the world. White spot syndrome virus (WSSV) and Aeromonas hydrophila (AH) are pathogens during mud crab mariculture. It has been reported that gut microbiota possessed a great impact on the host development, nutrition, immunity, and disease resistance. However, little information was known about the impacts of WSSV or AH infection on the structure, composition, and function of the gut microbiotain of mud crabs. In this study, the gut microbiota of mud crabs infected with A. hydrophila and WSSV were characterized. The results showed that the composition and bacteria correlation of the gut microbiota were significantly decreased. During A. hydrophila infection, the pathogens played a major regulatory role in host. While in the mud crabs infected with WSSV, many beneficial strains had a great impact on the host expect for the pathogens. Therefore, our study revealed the effect of pathogens infection on gut microbiota of mud crabs and clarified the difference between viral infection and bacterial infection.

miR-9875 functions in antiviral immunity by targeting PDCD6 in mud crab (Scylla paramamosain).

Programmed cell death 6 (PDCD6) is a well-known apoptosis regulator that is involved in the immunity of mammals. However, the effects of miRNA-mediated regulation of PDCD6 expression on apoptosis and virus infection in organisms, especially in marine invertebrates, have not been extensively explored. In this study, PDCD6 of mud crab (Scylla paramamosain) (Sp-PDCD6) was characterized. The results showed that Sp-PDCD6 contains five EF-hands domains and could suppress virus infection via apoptosis promotion. It also presented that Sp-PDCD6 was directly targeted by miR-9875 in vitro and in vivo, miR-9875 served as a positive regulator during the virus invasion. The findings indicated that the miR-9875-PDCD6 pathway possessed fundamental effects on the immune response to virus infection in mud crab. Therefore, our research provided a novel insight into the roles of both miR-9875 and PDCD6 in the regulation of apoptosis and virus defense in mud crab.

Sp-CBL inhibits white spot syndrome virus replication by enhancing apoptosis in mud crab (Scylla paramamosain).

In mammals, casitas B-lineage lymphoma (CBL) family proteins, a RING-type E3 ubiquitin ligase, are involved in many signal transduction pathways. However, the functions of CBL in invertebrates are not well elucidated. In this study, Sp-CBL containing CBL-N, CBL-2, CBL-3 and RING domains was identified in mud crab Scylla paramamosain. Sp-CBL was widely expressed in all tissues tested and found to be significantly up-regulated in the hemocytes of mud crab challenged by white spot syndrome virus (WSSV). The RNA interference of Sp-CBL increased the copy number of WSSV and declined the apoptosis rate of hemocytes. In addition, Sp-CBL could affect the activities of caspase 3 and the mitochondrial membrane potential. Taken together, the results of this study revealed that Sp-CBL could restrict WSSV proliferation through enhancing the apoptosis of the hemocytes, which would provide a novel insight into the anti-viral response in the innate immunity system of mud crab.

Biodegradation of Microplastic Derived from Poly(ethylene terephthalate) with Bacterial Whole-Cell Biocatalysts.

At present, the pollution of microplastic directly threatens ecology, food safety and even human health. Polyethylene terephthalate (PET) is one of the most common of microplastics. In this study, the micro-size PET particles were employed as analog of microplastic. The engineered strain, which can growth with PET as sole carbon source, was used as biocatalyst for biodegradation of PET particles. A combinatorial processing based on whole-cell biocatalysts was constructed for biodegradation of PET. Compared with enzymes, the products can be used by strain growth and do not accumulated in culture solution. Thus, feedback inhibition of products can be avoided. When PET was treated with the alkaline strain under high pH conditions, the product concentration was higher and the size of PET particles decreased dramatically than that of the biocatalyst under neutral conditions. This shows that the method of combined processing of alkali and organisms is more efficient for biodegradation of PET. The novel approach of combinatorial processing of PET based on whole-cell biocatalysis provides an attractive avenue for the biodegradation of micplastics.