Time-Series Multi-Omics Reveal Metabolic Reprogramming, Oxidative Stress and Programmed Cell Death in M. bovis-Infected Bovine Mammary Epithelial Cells

Abstract

Mycoplasmopsis bovis is a major causative agent of bovine mastitis and causes substantial economic losses in the dairy industry because of the chronic and refractory nature of the infection. This study aimed to determine how M. bovis PG45 infection dynamically alters host metabolism and gene expression over time and whether these changes are accompanied by progressive redox imbalance and enrichment of multiple programmed cell death pathways. Transcriptomic and untargeted metabolomic profiles were analyzed at 8, 24, 48, and 72 h post-infection using three biological replicates for RNA sequencing and six biological replicates for metabolomic analysis. RNA-seq identified 779, 1777, 4631, and 4855 differentially expressed genes (DEGs) at 8, 24, 48, and 72 h, respectively, revealing a progressive expansion of transcriptional responses. KEGG enrichment analysis identified significant temporal shifts from early TLR, NF-κB, and TNF signaling toward cell-cycle regulation and DNA replication at later stages (FDR < 0.05). Metabolomic analysis identified 721, 1505, 1270, and 849 differentially abundant metabolites (DAMs), respectively, with prominent alterations in lipid, amino acid, and redox-related metabolism. Integrated transcriptomic–metabolomic analysis identified significant stage-specific gene–metabolite associations (|r| > 0.8, p < 0.05), including strong associations of (S)-Leucic acid with SAA3 and SGSH and of N-acetyl-L-aspartic acid with TANC1. A dynamic pattern of glutathione depletion, together with disruption of sphingolipid metabolism and enrichment of ferroptosis-related pathways, provided convergent correlative evidence of increasing redox and lipid metabolic stress during infection. Programmed cell death-related pathway analysis showed increasing prominence of apoptosis and cellular senescence at later stages. The RNA-seq findings were further supported by qRT-PCR validation, with strong concordance at all four time points (R = 0.99, p ≤ 1.62 × 10−4). Collectively, these findings support a time-dependent transition from inflammatory activation to metabolic reprogramming, redox imbalance, and programmed cell death, and provide correlative evidence that ferroptosis may contribute to injury of bovine mammary epithelial cells during M. bovis infection.