Klebsiella pneumoniae, a common cause of clinical mastitis (CM) in dairy cows, can cause severe clinical symptoms. However, its pathogenicity in the bovine mammary gland is not well understood. Our objectives were to establish an in vitro infection model of K. pneumoniae on bovine mammary epithelial cells (bMEC) to assess (1) cytopathogenicity (adhesive and invasive ability, damage and apoptosis, pro-inflammatory effects) of K. pneumoniae on bMEC and (2) the role of hypermucoviscous (HMV) phenotype on cytopathogenicity. Two K. pneumoniae isolates from CM cows, 1 HMV and 1 non-HMV, were used to infect bMEC. Adhesion and invasion ability, release of lactate dehydrogenase (LDH), ultrastructural morphology, apoptosis, transcriptional expression of pro-inflammatory genes and production of pro-inflammatory cytokines were characterized at various intervals. Both K. pneumoniae isolates rapidly adhered to and invaded bMEC within 1 h post infection (pi), causing ultrastructural damage (swelling of mitochondria and vesicle formation on cell surface) after 3 h pi and apoptotic death after 9 h pi. In addition, K. pneumoniae promoted transcriptional expression of pro-inflammatory genes IL-6, IL-8, IL-1β, and tumor necrosis factor (TNF)-α and production of IL-8, IL-1β, and TNF-α cytokines. Compared with non-HMV K. pneumoniae, the HMV isolate had lower adhesive and invasive abilities but caused more serious cellular damage. In conclusion, K. pneumoniae was cytopathogenic on bMEC and induced a pro-inflammatory response; however, the HMV phenotype did not have a key role in pathogenicity. Therefore, more attention should be paid to milk loss, and targeted prevention and treatment strategies should be implemented in Klebsiella mastitis episodes.
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.
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