Browsing by Author "Deng, Zhaoju"

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    Mycoplasma bovis subverts autophagy to promote intracellular replication in bovine mammary epithelial cells cultured in vitro
    (2021-10-14) Liu, Yang; Deng, Zhaoju; Xu, Siyu; Liu, Gang; Lin, Yushan; Khan, Sohrab; Gao, Jian; Qu, Weijie; Kastelic, John P.; Han, Bo
    Abstract Mycoplasma species are the smallest prokaryotes capable of self-replication. To investigate Mycoplasma induced autophagy in mammalian cells, Mycoplasma bovis (M. bovis) and bovine mammary epithelial cells (bMEC) were used in an in vitro infection model. Initially, intracellular M. bovis was enclosed within a membrane-like structure in bMEC, as viewed with transmission electron microscopy. In infected bMEC, increased LC3II was verified by Western blotting, RT-PCR and laser confocal microscopy, confirming autophagy at 1, 3 and 6 h post-infection (hpi), with a peak at 6 hpi. However, the M. bovis-induced autophagy flux was subsequently blocked. P62 degradation in infected bMEC was inhibited at 3, 6, 12 and 24 hpi, based on Western blotting and RT-PCR. Beclin1 expression decreased at 12 and 24 hpi. Furthermore, autophagosome maturation was subverted by M. bovis. Autophagosome acidification was inhibited by M. bovis infection, based on detection of mCherry-GFP-LC3 labeled autophagosomes; the decreases in protein levels of Lamp-2a indicate that the lysosomes were impaired by infection. In contrast, activation of autophagy (with rapamycin or HBSS) overcame the M. bovis-induced blockade in phagosome maturation by increasing delivery of M. bovis to the lysosome, with a concurrent decrease in intracellular M. bovis replication. In conclusion, although M. bovis infection induced autophagy in bMEC, the autophagy flux was subsequently impaired by inhibiting autophagosome maturation. Therefore, we conclude that M. bovis subverted autophagy to promote its intracellular replication in bMEC. These findings are the impetus for future studies to further characterize interactions between M. bovis and mammalian host cells.
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    Prototheca spp. induce an inflammatory response via mtROS-mediated activation of NF-κB and NLRP3 inflammasome pathways in bovine mammary epithelial cell cultures
    (2021-12-11) Zhao, Wenpeng; He, Fumeng; Barkema, Herman W.; Xu, Siyu; Gao, Jian; Liu, Gang; Deng, Zhaoju; Shahid, Muhammad; Shi, Yuxiang; Kastelic, John P.; Han, Bo
    Abstract Emergence of bovine mastitis caused by Prototheca algae is the impetus to better understand these infections. Both P. bovis and P. ciferrii belong to Prototheca algae, but they differ in their pathogenicity to induce inflammatory responses. The objective was to characterize and compare pathogenesis of inflammatory responses in bMECs induced by P. bovis versus P. ciferrii. Mitochondrial ultrastructure, activity and mtROS in bMECs were assessed with transmission electron microscopy and laser scanning confocal microscopy. Cytokines, including TNF-α, IL-1β and IL-18, were measured by ELISA and real-time PCR, whereas expressions of various proteins in the NF-κB and NLRP3 inflammasome pathways were detected with immunofluorescence or Western blot. Infection with P. bovis or P. ciferrii damaged mitochondria, including dissolution and vacuolation of cristae, and decreased mitochondrial activity, with P. bovis being more pathogenic and causing greater destruction. There were increases in NADPH production and mtROS accumulation in infected bMECs, with P. bovis causing greater increases and also inducing higher cytokine concentrations. Expressions of NF-κB-p65, p-NF-κB-p65, IκBα and p-IκBα proteins in the NF-κB pathway, as well as NLRP3, Pro Caspase1, Caspase1 p20, ASC, Pro IL-1β, and IL-1β proteins in the NLRP3 inflammasome pathway, were significantly higher in P. bovis-infected bMECs. However, mito-TEMPO significantly inhibited production of cytokines and decreased expression of proteins in NF-κB and NLRP3 inflammasome pathways in bMECs infected with either P. bovis or P. ciferrii. In conclusion, P. bovis or P. ciferrii infections induced inflammatory responses in bMECs, with increased mtROS in damaged mitochondria and activated NF-κB and NLRP3 inflammasome pathways, with P. bovis causing a more severe reaction.