Weighted gene co-expression network analysis reveals key genes and lncRNAs in Bos indicus chronically infected with...

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteric disease affecting cattle worldwide, with considerable economic implications for dairy producers. MAP is a robust, intracellular pathogen highly resilient to environmental challenges. To elucidate the molecular mechanisms underlying MAP infection in desi cattle (Bos indicus), we performed a comprehensive transcriptomic analysis.

Diseased and healthy female desi cattle were selected for RNA sequencing based on phenotype. Raw data were processed, and differential expression of mRNA and lncRNA was determined. Gene modules and hub genes significantly correlated with traits were identified using network analysis. Further analysis included functional enrichment, PPI network construction, and lncRNA-mRNA co-expression within the key module. The differential expression of final hub genes was validated via qPCR.

RNA sequencing revealed 1,905 differentially expressed protein-coding genes and, for the first time, comprehensively annotated 45,947 lncRNA genes in desi cattle, of which 3,123 lncRNAs were differentially expressed in MAP-infected samples. Weighted Gene Co-expression Network Analysis identified 11 co-expressed gene modules, among which the turquoise module, comprising 870 protein-coding genes and 934 lncRNAs, was highly correlated with clinical traits. Functional enrichment analysis of this key module revealed significant involvement in defense response, inflammatory processes, and natural killer cell-mediated immunity and cytotoxicity. Gene set enrichment analysis highlighted suppressed pathways related to natural killer cell immunity, lectin response, and protein-DNA complex assembly, while activated pathways included G-protein-coupled receptor signaling, circulatory system processes, and metabolic pathways. Using network-based approach, we identified 12 hub genes (IL7R, TLR4, KLRK1, IFNG, TGFB1, CD68, CXCR6, GZMB, KLRG1, MMP9, GZMA, and SELL) in the turquoise module associated with MAP infection in PBMC transcriptomes across a mixed-breed Bos indicus cohort. These genes were associated with immune suppression and evasion, inflammation, and tissue remodeling. Additionally, 22 differentially expressed lncRNAs co-expressed with hub genes were identified, suggesting potential roles in modulating immune response.

Our findings provide novel insights into the molecular mechanisms of MAP infection in desi cattle, identifying critical hub genes and lncRNAs as potential targets for developing innovative diagnostic and therapeutic strategies for Johne's disease.