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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.   Supplementary Information  The online version contains supplementary material available at 10.1186/s13567-021-01002-z.

Mycoplasma bovis subverts autophagy to promote intracellular replication in bovine mammary epithelial cells cultured in vitro