Molecular Mechanism Of Inflammatory And Iron Death Induced By Candida 14-3-3 Protein In Bovine Mammary Epithelial Cells

Candida krusei is a kind of opportunistic pathogenic fungus,which is common in both human and animals.Because of the use of non-standard antibiotics and immunosuppressive therapy,the detection rate of Candida in human and veterinary clinic has been increasing year by year.Epidemiological investigation revealed that Candida Krusei and other Candida albicans fungi were the main pathogenic fungi of mycotic mastitis in dairy cows in Yinchuan,Ningxia.Candida krusei is naturally resistant to fluconazole and it has strong cell surface hydrophobicity,biphasic transformation ability between yeast and hyphae,hydrolase secretion and biofilm production,the results showed that both phases of Candida flexneri could invade the damaged cells of the host.With the development of research,it has been reported that fungi can not only directly invade tissue cells,but also release virulence proteins or other factors to interact with host cells by secreting extracellular vesicles.Proteomic analysis showed that the soluble homoheterodimer 14-3-3 protein was significantly up-regulated in the exosomes of Candida Krusei.Therefore,it has great significance to investigate the damage of Candida 14-3-3 protein on host cells in order to study the mechanism of infection.In this study,we constructed the recombinant expression vector of Candida 14-3-3 protein,expressed and purified the 14-3-3 protein in vitro,and established the model of mammary epithelial cell injury induced by 14-3-3 protein,to study the molecular mechanism of inflammatory response,autophagy and iron death induced by Candida 14-3-3 protein in bovine mammary epithelial cells,the aim of this study was to explore the molecular mechanism of fungal mastitis induced by Candida krusei exosome target molecules.The results showed that the recombinant expression vector pET-2 1a-BMH1 was successfully constructed and the biological activity of rCK 14-3-3 protein was obtained after induced expression and purification.The purity of rCK14-3-3 protein was over 90%and the concentration of rCK14-3-3 protein was 0.2 mg/mL.The concentration of rCK 14-3-3 protein was 50 μg/mL for 1 h,3 h and 6 h.rCK 14-3-3 protein can activate TLR2 and TLR4,it also can activate p38,ERK,p65 and IκBα in MAPK and NFκB signaling pathways to phosphorylate,and the relative expressions of IL-1β,IFN-γ,IL-18,TNF-α,IL6 and IL-12 were increased.rCK 14-3-3 protein significantly increased the expression of p-PI3K,p-Akt,LC3,Beclin-1 and P62,and decreased the expression of p-mTOR in PI3K/AKT/mTOR signaling pathway of bovine mammary epithelial cells.rCK 14-3-3 protein increased the Fe2+content,MDA content,and the level of reactive oxygen species(ROS)related to iron death,GSH content was decreased,the expression levels of iron death-related proteins NOCA4,GPX-4 and FTH1 were significantly decreased.In this study,rCK 14-3-3 protein with biological activity was obtained by prokaryotic expression,rCK 14-3-3 protein could activate TLR2/4-MAPK/NF-κB signaling pathway to induce effective immune response and inflammatory response in bovine mammary epithelial cells,it causes elevated levels of proinflammatory cytokine expression.rCK 14-3-3 protein can induce autophagy in bovine mammary epithelial cells by activating PI3K/AKT/mTOR signaling pathway.rCK14-3-3 protein may also induce iron death in cells through NOCA4-mediated autophagy of ferritin and through lipid peroxidation induction by Fenton reaction with iron ions.