Abstract
Klebsiella pneumoniae, an important cause of bovine mastitis worldwide, is strongly pathogenic to bovine mammary epithelial cells (bMECs). Our objective was to determine the role of mitochondrial damage in the pathogenicity of K. pneumoniae on bMECs, by assessing several classical indicators of mitochondrial dysfunction, as well as differentially expressed genes (DEGs). Two K. pneumoniae strains (HLJ-D2 and HB-AF5), isolated from cows with clinical mastitis (CM), were used to infect bMECs (MAC-T line) cultured in vitro. In whole-transcriptome analysis of bMECs at 6 h post-infection (hpi), there were 3453 up-regulated and 3470 down-regulated genes for HLJ-D2, whereas for HB-AF5, there were 2891 up-regulated and 3278 down-regulated genes (P < 0.05). Based on GO term enrichment of differentially expressed genes (DEGs), relative to the controls, the primary categories altered in K. pneumoniae-infected bMECs included cellular macromolecule metabolism, metabolic process, binding, molecular function, etc. Infections increased (P < 0.05) malondialdehyde concentrations and formation of reactive oxygen species in bMECs. Additionally, both bacterial strains decreased (P < 0.05) total antioxidant capacity in bMECs at 6 and 12 hpi. Furthermore, infections decreased (P < 0.05) mitochondrial membrane potential and increased (P < 0.01) mitochondrial calcium concentrations. Finally, severe mitochondrial swelling and vacuolation, as well as mitochondrial rupture and cristae degeneration, were detected in infected bMECs. In conclusion, K. pneumoniae infections induced profound mitochondrial damage and dysfunction in bMECs; we inferred that this caused cellular damage and contributes to the pathogenesis of K. pneumoniae-induced CM in dairy cows.
Highlights
Bovine mastitis caused by pathogenic microorganisms is an important economic threat to the dairy industry [1]
K. pneumoniae up‐ or down‐regulated differentially expressed genes (DEGs) associated with mitochondrial damage in bovine mammary epithelial cells (bMECs) Gene Ontology (GO) enrichment analysis of DEGs A total of 6923 and 6169 genes were differentially expressed with fold-change ≥ 2 or ≤ 0.5 (P-value < 0.05) after infection with HLJ-D2 or HB-AF5 K. pneumoniae (Figure 1A, B)
The most common up-regulated DEGs in bMECs after HLJ-D2 infection were associated with DNA (22), ADP (20), mitochondria (16) and interleukin (13), whereas mitochondria (100) and DNA (33) were frequently detected as down-regulated DEGs (Figure 3A)
Summary
Bovine mastitis caused by pathogenic microorganisms is an important economic threat to the dairy industry [1]. Klebsiella pneumoniae, a major pathogen causing clinical mastitis (CM), induces a strong immune response, altering milk quality and causing inflammatory changes in the udder [2,3,4]. Mitochondria are intracellular organelles with multiple features due to their bacterial ancestry [7]. In addition to being a cell’s energy powerhouse, they are a reservoir for cellular Ca2+, which is crucial for cell life and death [8]. Mitochondria have a role in the innate immune system by harboring innate sensors that can detect bacteria and trigger immune activation, making them an attractive target for pathogens [9, 10]. During co-evolution of pathogens and hosts, the
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