RNA-Seq Analysis Revealed the Virulence Regulatory Network Mediated by the Ferric Uptake Regulator (Fur) in Apostichopus japonicus Pathogenesis Induced by Vibrio splendidus

Abstract

The uptake and utilization of iron by bacteria must be strictly controlled. The ferric uptake regulator (Fur) is a global transcription factor widely present in bacteria that can perceive cellular iron levels and adjust the expressions of various genes accordingly. Our earlier research demonstrated that the knockdown of the fur gene in Vibrio splendidus significantly reduced its lethality to Apostichopus japonicus. Although the functions and mechanisms of Fur in regulating bacterial virulence genes have been extensively studied, its virulence regulatory network during V. splendidus pathogenesis in A. japonicus remains unclear. In this article, transcriptome sequencing analysis of V. splendidus under different iron conditions reveals substantial differential gene expressions in the simulated pathogenic environments, identifying 1185 differentially expressed genes, including 198 downregulated and 987 upregulated genes. Comparative analysis between wild-type and Vsfur knockdown strains shows that Vsfur knockdown altered the expression of 3593 genes in V. splendidus, with the most significant differential expression observed under simulated pathogenic conditions (1030 upregulated and 72 downregulated). KEGG enrichment analysis indicates that Vsfur knockdown caused significant gene enrichment in the flagellar assembly pathway and bacterial secretion system, critically impairing flagellar synthesis and secretion system function in V. splendidus. Eight genes selected for qRT-PCR validation showed expression levels in line with the RNA-seq results. Consistent with the transcriptomic results, Vsfur knockdown resulted in reduced antioxidant capacity, bacterial competitiveness, and cytotoxicity in V. splendidus. These findings elucidate the virulence regulatory mechanism of Fur in V. splendidus and provide a reference for understanding the occurrence of A. japonicus skin ulcer syndrome.

Keywords: ferric uptake regulator; flagellar assembly; skin ulcer syndrome; transcriptome sequencing; virulence.

Figure 1

Differential gene expression analysis results. (A) The figure is a statistical bar chart that displays the names of various differential comparisons and their distribution in the number of up- and downregulated differential genes. Green and red represent down- and upregulated genes, respectively. (B) Differential gene Venn diagram. The numerical markers in the figure reveal the number of specific or shared differentially expressed genes (DEGs). (C) Cluster heatmap of differential gene expression levels. The vertical and horizontal axes in the graph represent each gene and different samples, respectively. The depth of color intuitively reflects gene expression strength in a specific sample. Red and green indicate relatively high and low expression levels of the gene in the corresponding sample, respectively. The left side of the graph shows the clustering relationships between genes, presented in the form of a tree diagram.

Figure 2

Volcanic maps of the comparison groups. The figure shows the statistical results of gene expression differences between different groups of samples. The horizontal axis represents the multiple changes in gene expression, calculated as the fold change and presented in log (B/A) form. The vertical axis displays the statistical significance of changes in gene expression, i.e., p value. The red and green dots represent up- and downregulated genes, respectively. The black dots represent non-DEGs. (A) H2 vs. H1; (B) L2 vs. L1; (C) C2 vs. C1; (D) N2 vs. N1.

Figure 3

(A) Venn diagram of differentially expressed genes. The various parts in the figure are distinguished by different colors to better display different comparison groups. The numbers in the figure are used to represent the specific or shared number of DEGs. Among them, overlapping and non-overlapping regions represent the common and unique number of DEGs among different comparison groups, respectively. (B) Each row and column represent a unique gene and a specific sample, respectively. Red and green represent relatively high and low expression levels of genes in the sample, respectively. The tree chart reveals the similarity between genes: when the branches of two genes on the tree chart are close to each other, it indicates that their expression levels are very close. The names of all samples are listed below the figure for easy identification and tracking of information for each sample.

Figure 4

Column-like diagram of different gene GO comment classification. The horizontal axis represents various functional classifications, while the vertical axis displays the number of genes belonging to each classification (on the right) and the percentage of these genes in the total annotated genes (on the left). By using different colors, we can distinguish different classifications. The figures shows the functional annotation of DEGs between the (A) H2 and H1 groups; (B) L2 and L1 groups; (C) C2 and C1 groups; and (D) N2 and N1 groups.

Figure 5

Significant enrichment function scatter map. The vertical axis displays feature annotation information, while the horizontal axis represents the rich factor value of the feature. The color of the dot represents the size of the Qvalue, where the closer the color is to red, the smaller the Qvalue. The number of DEGs included in each function is reflected by the dot size. The figure shows the scatter plots of significant enrichment functions between the (A) H2 and H1 groups; (B) L2 and L1 groups; (C) C2 and C1 groups; and (D) N2 and N1 groups.

Figure 6

Bar chart of COG classification for DEGs. The horizontal axis represents COG functional categories, while the vertical axis displays the number of genes belonging to each classification (on the right) and the percentage of these genes in the total annotated genes (on the left). The figure illustrates the COG functional annotation of DEGs between the (A) H2 and H1 groups; (B) L2 and L1 groups; (C) C2 and C1 groups; and (D) and N1 groups.

Figure 7

(A) Temporal expression analysis of VsFldA, VsSecA, VsDusB, VsAspA, VsCrsA, VsPurB, VsFlgC, and VsCarB in the H, L, C, and N groups. Values are presented as mean ± SD (n = 5). Asterisks indicate significant differences: ** p < 0.01. (B) Oxidative stress capacity of the two experimental strains AJ01 and ΔVsfur. (C) Interbacterial competitiveness of the two experimental strains AJ01 and ΔVsfur. (D) Cytotoxicity of the two experimental strains AJ01 and ΔVsfur. The error line is the standard deviation (n = 3). The asterisks represent significant differences (** p < 0.01).