The nuclear receptor coactivator 4 regulates ferritinophagy induced by vibrio splendidus in coelomocytes of Apostichopus japonicus.

Iron homeostasis is vital for the host's defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.

Novel ApeC-containing protein mediates the recognition and internalization of Vibrio splendidus in Apostichopus japonicus.

Pattern recognition receptors (PRRs) mediate the innate immune responses and play a crucial role in host defense against pathogen infections. Apextrin C-terminal (ApeC)-containing proteins (ACPs), a newly discovered class of PRRs specific to invertebrates, recognize pathogens through their ApeC domain as intracellular or extracellular effectors. However, the other immunological functions of ACPs remain unclear. In this study, a membrane-localized ACP receptor was identified in the sea cucumber Apostichopus japonicus (denoted as AjACP1). The ApeC domain of AjACP1, which was located outside of its cell membrane, exhibited the capability to recognize and aggregate Vibrio splendidus. AjACP1 was upregulated upon V. splendidus infection, internalizing into the cytoplasm of coelomocytes. AjACP1 overexpression enhanced the phagocytic activity of coelomocytes against V. splendidus, while knockdown of AjACP1 by RNA interfere inhibited coelomocyte endocytosis. Inhibitor experiments indicated that AjACP1 regulated coelomocyte phagocytosis through the actin-dependent endocytic signaling pathway. Further investigation revealed that AjACP1 interacted with the subunit of the actin-related protein 2/3 complex ARPC2, promoting F-actin polymerization and cytoskeletal rearrangement and thereby affecting the coelomocyte phagocytosis of V. splendidus via the actin-dependent endocytic signaling pathway. As a novel membrane PRR, AjACP1 mediates the recognition and phagocytic activity of coelomocytes against V. splendidus through the AjACP1-ARPC2-F-actin polymerization and cytoskeletal rearrangement pathway.

Calreticulin from Apostichopus japonicus relieves endoplasmic reticulum stress induced by Vibrio splendidus through autophagy.

When organisms are exposed to external stimuli, misfolded proteins accumulate continuously, resulting in endoplasmic reticulum (ER) stress. Autophagy is of great significance for eliminating aggregated proteins and maintaining cellular homeostasis. However, the molecular mechanism of activating autophagy in response to ER stress in sea cucumber is remain unclear. In the current study, we demonstrated that the pathogen Vibrio splendidus can cause ER stress in Apostichopus japonicus coelomocytes and identified a Ca2+ binding partner calreticulin (designated as AjCRT), which increased with the occurrence of ER stress. The nucleotide sequence analysis showed that the open reading frame of AjCRT was 1242 bp and encoded a 413-amino-acid residue polyprotein with calreticulin domains. The spatial expression analysis revealed that AjCRT was ubiquitously expressed in all examined tissues with large magnitude in the coelomocytes and was minimally expressed in muscle. Furthermore, silencing AjCRT in vivo could significantly exacerbate ER stress induced by V. splendidus and resulted in the significant reduction of coelomocyte autophagy. These findings indicate a calreticulin-based mechanism that positively regulates autophagy in response to ER stress induced by pathogen infection. The results will provide a basis for understanding the way of host alleviating ER stress through autophagy, and pharmacological approaches may have potential for managing ER stress induced by pathogen and related cellular disorders.

Bacillus coagulans restores pathogen-induced intestinal dysfunction via acetate-FFAR2-NF-κB-MLCK-MLC axis in Apostichopus japonicus.

Skin ulceration syndrome (SUS) is currently the main disease threatening Apostichopus japonicus aquaculture due to its higher mortality rate and infectivity, which is caused by Vibrio splendidus. Our previous studies have demonstrated that SUS is accompanied by intestinal microbiota (IM) dysbiosis, alteration of short-chain fatty acids (SCFAs) content and the damage to the intestinal barrier. However, the mediating effect of IM on intestine dysfunction is largely unknown. Herein, we conducted comprehensive intestinal microbiota transplantation (IMT) to explore the link between IM and SUS development. Furthermore, we isolated and identified a Bacillus coagulans strain with an ability to produce acetic acid from both healthy individual and SUS individual with IM from healthy donors. We found that dysbiotic IM and intestinal barrier function in SUS recipients A. japonicus could be restored by IM from healthy donors. The B. coagulans strain could restore IM community and intestinal barrier function. Consistently, acetate supply also restores intestinal homeostasis of SUS-diseased and V. splendidus-infected A. japonicus. Mechanically, acetate was found to specifically bind to its receptor-free fatty acid receptor 2 (FFAR2) to mediate IM structure community and intestinal barrier function. Knockdown of FFAR2 by transfection of specific FFAR2 siRNA could hamper acetate-mediated intestinal homeostasis in vivo. Furthermore, we confirmed that acetate/FFAR2 could inhibit V. splendidus-activated NF-κB-MLCK-MLC signaling pathway to restore intestinal epithelium integrity and upregulated the expression of ZO-1 and Occludin. Our findings provide the first evidence that B. coagulans restores pathogen-induced intestinal barrier dysfunction via acetate/FFAR2-NF-κB-MLCK-MLC axis, which provides new insights into the control and prevention of SUS outbreak from an ecological perspective.IMPORTANCESkin ulceration syndrome (SUS) as a main disease in Apostichopus japonicus aquaculture has severely restricted the developmental A. japonicus aquaculture industry. Intestinal microbiota (IM) has been studied extensively due to its immunomodulatory properties. Short-chain fatty acids (SCFAs) as an essential signal molecule for microbial regulation of host health also have attracted wide attention. Therefore, it is beneficial to explore the link between IM and SUS for prevention and control of SUS. In the study, the contribution of IM to SUS development has been examined. Additionally, our research further validated the restoration of SCFAs on intestinal barrier dysfunction caused by SUS via isolating SCFAs-producing bacteria. Notably, this restoration might be achieved by inhibition of NF-κB-MLCK-MLC signal pathway, which could be activated by V. splendidus. These findings may have important implications for exploration of the role of IM in SUS occurrence and provide insight into the SUS treatment.

Vibrio splendidus Fur regulates virulence gene expression, swarming motility, and biofilm formation, affecting its pathogenicity in Apostichopus japonicus.

Vibrio splendidus is an opportunistic pathogen that causes skin ulcer syndrome and results in huge losses to the Apostichopus japonicus breeding industry. Ferric uptake regulator (Fur) is a global transcription factor that affects varieties of virulence-related functions in pathogenic bacteria. However, the role of the V. splendidus fur (Vsfur) gene in the pathogenesis of V. splendidus remains unclear. Hence, we constructed a Vsfur knock-down mutant of the V. splendidus strain (MTVs) to investigate the role of the gene in the effect of biofilm, swarming motility, and virulence on A. japonicus. The result showed that the growth curves of the wild-type V. splendidus strain (WTVs) and MTVs were almost consistent. Compared with WTVs, the significant increases in the transcription of the virulence-related gene Vshppd mRNA were 3.54- and 7.33-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. Similarly, compared with WTVs, the significant increases in the transcription of Vsm mRNA were 2.10- and 15.92-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. On the contrary, the mRNA level of the flagellum assembly gene Vsflic was downregulated 0.56-fold in MTVs at the OD600 of 1.0 compared with the WTVs. MTVs caused delayed disease onset time and reduced A. japonicus mortality. The median lethal doses of WTVs and MTVs were 9.116 × 106 and 1.658 × 1011 CFU·ml-1, respectively. Compared with WTVs, the colonization abilities of MTVs to the muscle, intestine, tentacle, and coelomic fluid of A. japonicus were significantly reduced. Correspondingly, the swarming motility and biofilm formation in normal and iron-replete conditions were remarkably decreased compared with those of WTVs. Overall, these results demonstrate that Vsfur contributes to the pathogenesis of V. splendidus by regulating virulence-related gene expression and affecting its swarming and biofilm formation abilities.

CHOP promotes coelomocyte apoptosis through p38-MAPK pathway in Vibrio splendidus-challenged sea cucumber Apostichopus japonicus.

CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) belongs to the C/EBP family of transcription factors that has been proven to regulate apoptosis in many vertebrate species. However, the functional role of CHOP in invertebrates is largely unknown. In this paper, the open reading frame of CHOP was cloned and characterized in the sea cucumber Apostichopus japonicus (AjCHOP). The deuced amino acid of AjCHOP shared a conserved RTP801_C domain from 63 to 171 aa. Phylogenetic analysis indicated that AjCHOP clustered with CHOPs from Lytechinus variegatus and Strongylocentrotus purpuratus. To confirm the immune function of AjCHOP, the time-course expression profiles of AjCHOP were investigated, and the findings revealed AjCHOP was significantly induced in coelomocytes at mRNA and protein levels after Vibro splendidus challenge. Furthermore, knockdown of AjCHOP in coelomocyes by siRNA transfection significantly decreased the apoptosis level induced by V. splendidus. Mechanically, AjCHOP-mediated apoptosis was dependent on the activation of p38-MAPK pathway but not JNK/ERK-MAPK. Overall, our results supported that V. splendidus triggers apoptosis among the coelomocytes, whereas AjCHOP mediates through the p38-MAPK pathway in A. japonicus.

METTL3 activates PERK-eIF2α dependent coelomocyte apoptosis by targeting the endoplasmic reticulum degradation-related protein SEL1L in echinoderms.

N6-methyladenosine (m6A) plays an important role in regulating many physiological and disease processes in vertebrates, in which methyltransferase-like 3 (METTL3) is the best-known m6A methyltransferase. However, the functional roles of invertebrate METTL3 have not yet been highlighted. In this study, we found that METTL3 from Apostichopus japonicus (AjMETTL3) was significantly induced in coelomocytes accompanied by higher levels of m6A modification in response to Vibrio splendidus challenge. Overexpression or silencing of AjMETTL3 in coelomocytes increased or decreased the m6A levels and promoted or inhibited V. splendidus-induced coelomocyte apoptosis, respectively. To further explore the molecular mechanism of AjMETTL3-mediated coelomic immunity, m6A-seq analysis revealed that the endoplasmic reticulum-related degradation (ERAD) pathway was significantly enriched, in which suppressor/enhancer of Lin-12-like (AjSEL1L) was suggested to be a target of AjMETTL3 in a negative regulatory manner. Functional analysis revealed that the increased AjMETTL3 reduced the stability of AjSEL1L mRNA by targeting the m6A modification site of 2004 bp-GGACA-2008 bp. The decreased AjSEL1L was further confirmed to be involved in AjMETTL3-mediated coelomocyte apoptosis. Mechanistically, the inhibited AjSEL1L increased the transcription of AjOS9 and Ajp97 in the EARD pathway to promote ubiquitin protein accumulation and ER stress, which further activated AjPERK-AjeIF2α pathway dependent coelomocyte apoptosis, but not the AjIRE1 or AjATF6 pathway. Taken together, our results supported invertebrate METTL3-mediated coelomocyte apoptosis by regulating the PERK-eIF2α pathway.

Exogenous adenosine and/or guanosine enhances tetracycline sensitivity of persister cells.

Vibrio splendidus is an opportunistic pathogen, its pathogenicity continues to be a major aquaculture disease infection problem in many parts of the world. Bacteria can form dormant and persister cells, which may be responsible for the difficulty in treating latent infections. Bacterial persister cells are a small subpopulation with high phenotypic heterogeneity that have the ability to persist in response to high concentrations of antibiotics. In our previous work, we have confirmed tetracycline could induce V. splendidus AJ01 persister cells formation. Here, we show that exogenous adenosine and/or guanosine supply restores susceptibility of AJ01 persister cells to tetracycline, leading to effective killing of this persist subpopulation upon wake-up. Mechanistically, exogenous adenosine and/or guanosine promotes the intracellular ATP level, reduces percentage of cells with protein aggresomes, and destroys membrane stability. In addition, when cells were exposed to tetracycline, we found that cells with small nucleocytoplasmic ratio is easy to survive. Overall, our results support that exogenous adenosine or guanosine could be an effective strategy for treating infections with antibiotic-persist bacteria via regulating persisters cells formation.

Molecular Mechanisms Underlying Metabolic Resistance to Cyflumetofen and Bifenthrin in Tetranychus urticae Koch on Cowpea.

Tetranychus urticae Koch (T. urticae) is one of the most tremendous herbivores due to its polyphagous characteristics, and is resistant to most acaricides. In this study, enzyme-linked immunosorbent assay (ELISA), transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) were carried out to analyze the mechanisms of T. urticae metabolic resistance to cyflumetofen and bifenthrin on cowpea. The enzyme activity of UDP-glucuronosyltransferases (UGTs) and carboxylesterases (CarEs) in the cyflumetofen-resistant (R_cfm) strain significantly decreased, while that of cytochrome P450 monooxygenases (P450s) significantly increased. Meanwhile, the activities of glutathione-S-transferases (GSTs), CarEs and P450s in the bifenthrin-resistant (R_bft) strain were significantly higher than those in the susceptible strain (Lab_SS). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses, in the R_cfm mite strain, two carboxyl/cholinesterase (CCE) genes and two P450 genes were upregulated and one gene was downregulated, namely CYP392E7; in the R_bft mite strain, eleven CCE, nine UGT, two P450, four GST and three ABC genes were upregulated, while four CCE and three P450 genes were downregulated. Additionally, 94 differentially expressed genes (DEGs) were common to the two resistant groups. Specifically, TuCCE46 and TuCCE70 were upregulated in both resistant groups. Furthermore, the qRT-PCR validation data were consistent with those from the transcriptome sequencing analysis. Specifically, TuCCE46 (3.37-fold) was significantly upregulated in the R_cfm strain, while in the R_bft strain, TeturUGT22 (5.29-fold), teturUGT58p (1.74-fold), CYP392A11 (2.89-fold) and TuGSTd15 (5.12-fold) were significantly upregulated and TuCCE01 (0.13-fold) and CYP392A2p (0.07-fold) were significantly downregulated. Our study indicates that TuCCE46 might play the most important role in resistance to cyflumetofen, and TuCCE01, teturUGT58p, teturUGT22, CYP392A11, TuGSTd15, TuGSTm09 and TuABCG-13 were prominent in the resistance to bifenthrin. These findings provide further insight into the critical genes involved in the metabolic resistance of T. urticae to cyflumetofen and bifenthrin.

Genome-wide identification m6A modified circRNAs revealed their key roles in skin ulceration syndrome disease development in Apostichopus japonicus.

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs (ncRNAs) and can be acted as competing endogenous RNAs (ceRNAs) to regulate microRNA (miRNA) and downstream gene expression. Recently, m6A modification has been found in circRNA, and m6A circRNAs also play important roles in various biological processes and a variety of diseases. Our previous study had been demonstrated that circRNAs were differentially expressed in skin ulceration syndrome (SUS) diseased sea cucumber Apostichopus japonicus. However, whether the function of circRNAs are dependent on m6A levels are largely unknown. Here, we firstly investigated the genome-wide map of m6A circRNAs in sea cucumbers with different stages of Vibrio splendidus challenge, that's Control group, SUS-diseased group, and SUS-resistant group. MeRIP-seq revealed that m6A abundances were enriched in circRNAs in all three groups, especially for SUS-resistant group. Among them, more than 62% of modified circRNAs harbor only a single m6A peak and about 55% of m6A sites in circRNAs were derived from sense overlapping in each group. After V. splendidus infection, we found that most of m6A peaks in circRNAs were upregulated and less were downregulated in both SUS-diseased and SUS-resistant groups when compared with Control. Furthermore, GO analysis indicated that the host genes of circRNAs with dysregulated m6A peaks in SUS-diseased and SUS-resistant groups were both mainly enriched in the adhesion pathway. More importantly, we discovered that more than 50% m6A circRNAs showed a positive correlation between the circRNAs expression and m6A methylation levels both in SUS-diseased and SUS-resistant groups. Therefore, a core circRNA-miRNA-mRNA (ceRNA) network whether influenced by m6A modification was constructed based on conjoint analysis. Our results indicated that several selected m6A circRNAs bind with miRNAs were mainly targeting to ubiquitylation system and adhesion pathway. What's more, three candidate m6A circRNAs and three target genes were validated by MeRIP-qPCR and qPCR, whose m6A levels in circRNA and mRNA expressions were consistent with disease occurrence or disease resistance. All of our current findings suggested that m6A circRNAs could play important roles during pathogen infection and might be served as a new molecular biomarker in SUS disease diagnose of A. japonicus.

Vibrio splendidus virulence to Apostichopus japonicus is mediated by hppD through glutamate metabolism and flagellum assembly.

Vibrio splendidus is the main opportunistic pathogen that causes skin ulcer syndrome in Apostichopus japonicus. hppDIn the present study, mutant V. splendidus with an in-frame deletion of hppDV.s. (MTVs) was constructed. The median lethal doses of wild-type V. splendidus (WTVs) and MTVs were 5.129 × 106 and 2.606 × 1010 CFU mL-1, respectively. RNA-Seq was performed using WTVs and MTVs cells at different growth stages to explore the mechanisms of the pathogenesis mediated by hppDV.s. Gene Ontology analysis showed that the expression levels of 105 genes involved in amino acid metabolism and protein binding were remarkably different between MTVs and WTVs. Kyoto Encyclopedia of Genes and Genomes analysis showed that the pathways of glutamate metabolism and flagellum assembly involved in biofilm formation and swarming motility were suppressed in MTVs. Correspondingly, the swarming motility, biofilm formation and colonisation of MTVs were remarkably decreased compared with those of WTVs. The results showed that 4-hppD catalyses tyrosine into fumarate, which could enhance glutamate metabolism and ATP production; promote flagellum assembly through the TCA cycle and lead to higher swarming, biofilm formation and colonisation abilities, to contribute to the pathogenesis of V. splendidus.

4-Hydroxyphenylpyruvate dioxygenase from sea cucumber Apostichopus japonicus negatively regulates reactive oxygen species production.

As a wide distribution molecule, 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) catalyzes the second step in the tyrosine catabolism pathway. This process commonly occurs in all aerobic life forms. The broad distribution of these metabolites suggests that they have an important role in many organisms. A portion of the 4-HPPD homology sequence was also identified in Apostichopus japonicus transcriptome. However, the functional roles of A. japonicus 4-HPPD remain unclear. In the current study, a 4-HPPD homolog was cloned from A. japonicus (designated as AjHPPD). The nucleotide sequence analysis showed that the open reading frame of AjHPPD was 1149 bp and encoded a 382-amino-acid residue polyprotein with glyoxalase_4 (residues 20-133) and glyoxalase (residues 180-335) domains. The spatial expression analysis revealed that AjHPPD was ubiquitously expressed in all examined tissues with large-magnitude in the respiratory tree and was minimally expressed in coelomocytes. Compared with a control group, the significant increase in transcription of AjHPPD mRNA in the Vibrio splendidus-challenged sea cucumber was 2.10-fold (p < 0.01) at 48 h and returned to the normal level at 72 and 96 h. Similarly, compared with a control group, the significant increase in the transcription of AjHPPD mRNA was 3.36-fold (p < 0.01) at 24 h after stimulation with 10 mg mL-1 of LPS. On the one hand, silencing AjHPPD in vitro could inhibit the expression of pentose phosphate pathway (PPP) flux enzyme glucose-6-phosphate dehydrogenase (G6PD) at the mRNA level and prevent the clearance of reactive oxygen species (ROS) in sea cucumbers. On the other hand, interference of AjHPPD by using specific siRNA can result in the significant promotion of coelomocyte apoptosis with a 1.61-fold increase in vitro. AjHPPD negatively regulated ROS levels by modulating tyrosine catabolism on AjG6PD expression and coelomocyte apoptosis in response to pathogen infection.

A sigma factor RpoD negatively regulates temperature-dependent metalloprotease expression in a pathogenic Vibrio splendidus.

Vibrio splendidus is an important aquatic pathogen that can cause typical symptoms of skin ulceration syndrome (SUS) in sea cucumber Apostichopus japonicus. A metalloprotease Vsm from V. splendidus, has been reported to be an important virulence factor for SUS outbreak. In the present study, the mRNA expression level of vsm was found to correlate to temperature with a peak expression at 28 °C. In contrast, the expression of a sigma factor rpoD, was significantly repressed at 28 °C. A predicted RpoD binding site in the promoter region of vsm revealed the potential regulation of RpoD on vsm expression. Electrophoretic mobility shift assay showed that the purified recombinant RpoD could specifically bind to the promoter region of vsm. Co-transfection of vsm promotor and pT3-rpoD into E. coli significantly inhibited the ß-Galactosidase activities in a temperature-dependent manner, and the activities were 0.41-, 0.89- and 0.18-fold at 10 °C, 28 °C and 37 °C compared to the control DH5α/pT3. A rpoD overexpression strain Vs/JRTcrpoD was constructed to further examine the effect of RpoD on the expression of vsm in vivo. By real time RT-PCR analysis, vsm expression level was 0.47-fold in Vs/JRTcrpoD compared to that in Vs/JRTc. Consistently, the metalloprotease activities in Vs/JRTcrpoD was decreased by 18% compared to that in Vs/JRTc. All the results suggested that the sigma factor RpoD, showed a negative regulation on expression of vsm gene by directly interacting with the promoter region of vsm.

Dual functions of a 4-hydroxyphenylpyruvate dioxygenase for Vibrio splendidus survival and infection.

Vibrio splendidus is a well-documented pathogenic bacterium that can trigger different diseases, including skin ulcer syndrome in Apostichopus japonicus. In our previous study, a gene named Vshppd encoding a 4-hydroxyphenylpyruvate dioxygenase homologue was cloned from pathogenic V. splendidus, and validated to be responsible for the haemolysis activities of V. splendidus. In this study, Vshppd was determined to participate in the catabolism of tyrosine and promote pyomelanin production in Escherichia coli BL21 (DE3) harboring Vshppd. The purified melanin pigment displayed obvious antimicrobial activity against E. coli and Micrococcus luteus and protective effect on V. splendidus under ultraviolet irradiation. As an important virulence factor, Vshppd was further determined to be cytotoxic to the coelomocyte of A. japonicus and cell viability decreased to approximately 68%, 77%, 54% and 44% when 50, 60, 80 and 100 µL of purified rVshppd was present, respectively. To better understand the potential effect of Vshppd mediated oxidative stress, we injceted A. japonicus with the rVshppd, which showed significantly stimulatory effects on the expression of oxidative stress related genes catalase (cat), glutathione S-transferase (gst), glutathione peroxidase (gpx), heat shock protein 70 (hsp70) of A. japonicus. At 48 h, the expression level of cytochrome P450 (cyp450) was down-regulated compared with that treated with BSA. It was suggested that Vshppd exhibited cytotoxicity via altering the oxidative stress. Our result indicated that Vshppd was not only involved in the self-protection, but also contributed to the pathogenesis of V. splendidus by modulating the oxidative stress imbalance in A. japonicus.

Quorum sensing-disrupting coumarin suppressing virulence phenotypes in Vibrio splendidus.

In the present study, the effects of an environmental friendly natural reagent coumarin, on the growth and potential virulence factors, as well as its ability to interfere the infection of Vibrio splendidus (Vs), were determined. Coumarin showed no effects on the maximal growth of Vs, and biofilm formation of Vs, while it significantly decreased protease activity and hemolytic activity by 43 and 80%, respectively. Correspondingly, coumarin exhibited an obviously protective effect, with a relative percent survival of 60% upon Apostichopus japonicus from infection by Vs. To preliminarily investigate the mechanism underlining the inhibitory effects, regulation of genes Vsm and Vsh respectively related to protease activity and hemolytic activity by supernatant and supernatant extract containing acyl-homoserine lactones (AHLs) and coumarin was determined. Cell-free supernatant from higher density and its ethyl acetate extract containing AHL signal molecules could respectively upregulate the mRNA level of Vsm by 17.4- and 2.3-fold and Vsh by 7.2- and 5.0-fold, when Vs was at lower cell density. However, coumarin could reduce the stimulatory effects of both the supernatant and its ethyl acetate extract. Combining all the results in our study, it was suggested that coumarin could be considered as an alternative to be used for controlling infection of Vs, downregulating the expression of potential virulence factors through interfering the AHL-mediated pathways.

Characterization of a metalloprotease involved in Vibrio splendidus infection in the sea cucumber, Apostichopus japonicus.

Vibrio splendidus is an important aquatic pathogen that infects a broad range of hosts, leading to typical symptoms of "skin ulceration syndrome" in the sea cucumber Apostichopus japonicus. However, there are few reports on the virulence factors and pathogenic mechanism of V. splendidus. In the present study, V. splendidus could survive in the coelomic fluid of A. japonicus but poorly internalized into the coelomocyte under the tested conditions. It was thus postulated that V. splendidus was pathogenic to A. japonicus, mainly due to its extracellular products. The main extracellular proteins of V. splendidus were detected using MALDI-TOF-TOF/MS, and a metalloprotease was identified. The gene encoding a metalloprotease belonging to the thermolysin family, named vsm, was cloned and characterized. Furthermore, the expression of vsm was growth dependent and the supernatant of V. splendidus also showed high protease activity. The vsm gene was conditionally expressed in Escherichia coli BL21(DE3). Enzyme activity analysis showed that the optimal temperature and pH for purified recombinant Vsm were approximately 40 °C and 7.0, respectively. Furthermore, Vsm was determined to be associated with the pathogenesis of V. splendidus due to the following aspects: (1) real-time reverse transcriptase PCR showed that expression of vsm was significantly up-regulated when V. splendidus was co-incubated with the coelomic fluid of A. japonicus; and (2) purified recombinant Vsm showed obvious cytotoxicity to the coelomocyte of A. japonicus. Our results indicated that Vsm is involved in the interaction between V. splendidus and A. japonicus and also contributed to the cytotoxic effects on the coelomocyte of A. japonicus.

Inhibition of marine Vibrio sp. by pyoverdine from Pseudomonas aeruginosa PA1.

Siderophores are low-molecular-weight chemicals that are secreted by many microorganisms to chelate iron from the external environment in order to facilitate their growth and diverse metabolisms. In this study, a fluorescent siderophore, pyoverdine, secreted by Pseudomonas aeruginosa PA1 was purified by affinity chromatography using Cu-sepharose. Pyoverdine was determined to have a molecular mass of 1333.54 Da, as determined by MALDI-TOF/TOF, and belong to type I pyoverdine, as determined by PCR analysis of its corresponding outer membrane ferri-pyoverdine receptor. Pyoverdine showed different degrees of inhibitory effects on the growth of marine Vibrio sp. strains. It was also shown that the biofilm developed by Vibrio parahaemolyticus WzW1 and Wz2121 and Vibrio cyclitrophicus HS12 was significantly reduced, alone with the repressed growth in the presence of pyoverdine. Siderophore production was determined in the strains of Vibrio sp. in response to the pyoverdine-induced iron-limited conditions. The siderophore production of most Vibrio sp. was up-regulated, with the exception of the bacteria that produced little siderophore. Furthermore, Apostichopus japonicus cultured in pyoverdine pretreated seawater showed a relative percent of survival of 89% when they were challenged by Vibrio splendidus. Our results demonstrated that pyoverdine may be a promising agent that could be potentially applied to treat vibriosis.