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.

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.

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.

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.