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Vibrio splendidus is a pathogen that infects a wide range of hosts, especially the sea cucumber species Apostichopus japonicus. Previous studies showed that the level of L-glutamic acid (L-Glu) significantly increased under heat stress, and it was found to be one of the best carbon sources used by V. splendidus AJ01. In this study, the effects of exogenous L-Glu on the coelomocyte viability, tissue status, and individual mortality of sea cucumbers were analyzed. The results showed that 10 mM of L-Glu decreased coelomocyte viability and increased individual mortality, with tissue rupture and pyknosis, while 0.1 mM of L-Glu slightly affected the survival of sea cucumbers without obvious damage at the cellular and tissue levels. Transcriptomic analysis showed that exogenous L-Glu upregulated 343 and downregulated 206 genes. Gene Ontology (GO) analysis showed that differentially expressed genes (DEGs) were mainly enriched in signaling and membrane formation, while a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in the upregulated endocytosis and downregulated lysosomal pathways. The coelomocyte viability further decreased by 20% in the simultaneous presence of exogenous L-Glu and V. splendidus AJ01 compared with that in the presence of V. splendidus AJ01 infection alone. Consequently, a higher sea cucumber mortality was also observed in the presence of exogenous L-Glu challenged by V. splendidus AJ01. Real-time reverse transcriptase PCR showed that L-Glu specifically upregulated the expression of the fliC gene coding the subunit protein of the flagellar filament, promoting the swimming motility activity of V. splendidus. Our results indicate that L-Glu should be kept in a state of equilibrium, and excess L-Glu at the host-pathogen interface prompts the virulence of V. splendidus via the increase of bacterial motility.
LR20C190001 Zhejiang Provincial Natural Science Foundation, 31972833 the National Natural Science Foundation of China, N/A the K.C. Wong Magna Fund at Ningbo University
Figure 1. Effects of different concentrations of L-Glu on sea cucumbers at both the cellular and individual levels. (A) Viability of coelomocytes treated with different concentrations of L-Glu for 12 h. Data are means and standard deviations from three independent experiments. ** p < 0.01. (B) The survival percentages of sea cucumbers treated with different concentrations of L-Glu. The dead sea cucumbers in each group were recorded and removed to calculate the survival percentages.
Figure 2. Tissue structure of the body wall of the L-Glu-treated individuals. (A) Control sea cucumbers (NC). (B–D) represent the slices of individual morphological changes and body wall tissues of sea cucumbers treated with different concentrations of L-Glu. The assays were repeated in triplicate, and one representative figure is shown for each sample. Red arrows indicate obvious tissue damage of pyknosis and loosening of tissue connections.
Figure 3. Transcriptomic analysis of sea cucumbers treated with 0.1 mM L-Glu. (A) The volcano map of DEGs. The abscissa coordinate is the log2FoldChange value, and the ordinate is −log10padj. The dashed gray line represents the threshold line of DEGs’ screening criteria. (B) Histogram based on the GO analysis. The abscissa is the GO element, and the ordinate is the significance level of GO element enrichment, represented by −log10(padj). (C) Scatter plot based on KEGG analysis of upregulated DEGs. (D) Scatter plot based on KEGG analysis of downregulated DEGs. The abscissa is the ratio of the number of DEGs annotated in the KEGG pathway to the total number, and the ordinate is the KEGG pathway.
Figure 4. The effects of L-Glu on cell viability and individual survival. (A) Cell viability in the presence of V. splendidus AJ01 and the V. splendidus AJ01 plus L-Glu. Data are means and standard deviations from three independent experiments. * p < 0.05 and *** p < 0.001. (B) The survival percentage of sea cucumbers in the presence of L-Glu when sea cucumbers were treated with V. splendidus AJ01. Each growth was performed in triplicate. Data are means and standard deviations from three independent experiments.
Figure 5. (A) Effects of different concentrations of L-Glu on growth of V. splendidus AJ01. (B) Growth curve of V. splendidus AJ01 in the M9 minimal medium with 3% salinity simultaneously with L-Glu and coelomic fluid. Each growth was performed in triplicate. Data are means and standard deviations from three independent experiments.
Figure 6. The effects of L-Glu on the expression of virulence-related factors in the matrix of coelomic fluid. (A) Expression of virulence-related genes in the presence of L-Glu and coelomic fluid. (B) V. splendidus AJ01 swimming in the M9 minimal medium with 3% salinity simultaneously with L-Glu and coelomic fluid. (C) Statistical analysis of the diameters of the swimming circles. Data are means and standard deviations from three independent experiments. * p < 0.05 and ** p < 0.01.
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