Mycoplasma bovis inhibits autophagy in bovine mammary epithelial cells via a PTEN/PI3K-Akt-mTOR-dependent pathway

Abstract

Although autophagy can eliminate some intracellular pathogens, others, e.g., Staphylococcus aureus, Salmonella, Mycoplasma bovis, can evade it. The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway, a key regulator of autophagy, is involved in initiation and promotion of a range of pathological diseases. As the effects of M. bovis on the autophagic pathway are not well documented, our objective was to elucidate the effects of M. bovis infection on the PI3K-Akt-mTOR cellular autophagic pathway in bovine mammary epithelial cells (bMECs). Ultrastructure of bMECs infected with M. bovis was assessed with transmission electron microscopy, co-localization of LC3 puncta with M. bovis was confirmed by laser confocal microscopy, and autophagy-related indicators were quantified with Western blotting and RT-PCR. In M. bovis-infected bMECs, intracellular M. bovis was encapsulated by membrane-like structures, the expression level of LC3-II and Beclin1 protein decreased at the middle stage of infection, degradation of SQSTM1/P62 was blocked, autophagy of bMECs was inhibited, and PI3K-Akt-mTOR protein was activated by phosphorylation. Furthermore, the tumor suppressor PTEN can inhibit the PI3K-Akt signaling pathway through dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate and may be important for cellular resistance to infection. In the present study, the number of intracellular M. bovis was inversely related to the change in the level of autophagy markers (e.g., LC3-II, SQSTM1/P62) within host cells induced by the low knockdown of Akt or PTEN. We concluded that M. bovis-infected bMECs alleviated cellular autophagy through a PI3K-Akt-mTOR pathway, and that PTEN acted as a protective gene regulating autophagy, a key step in controlling infection.

Keywords: Mycoplasma bovis; PI3K-Akt-mTOR pathway; PTEN; autophagy; bovine mammary epithelial cells.

Figure 1

M. bovis infection induced autophagy in bMECs. (A) Ultra-microstructure observations of intracellular M. bovis, bMECs control (a) or M. bovis strain PG45 infected at an MOI of 30 for 3 h (c); the black arrow is an autophagosome-like membrane vesicle. Control and M. bovis-infected cells were fixed and processed for electron microscopy. Scale bars, 2  μm (a,c) and 1  μm (b,d). (B,C) bMECs were control or infected with M. bovis (MOI = 30) for 0, 3, 6, 9, 12, and 24  h. At the end of the infection, expression levels of LC3, SQSTM1, Beclin1, ATG5, and β-actin (loading control) were analyzed by Western blotting with specific antibodies. (D–G) Relative quantification of LC3-II, SQSTM1, Beclin1, ATG5 protein levels compared to LC3-I or β-actin protein levels was determined by densitometry, and the ratio of M. bovis infected group to control group protein levels was calculated. (H) Ad-GFP-LC3B transfected bMECs for 24  h and then cells were infected with M. bovis (stained red with DiI, MOI = 30). Cells were fixed and nuclei counterstained with DAPI prior to confocal laser microscopy. Scale bars: 20  μm. Data represent mean ± SD of 3 independent experiments. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 2

The role of PTEN/PI3K-Akt-mTOR pathway in M. bovis affecting autophagy in bMECs. (A,B) bMECs were control or infected with M. bovis (MOI = 30). Cells were harvested at 0, 3, 6, 9, 12, and 24  h and then Western blotted with anti-PTEN, anti-p-PI3K, anti-p-mTOR, anti-mTOR, anti-p-Akt, anti-Akt, and anti-β-actin (loading control) antibodies. (C) PTEN protein levels relative to β-actin were determined by densitometry. (D) p-PI3K protein levels relative to β-actin were determined by densitometry. (E) p-mTOR levels relative to mTOR were determined by densitometry. (F) p-Akt levels relative to Akt were determined by densitometry. The ratio of M. bovis infected group to control group protein levels was calculated. Data represent the mean ± SD of 3 independent experiments. ^*p < 0.05; ^**p < 0.01.

Figure 3

PTEN promoted autophagy of bMECs by inhibiting PI3K-Akt–mTOR pathway to suppress replication of M. bovis. (A) bMECs were transfected with siRNA-Akt (25  nM) and siRNA-PTEN (25  nM) for 48  h and then infected with M. bovis (MOI = 30). Co-localization of LC3B (green) with M. bovis (red) was detected by confocal microscopy at 6  h post-infection. Cell nuclei were counterstained with DAPI. Scale bar: 10 and 20  μm. (B) Comparison of intracellular replication of M. bovis in bMECs with downregulated Akt or PTEN status. bMECs were transfected with siRNA-Akt (25  nM) and siRNA-PTEN (25  nM) for 48  h and then infected with M. bovis (MOI =30); the intracellular M. bovis load was measured at 3, 6, 12, and 24  h postinfection. Data represent mean ± SD of 3 independent experiments. ^*p < 0.05; ^***p < 0.001.

Figure 4

Phosphorylation of Akt was positively related to inhibition of autophagy by M. bovis. (A) Effects of CI-1033 on viability of bMECs in DMEM medium containing 10% FBS based on CCK-8 assays. (B) Cells were pre-treated with CI-1033 (1  μm) for 1  h and then control with DMSO, and cells infected with M. bovis (MOI = 30) were further cultured for 6  h. Thereafter, cell samples were analyzed by Western blotting with anti-Akt, anti-p-Akt, anti-LC3, anti-SQSTM1, and anti-GAPDH (loading control) antibodies. (C) Relative quantification of p-Akt protein levels compared to Akt protein, LC3-II protein levels compared to LC3-I protein, and SQSTM1 protein levels compared to GAPDH protein was determined by densitometry in M. bovis-infected bMECs in the absence or presence of CI-1033. (D) Transcriptional levels of PI3K-Akt–mTOR signaling pathway and autophagy-related genes including Akt, LC3-II and SQSTM1, were detected by real-time PCR in bMECs. The data represent the mean ± SD of 3 independent experiments. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 5

Inhibition of Akt with specific siRNAs targeting Akt reversed inhibition of autophagy in bMECs by M. bovis. (A) bMECs were transfected with siRNA-control (NC; 25  nM) and siRNA-Akt-1/2/3 (25  nM) for 48  h. Akt protein expression were analyzed by Western blotting. (B) bMECs were transfected with siRNA-Akt-1 targeting Akt for 48  h; then, cells were infected with M. bovis (MOI = 30) for 6  h. Thereafter, cell samples were analyzed by Western blotting with anti-Akt, anti-p-Akt, anti-LC3, anti-SQSTM1, and anti-β-actin (loading control) antibodies. (C) Relative quantification of target protein levels compared to β-actin protein was determined by densitometry in transfected siRNA-Akt-1 cells. (D) Transcriptional levels of PI3K-Akt–mTOR signaling pathway and autophagy-related genes including Akt, LC3-II and SQSTM1, were detected by real-time PCR in bMECs. Data represent mean ± SD of 3 independent experiments. ^#p > 0.05; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 6

Absence of PTEN expression exacerbated inhibition of autophagy of bMECs by M. bovis. (A) bMECs were transfected with siRNA-control (NC; 25  nM) and siRNA-PTEN-1/2/3 (25  nM) for 48  h. PTEN protein expression was analyzed by Western blotting. (B) bMECs were transfected with siRNA-PTEN-1 targeting PTEN for 48  h; then, cells were infected with M. bovis (MOI = 30) for 6  h. Thereafter, cell samples were analyzed by Western blotting with anti-PTEN, anti-Akt, anti-p-Akt, anti-Beclin1, anti-LC3, and anti-β-actin (loading control) antibodies. (C) Relative quantification of target protein levels compared to β-actin protein was determined by densitometry in transfected siRNA-PTEN-1 cells. (D) Transcriptional levels of PI3K-Akt–mTOR signaling pathway and autophagy-related genes including Akt, Beclin1 and LC3-II, were detected by real-time PCR in bMECs. Data represent the mean ± SD of 3 independent experiments. ^#p > 0.05; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 7

Proposed model for inhibition of autophagy by M. bovis via the PTEN/PI3K-Akt-mTOR pathway. Infection of invading bMECs by M. bovis activated phosphorylation of Akt and inhibited the onset of downstream autophagic activity, preventing autophagosomes from delivering engulfed M. bovis to lysosomes for degradation, thereby enabling M. bovis to survive. Yellow arrow and red bar indicate stimulation and inhibition, respectively. This figure was partially made using Servier Medical Art (smart.servier.com).