Transcriptomic in silico analysis of bovine Escherichia coli mastitis highlights its immune-related expressed genes as an effective biomarker

Abstract

Background: Mastitis is one of the major diseases causing economic loss to the dairy industry by reducing the quantity and quality of milk. Thus, the objective of this scientific study was to find new biomarkers based on genes for the early prediction before its severity.

Methods: In the present study, advanced bioinformatics including hierarchical clustering, enrichment analysis, active site prediction, epigenetic analysis, functional domain identification, and protein docking were used to analyze the important genes that could be utilized as biomarkers and therapeutic targets for mastitis.

Results: Four differentially expressed genes (DEGs) were identified in different regions of the mammary gland (teat cistern, gland cistern, lobuloalveolar, and Furstenberg's rosette) that resulted in 453, 597, 577, and 636 DEG, respectively. Also, 101 overlapped genes were found by comparing 27 different expressed genes. These genes were associated with eight immune response pathways including NOD-like receptor signaling pathway (IL8, IL18, IL1B, PYDC1) and chemokine signaling pathway (PTK2, IL8, NCF1, CCR1, HCK). Meanwhile, 241 protein-protein interaction networks were developed among overlapped genes. Fifty-seven regulatory events were found between miRNAs, expressed genes, and the transcription factors (TFs) through micro-RNA and transcription factors (miRNA-DEG-TF) regulatory network. The 3D structure docking model of the expressed genes proteins identified their active sites and the binding ligands that could help in choosing the appropriate feed or treatment for affected animals.

Conclusions: The novelty of the distinguished DEG and their pathways in this study is that they can precisely improve the detection biomarkers and treatments techniques of cows' Escherichia coli mastitis disease due to their high affinity with the target site of the mammary gland before appearing the symptoms.

Keywords: Dairy cow; Differentially expressed genes; Escherichia coli; Mastitis; Microarray; Transcriptomic analysis.

Fig. 1

Volcano plot of DEGs in teat cistern (a), gland cistern (b), lobuloalveolar (c), and Furstenberg’s rosette (d), regions. Red horizontal dot line means FDR = 0.05 cutoff line, two red vertical dot lines mean logFC = 1 and logFC = − 1 cutoff line. Gene dots that were distributed above the FDR = 0.05 red line and out of |logFC| = 1 were the DEGs we identified

Fig. 2

a Venn diagram of DEGs in the four different regions. b Hierarchical clustering of 101 overlapping differentially expressed genes (ODEGs)

Fig. 3

miRNA-DEG-TF regulatory network. Red rounded nodes represent DEGs, yellow trigonal nodes represent miRNAs, and blue quadrate nodes represent significantly related TFs; red arrow lines mean TF-DEGs regulatory relationships, green arrow lines mean miRNA-DEGs regulatory relationships

Fig. 4

a Heatmap of selected 4 DEGs. b Barplot of selected DEGs. * Refers to 0.01 ≤ p < 0.05, ** refers to 0.005 ≤ p < 0.01, *** refers to p < 0.005. c CpG island in the promoter region of CTST. The blue-filled region is CpG Island. d Presents the active sites in the differentially expressed gene (DEG) of CTSC. e Alignment of CTSC protein among 5 different species. The highlight sequence parts with yellow were consensus sequences of different species

Fig. 5

3D structure of four selected proteins. Ligand 1 and 2 were ligand molecular which can dock and bind to the active sites of proteins (a, b). Words in the yellow boxes were the information about the active binding sites. In the 3D structures, red ones mean α-helix, the orange ones mean β-fold. a–e Represents CTSC, IL10, IL8, and IL18