Prototheca bovis induces autophagy in bovine mammary epithelial cells via the HIF-1α and AMPKα/ULK1 pathway

Abstract

Prototheca bovis, a highly contagious pathogen, causes bovine mastitis, resulting in premature culling of affected cows and severe economic losses. Infection with P. bovis caused oxidative stress and apoptosis in bovine mammary epithelial cells (bMECs); however, mechanisms underlying P. bovis-induced autophagy remain unclear. Therefore, the autophagy flux induced by P. bovis in bMECs was analyzed by Western blot and laser scanning confocal microscopy. Expression levels of proteins in the HIF-1α and AMPKα/ULK1 pathway, including HIF-1α, AMPKα, p-AMPKα, ULK1, p-ULK1, mTOR, and p-mTOR, plus expression of autophagy-related genes including SQSTM1/p62, Atg5, Beclin1, and LC3II/LC3I, were quantified with Western blot. Infection with P. bovis induced autophagosomes and LC3 puncta in bMECs that were detected using transmission electron microscopy and laser scanning confocal microscopy, respectively. In addition, lysosome-associated proteins Rab7 and LAMP2a, and lysosomal activity were measured with Western blot and laser scanning confocal microscopy. Infection with P. bovis induced an unobstructed autophagic flux, increased protein expression of LC3II/LC3I, and decreased SQSTM1/p62 protein expression at 6 hpi. Furthermore, P. bovis upregulated protein expression in the HIF-1α and AMPKα/ULK1 pathway and increased the ratio of LC3II/LC3I, implying autophagy was activated in bMECs. However, deletion of AMPKα or ULK1 decreased LC3II/LC3I expression levels and LC3 puncta numbers, suggesting that autophagy was inhibited in bMECs. Additionally, deficiency of HIF-1α decreased protein expression of AMPKα and ULK1 as well as LC3 puncta numbers, and autophagy induced by P. bovis was also inhibited in bMECs. At 6 hpi, lysosome-associated protein Rab7 was decreased and LAMP2a was increased, indicating normal autophagy. In contrast, at 12 hpi, expression of Rab7 and LAMP2a proteins indicated that autophagy was inhibited in bMECs at that time. Therefore, we confirmed that P. bovis infection induced autophagy in bMECs via the HIF-1α and AMPKα/ULK1 pathway, with involvement of lysosome-associated protein Rab7 and LAMP2a.

Antioxidative Sirt1 and the Keap1-Nrf2 Signaling Pathway Impair Inflammation and Positively Regulate Autophagy in Murine Mammary Epithelial Cells or Mammary Glands Infected with Streptococcus uberis

“…We identified autophagy as a crucial component of epithelial cell defense against mastitis pathogens, with the involvement of HIF-1α, AMPKα/ULK1 and the PTEN/PI3K-Akt-mTOR pathway [29][30][31]. Furthermore, we also reported that S. uberis infection induced autophagy by decreasing synthesis of pro-inflammatory cytokines [32].…”

Preventive Effects of Apple Polyphenol Extract on High-Fat-Diet-Induced Hepatic Steatosis Are Related to the Regulation of Hepatic Lipid Metabolism, Autophagy, and Gut Microbiota in Aged Mice

“…Both LAMP2 and RAB7 are lysosomeassociated proteins. 42 The expressions of Lamp2 and Rab7 were reduced in the aged HFD group, and RAB7 was reduced in the young HFD group, indicating that autophagy was inhibited by HFD, but increased Rab7 expression after APE intervention indicated the promotion of autophagy in the liver of aged mice in HAP. However, APE intervention did not affect the protein expression of RAB7.…”

“…In this study, the upregulated expression of LAMP1 in aged mice supported the increased autophagic flux by APE intervention. Both LAMP2 and RAB7 are lysosome-associated proteins . The expressions of Lamp2 and Rab7 were reduced in the aged HFD group, and RAB7 was reduced in the young HFD group, indicating that autophagy was inhibited by HFD, but increased Rab7 expression after APE intervention indicated the promotion of autophagy in the liver of aged mice in HAP.…”

In Vitro Activity of Quaternary Ammonium in Prototheca Isolated from Clinical Bovine Mastitis Identified by Mass Spectrometry and PCR Sequencing of the cytb Gene Marker

“…However, only a few of the compounds approached have been assessed in vivo and provided only a temporary resolution of clinical signs [37,40]. The refractoriness of Prototheca species to conventional treatment under in vivo conditions can be attributed to the development of pyogranulomatous reactions [46], high genetic diversity [40], and mechanisms of virulence and evasion of the immune re-sponse [47], such as biofilm production [23,24] and the induction of cellular apoptosis in mammary cells [48,49].…”