Multi-omics analysis of miRNA-mediated intestinal microflora changes in crucian carp Carassius auratus infected with Rahnella aquatilis

Abstract

Infection by an emerging bacterial pathogen Rahnella aquatilis caused enteritis and septicemia in fish. However, the molecular pathogenesis of enteritis induced by R. aquatilis infection and its interacting mechanism of the intestinal microflora associated with microRNA (miRNA) immune regulation in crucian carp Carassius auratus are still unclear. In this study, C. auratus intraperitoneally injected with R. aquatilis KCL-5 was used as an experimental animal model, and the intestinal pathological changes, microflora, and differentially expressed miRNAs (DEMs) were investigated by multi-omics analysis. The significant changes in histopathological features, apoptotic cells, and enzyme activities (e.g., lysozyme (LYS), alkaline phosphatase (AKP), alanine aminotransferase (ALT), aspartate transaminase (AST), and glutathione peroxidase (GSH-Px)) in the intestine were examined after infection. Diversity and composition analysis of the intestinal microflora clearly demonstrated four dominant bacteria: Proteobacteria, Fusobacteria, Bacteroidetes, and Firmicutes. A total of 87 DEMs were significantly screened, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the potential target genes were mainly involved in the regulation of lipid, glutathione, cytosine, and purine metabolism, which participated in the local immune response through the intestinal immune network for IgA production, lysosome, and Toll-like receptor (TLR) pathways. Moreover, the expression levels of 11 target genes (e.g., TLR3, MyD88, NF-κB, TGF-β, TNF-α, MHC II, IL-22, LysC, F2, F5, and C3) related to inflammation and immunity were verified by qRT-PCR detection. The correlation analysis indicated that the abundance of intestinal Firmicutes and Proteobacteria was significantly associated with the high local expression of miR-203/NF-κB, miR-129/TNF-α, and miR-205/TGF-β. These findings will help to elucidate the molecular regulation mechanism of the intestinal microflora, inflammation, and immune response-mediated miRNA-target gene axis in cyprinid fish.

Keywords: Carassius auratus; Rahnella aquatilis; inflammation; intestinal microflora; miRNA.

Figure 1

Histopathological changes and analysis of enzyme activity in the intestine of Carassius auratus infected with Rahnella aquatilis. (A–D) Intestinal tissues of control group fish. (A) Intestinal mucosal epithelial cells were necrotic and exfoliated by H&E staining (upward arrow), and intestinal villi were severely damaged (left arrow). (B) Mucous cells in intestinal tissue increased in Alcian blue-Periodic acid Schiff (AB-PAS) staining (left arrow). (C) Alkaline phosphatase staining-positive reaction increased (downward arrow). (D) TUNEL fluorescence staining of apoptotic cells (left arrow). (E) Activity analysis of immune-related enzymes. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 2

Analysis of intestinal microflora diversity of Carassius auratus infected with Rahnella aquatilis. (A) Box diagram of alpha-diversity index among groups. (B) Principal coordinates analysis (PCoA) of the intestinal microflora. (C) Unweighted pair group method with arithmetic mean (UPGMA) cluster tree analysis of intestinal microflora. Note: Groups a, b, and c were infected for 0, 36 and 72 hpi, respectively, the same as below.

Figure 3

Analysis of the composition of intestinal microflora in Carassius auratus infected with Rahnella aquatilis. (A) Operational taxonomic unit (OTU) Venn diagram of intestinal microflora. (B) Species distribution map of intestinal microflora at phylum level. (C) Species distribution map of intestinal microflora at genus level. (D) Histogram of linear discriminant analysis (LDA) value distribution of intestinal microflora. *P<0.05.

Figure 4

Analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) function (A) and correlation (B) of intestinal microflora in Carassius auratus infected with Rahnella aquatilis.

Figure 5

Analysis of differentially expressed miRNAs (DEMs) in the intestine of Carassius auratus infected with Rahnella aquatilis. (A) Volcanic map of DEMs. Each point in the figure represents a miRNA. (B) Cluster diagram of DEMs. The column represents different samples, and the row represents different miRNAs, which were clustered by log10 (TPM + 1e−6) value. Red stands for high expression of miRNAs and blue for low expression of miRNAs.

Figure 6

Functional analysis of differentially expressed miRNA (DEM) target genes in Carassius auratus intestine infected with Rahnella aquatilis. (A) Gene Ontology (GO)-annotated classification chart. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and scattered point graph.

Figure 7

Expression analysis of miRNA immune-related genes in Carassius auratus intestine infected with Rahnella aquatilis. (A) Network diagram of the interaction between key differentially expressed miRNAs and target genes. Blue represents downregulation, and red represents upregulation. (B) Temporal changes in the expression of target genes. *P<0.05, **P<0.01, ***P<0.001. (C) Analysis of the interaction between genes and proteins related to intestinal immunity. Dark blue lines represent known interactions from curated databases. Purple lines represent experimentally determined known interactions. Green lines represent predicted interactions with neighboring genes. Red lines represent gene fusions. Blue lines represent gene clusters. Yellow lines represent text-mining evidence. Black lines represent co-expression. Light blue lines represent protein homology-based interactions.

Figure 8

Molecular characteristics of potential interaction between miRNAs and target genes proteins. (A) Three binding sites in miR-129, miR-203, and miR-205 to their target genes. (B) Tertiary structure of TNF-α, NF-κB, and TGF-β proteins. (C) Species genetic relationship of miR-205 sequences. (D) Phylogenetic tree analysis of pre-miR-205 from the 26 species.

Figure 9

Analysis of the interaction network of regulatory mechanisms of key miRNAs/intestinal microflora/target genes in Carassius auratus intestinal infected with Rahnella aquatilis. (A) Heatmap of Spearman’s correlation between the intestinal microflora and key miRNAs. (B) Potential molecular regulatory mechanism of intestinal microflora-mediated miRNA–target gene axis.