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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.
Figure 1. (a) Growth curves of WTVs and MTVs. WTVs and MTVs were spread onto 2216E solid plates at 28°C overnight. Two single colonies were inoculated into flasks with 100 mL of fresh 2216E medium and incubated at 28°C with shaking at 180 rpm. Overnight cultures were diluted to the same concentration, and 200 μL aliquots of WTVs and MTVs were transferred into flasks with 100 mL of fresh 2216E medium. OD600 values were measured at different time points. (b) Hemolysis ring on blood agar plate. [a] WTVs and [b] MTVs were spread on 2216E sheep blood agar plate and incubated at 28°C overnight. (c) Colour contrasts of [a] WTVs and [b] MTVs cells cultured in 2216E medium at 28°C until OD600 reached 1.5. (d) Bacterial survival rate under UV irradiation (OD600 = 0.6). Cell number on solid plates was counted after UV light exposure for 0, 5, 10, 15 and 30 min. The total number of WTVs and MTVs was the same at the beginning, which regarded as 100 and the survival rate was expressed as a percentage. The Y-axes was shown as a log scale. (e) Bacterial survival rate under UV irradiation (OD600 = 1.5). Cell number on solid plates was counted after UV light exposure for 0, 5, 10, 15 and 30 min. The total number of WTVs and MTVs was the same at the beginning, which regarded as 100 and the survival rate was expressed as a percentage. The Y-axes was shown as a log scale.
Figure 2. (a) Lethality of WTVs and MTVs. A. japonicus was randomly divided into 13 tanks with 20 individuals each. The WTVs and MTVs strains used for infection were cultured in 2216E medium (24 h, 28 °C) until OD600 was approximately 1.0. The strains were then washed and re-suspended in PBS (28 °C). For survival assays, weight-matched A. japonicus individuals were infected with 5 × 107, 1 × 107, 5 × 106, 1 × 106, 5 × 105 and 1 × 105 CFU mL−1V. splendidus (WTVs or MTVs). A. japonicus infected with PBS was used as the negative control. The water temperature during infection was 16°C. The daily mortality of infected A. japonicus was recorded. (b) The observed symptoms of [a] WTVs, [b] MTVs and [c] negative control group (PBS). Dead A. japonicus were removed in a timely manner and photographed to observe symptoms.
Figure 3. (a) DEG distribution between WTVs and MTVs. Log2 indicates the mean expression level for each gene, and the vertical axis represents the statistical significance of the difference in gene expression. Each dot represents one gene. Red and green dots represent up-regulated and down-regulated DEGs, respectively. Blue dots represent no DEGs. [a] OD600 = 0.6; [b] OD600 = 1.0; [c] OD600 = 1.5. (b) GO enrichment of down-regulated DEGs (OD600 = 1.0). (c) KEGG enrichment of down-regulated DEGs (OD600 = 1.0).
Figure 4. GO enrichment of DEGs. The method used for GO enrichment analysis was GOseq, which can accurately calculate the probability of a GO term enriched by differential genes. The ordinate is the enriched GO term, and the abscissa is the number of differential genes in the term. Green bars represent biological processes, orange bars represent cellular components, and blue bars represent molecular functions. (a) MTVs 0.6 vs WTVs 0.6; (b) MTVs 1.0 vs WTVs 1.0; (c) MTVs 1.5 vs WTVs 1.5. (d) Temporal expression analyses of [a] flgDV.s., [b] tpPV.s., [c] clpBV.s., [d] hAV.s., [e] hsp20V.s, and [f] lolBV.s. in WTVs or MTVs at the OD600 of 1.0. Values are presented as mean ± SD (n = 5). Asterisks indicate signiﬁcant differences: *P < 0.05 and **P < 0.01.
Figure 5. (a) Biofilm formation of WTVs, MTVs and hppDC. (b) Swimming motility of WTVs, MTVs and hppDC. [a] The same concentration of MTVs (5 μL) was dropped on low-agar 2216E medium and cultured for 48 h at 28°C. [b] The same concentration of hppDC (5 μL) was dropped on low-agar 2216E medium and cultured for 48 h at 28°C. [c] The same concentration of the WTVs (5 μL) was dropped on low-agar 2216E medium and cultured for 48 h at 28°C. (c) Bar graph of the swimming motility of WTVs, MTVs and hppDC. (d) The adhesive ability of WTVs and MTVs to different A. japonicus tissues was demonstrated by colony counting. A. japonicus was soaked in WTVs and MTVs (1.0 × 107 CFU mL−1) for 24 h for infection. The A. japonicus tissues were weighed and homogenised. The homogenised solution was diluted gradiently and then coated on 2216E plates. Colony count was determined on the next day. The error line represents the SD (n = 3). Asterisks represent the significance of difference (*P < 0.05, **P < 0.01).