Statement of ethics
This study was conducted in accordance with the ethical guidelines of China Agricultural University (CAU; Beijing, China). Prior to the beginning of the study, ethical approval was granted by the Departmental Committee of the College of Veterinary Medicine, CAU.
Antibody and reagents
Enhanced Cell Counting Kit-8 (CCK-8), Ad-mCherry-GFP-LC3B, Ad-GFP-LC3B, Bicinchoninic acid (BCA) protein assay kit, Lyso-Tracker red and radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology) were purchased from Beyotime (Shanghai, China). The PPLO broth was from BD Biosciences (San Jose, CA, USA), whereas horse serum, Fetal Bovine Serum (FBS), Dulbecco modified Eagle medium (DMEM) and Hank balanced salt solution with Ca2+ and Mg2+ (HBSS) were purchased from Hyclone (Logan, UT, USA). Penicillin G, streptomycin, gentamicin, amphotericin and bovine serum albumin (BSA) were from Coolaber (Beijing, China). Collagenase IV, 0.4 mg/mL DNAse I, 0.5 mg/mL hyaluronidase I-S, rapamycin and 3-methyladenine were from Sigma-Aldrich (St. Louis, MO, USA). Polyvinylidene difluoride membrane was from Millipore (Bedford, MA, USA). Coverslips, 4′, 6-Diamidine-2′-phenylindole dihydrochloride (DAPI) and Triton X-100 were all purchased from Solarbio (Beijing, China). An enhanced chemiluminescence (ECL) kit was obtained from Thermo Fisher Scientific Pierce (Rockford, IL, USA). Anti-p62 antibody and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody were purchased from Abcam (Cambridge, MA, USA). Anti-LAMP2 antibody and anti-β-actin antibody were obtained from Beyotime. Anti-LC3B antibody, anti-Beclin1 antibody and anti-cytokeratin 18 (CK18) were from Proteintech (Chicago, IL, USA). Peroxidase-conjugated goat anti-mouse IgG were from ZSGB-BIO (Beijing, China). Goat anti-rabbit IgG were from Beyotime. Alexa fluor conjugated antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and Freund incomplete adjuvant was from Sigma-Aldrich.
M. bovis strain and growth conditions
M. bovis strain PG45 (ATCC 25,523) was purchased from the ATCC. For infection experiments, M. bovis were cultured in PPLO broth with 20% horse serum and penicillin (100 IU/L) in 5% CO2 at 37 °C for 72 h. The PPLO broth was prepared by dissolving 21 g of Difco PPLO medium (BD Biosciences) and 2.5 g of yeast extract (BD Biosciences) in 700 mL of ultrapure water, then autoclaving it at 121 °C for 30 min. M. bovis were collected by centrifugation (8000 × g for 40 min) and then washed with phosphate-buffered saline (PBS). The number of colony forming units was determined by performing ten-fold serial dilutions in PBS and subsequently spotting on PPLOA plates , prepared by supplementing PPLO broth with 20% horse serum and 0.75% agar. The bacteria were suspended in PBS to a cell density of 108 colony-forming units per milliliter (CFU/mL), and the suspension was stored at −70 °C until use .
Mouse anti-M. bovis serum preparation
BALB/c mice, 6–8 weeks old, were immunized with 1 × 106 CFU M. bovis strain PG45 mixed with an equal volume of Freund incomplete adjuvant and delivered by multipoint subcutaneous injections. Mice were immunized 3 times at 2-week intervals. At 2 weeks after the last immunization, mice were euthanized and blood was collected and incubated at 37 °C for 0.5 h, followed by incubation at 4 °C for 0.5 h. Serum from all mice were combined to form a common pool and stored at −20 °C.
Primary bovine mammary gland epithelial cells were collected as described [22, 23] from lactating Holstein cows with clinically healthy udders (milk somatic cell count < 105 cells/mL). Briefly, ~ 100 g of mammary gland tissue was obtained within 30 min after slaughter and transported to the laboratory at room temperature (23 ± 3 °C) in 500 mL Hank balanced salt solution with Ca2+ and Mg2+ (HBSS) supplemented with 500 μL penicillin G (100 mg/mL), 500 μL streptomycin (100 mg/mL), 500 μL gentamicin (100 mg/mL), and 500 μL amphotericin (5 mg/mL).
The following were conducted in a laminar flow hood under sterile conditions. Tissue was washed with HBSS and surface tissue excised and discarded. Interior tissue was minced into 1 cm3 pieces, followed by incubation for 1 h in 500 mL HBSS supplemented with 500 μL penicillin G (100 mg/mL), 500 μL streptomycin (100 mg/mL), 500 μL gentamicin (100 mg/mL), and 500 μL amphotericin (5 mg/mL) at 37 °C. Then, tissue blocks were further minced into 1–5 mm3 pieces that were rinsed several times with HBSS to remove milk and blood. These tissue blocks were transposed into 250 mL HBSS with final concentrations of 0.5 mg/mL collagenase IV, 0.4 mg/mL DNAse I, 0.5 mg/mL hyaluronidase I-S, penicillin G (100 μg/mL), streptomycin (100 μg/mL), gentamicin (100 μg/mL), and amphotericin (5 μg/mL). Digestion was performed for 3 h at 37 °C, with the liquid swirled every 10 min. Then, the suspension was filtered through sterile metal strainers (pore size ~ 1 mm2) and the cells were subsequently collected by centrifugation for 10 min at 1000 g. Pellets were resuspended in 1/2 volume HBSS, filtered through sterile metal strainers (pore size ~ 0.5 mm2), and centrifuged for 10 min at 1000 g. Pellets were re-suspended in ½ volume HBSS, filtered through sterile metal strainers (pore size ~70 μm2), and centrifuged for 10 min at 1000 g. The precipitate was re-suspended in 1 L full medium with 10% FBS and 1 mL penicillin G (100 mg/mL), 1 mL streptomycin (100 mg/mL), and 1 mL amphotericin (5 mg/mL). Cells were purified by differential adhesion, as follows [22, 23]. Cells were transferred into a 150 T flask and incubated in 5% CO2 at 37 °C for 30 min to allow fibroblasts to attach. Epithelial cells were decanted and counted after staining by trypan blue. Cells were seeded in 25 T flasks at a concentration of 1 × 105 cells/mL for routine subculture. One passage was defined as one time subculture and purification was done in passages 1–3. The bMEC in passages 6, 7, 8 and 9 were stained with CK18, a marker of luminal epithelial cells [24, 25], and identified by fluorescence microscopy. Briefly, bMEC were seeded in 6 well-plates with coverslips. When cells reached 60–70% confluence, they were fixed in 4% paraformaldehyde at room temperature for 15 min. Then cells were washed twice in PBS, resuspended in 1 mL PBS, and incubated with anti-CK18 antibody (at a dilution of 1:200 in PBS) at 4 °C overnight (14–16 h). Cells were washed twice in PBS and resuspended in 1 mL PBS, and incubated with Alexa Fluor-conjugated secondary antibodies for 0.5 h at RT. Thereafter, cells were stained with DAPI and imaged with a Nikon A1 LFOV confocal microscope at laser wavelengths of 405 and 488 nm. The purity of bMEC was defined as the proportion of CK18-positive cells among total cells (stained by DAPI). A random selection of 5 fields per sample was analyzed, with 3 repeats conducted for cells in each passage.
Cell infection and gentamicin protection assay
The PG45 strain was used to explore intracellular replication of Mycoplasma in bMEC, following a protocol presented in Figure 1A. Viable M. bovis in the inoculum were counted, as described above. Specifically, cells were seeded at a concentration of 1 × 105 cells/mL in 6 well plates (2 mL per well) 24 h prior to experiments. The bMEC were inoculated (time = 0 h) with M. bovis at multiplicities of infection (MOI) of 1:30 when cells reached 60–70% confluence. After infection at 37 °C for 1 h, the inoculum was removed and cells were washed twice with sterilized PBS, 2 mL/well, to remove nonadherent M. bovis. Thereafter, all extracellular M. bovis were killed by the addition of 2 mL/well DMEM with 400 μg/mL gentamicin for 2 h at 37 °C (time = 1 h). Cells were again washed as described above. Finally, fresh DMEM with 10% fetal bovine serum (FBS) and 20 μg/mL gentamicin was added to the infected cells, 2 mL/well (time = 1 h).
Enumerating intracellular M. bovis
At designated time points, cells were washed thrice with PBS after treatment, as described above and the CFU were enumerated as described . Cells were detached from plates with a 23-gauge needle and syringe; thereafter, bacterial concentrations were confirmed by plating ten-fold serial dilutions . Fold changes in intracellular M. bovis loading were defined as the number of intracellular M. bovis at a subsequent time point, divided by the number of intracellular M. bovis at 3 hpi. The assay was repeated 3 times independently, with 3 biological replicates for each treatment. To enumerate CFU, M. bovis in each cell well were counted, with 6 repeats on plates.
The GFP-LC3B assay is used widely to monitor autophagy or colocalization with cargo [26, 27]. Ad-GFP-LC3B is an adenovirus expressing GFP-LC3B fusion protein, whereas Ad-mCherry-GFP-LC3B expresses mCherry-GFP-LC3B fusion protein ; both were used, in accordance with the manufacturer’s instructions, to transfect bMEC. Briefly, the bMEC were seeded on 6-well plates with coverslips, followed by incubation in 5% CO2 at 37 °C for 12 h. When bMEC density was 40–50% confluence, cells were infected with Ad-mCherry-GFP-LC3B or Ad-GFP-LC3B at MOI of 1:10 in DMEM containing 10% FBS. Then, cells were incubated in 5% CO2 at 37 °C for 24 h. Subsequently, these cells were used in infection experiments. Ad-mCherry-GFP-LC3B was used to demonstrate blockage of the autophagic flux in eukaryocytes . Infection of Ad-mCherry-GFP-LC3B resulted in overexpression of tandem mRFP/mCherry-GFP proteins. In cells lacking an autophagic state, mCherry-GFP-LC3B was present in the cytoplasm as diffuse yellow fluorescence (combined effects of mCherry and GFP). However, in infected cells under an autophagic state, mCherry-GFP-LC3B targeted the autophagic substrate to form mRFP/mCherry puncta. The GFP is sensitive to low pH and the GFP signal is quenched in an acidic or mature autophagosome. The mCherry is stable when exposed to an acidic environment; therefore, quenching the GFP signal causes an LC3B puncta with mCherry signal only. When imaged under confocal microscopy, the mCherry-GFP-LC3B puncta will yield a red shift signal, indicating the autophagy flux was complete. However, if the autophagy flux is blocked, autophagosome maturation is inhibited and mCherry-GFP-LC3B produces yellow puncta. The bMEC were scanned with a Nikon A1 LFOV confocal microscope and numbers of RFP and GFP puncta were calculated [26, 28]. Twenty cells for each sample and at least 60 cells in each group were used for statistical analyses.
Lyso-tracker red staining
Lyso-Tracker Red staining was used to detect acidification of lysosomes or mature autophagosomes . Lyso-Tracker Red is based on DND-99, which is sensitive to a low pH. Lysosomes or mature autophagosomes marked by Lyso-Tracker Red yield red structures when acidification is activated. At designated time points, bMEC were stained with Lyso-Tracker Red. Briefly, cells were incubated with 50 nM Lyso-Tracker Red at 37 °C for 30 min and the fluorescence signal of Lyso-Tracker Red was observed at 0, 1, 3, 6, 9, 12, and 24 hpi with a Nikon A1 LFOV confocal microscope. Randomly selected 5 fields per sample were analyzed for each sample and 3 repeats were conducted.
Induction and inhibition of autophagy
Following infection as described above, autophagy was induced by treatment with 20 μg/mL rapamycin or Hanks’ balanced salt solution (HBSS). Alternatively, autophagy was inhibited by pre-incubating bMEC with 5 mM 3-methyladenine (3-Ma) in full-nutrition medium for 3 h prior to infection. Moreover, effects of rapamycin and 3-Ma on M. bovis viability were determined. Specifically, 1 × 106 CFU/mL M. bovis was incubated in PPLO medium with 20% horse serum, with or without 1 of the 2 compounds. The CFU of M. bovis was determined as described above, after incubation in 5% CO2 at 37 °C for 24 h. Effects of rapamycin or 3-Ma on viability of bMEC was evaluated with CCK-8 assays . Briefly, bMEC were seeded in 96-well plates (1 × 104 cells/well) in 100 μL DMEM with 10% FBS. After incubation in 5% CO2 at 37 °C for 24 h, cells reached 60–70% confluence. Cells were treated with rapamycin (20 μg/mL in DMEM with 10% FBS), or 3-Ma (5 mM in DMEM with 10% FBS) or nothing (Control), followed by 24 h incubation in 5% CO2 at 37 °C. Then, fresh DMEM containing 10% CCK-8 was placed in the well. The cells were incubated in 5% CO2 at 37 °C for 2 h and optical density was determined with a microplate reader (Bio-Rad, Hercules, CA, USA) at 450 nm. The density of treated cells relative to control cells was calculated.
At designated time points, cells were washed 3 times with PBS and total protein extracted with RIPA lysis buffer on ice. The liquid was centrifuged at 12 000 × g for 15 min. Protein concentrations were determined with a BCA protein assay kit, according to the manufacturer’s instructions. For each sample, equal amounts of protein were separated by SDS-PAGE. Subsequently, proteins were transferred onto a PVDF using a semidry blotting system. Blots were first blocked with 5% skim milk in Tween-20/TBS (TBST) at room temperature for 2 h, followed by 3 washes in TBST for 10 min each; then, membranes were incubated with each specific primary antibody overnight at 4 °C. After 3 washes in TBST for 10 min each, membranes were incubated with secondary antibodies for 1 h at room temperature (RT). Signals were detected using an ECL-Plus Western blot detection system and band density analyzed with Image J (National Institutes of Health, Bethesda, MD, USA).
RNA extraction, cDNA synthesis and real-time PCR
At the various time points indicated, bMEC were harvested with 1 mL TransZol Up lysis solution (TransGen Biotech, Beijing, China). Total RNA was extracted with a total RNA extraction kit (TransGen Biotech), according to the manufacturer’s instructions. The cDNA was synthesized using TransScript® II All-in-One First-Strand cDNA Synthesis SuperMix for PCR (TransGen Biotech). Both RNA and cDNA were quantified with a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For autophagy-associated genes, mRNA expression levels were verified with real time PCR. This assay included the following steps: pre-denaturation at 94 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 60 s using the Applied Biosystems StepOnePlus Real Time PCR system (Thermo Fisher Scientific). For melt curve analysis, PCR products were heated from 55 to 95 °C, with the fluorescence signal checked every 0.5 °C to verify the specificity of each pair of primers. Cycle threshold (Ct) values were determined with StepOne TM Software version 2.3 (Thermo Fisher Scientific). In this study, ΔCt = Ct target gene − Ct endogenous control (arithmetic mean of the reference gene), whereas ΔΔCt = ΔCt sample − Ct control (uninfected cells). To visualize impacts of M. bovis on the responses of target genes in bMEC, relative mRNA expression data were presented as 2−ΔΔCt. Real-time quantitative PCR was used to amplify 100 ng of cDNA using the following pairs of primers: cattle LC3B upstream primer 5′-atgccgtccgagaaaacctt-3′ and downstream primer 5′-ccgggattttggtaggatgc-3′; cattle P62 upstream primer 5′-tctgccctgactacgaccta-3′ and downstream primer 5′-ccatgtttcagcttccggag-3′; cattle Beclin1 upstream primer 5′-gacactcagctcaacgtcac-3′ and downstream primer 5′-gcttcctcctgatccaacct-3′; cattle Lamp2a upstream primer 5′-ccgtgtctggagcatttcag-3′ and downstream primer 5′-ggtgtcatcatccagcgaac-3′; cattle GAPDH upstream primer 5′-attgaccttcactacatggt-3′ and downstream primer 5′-acccttcaagtgagccccag-3′. For these studies, GAPDH was the reference gene.
Transmission electron microscopy
The bMEC were digested with trypsin and centrifuged at 1000 × g for 5 min. The cells were washed twice with PBS, fixed with 2.5% glutaraldehyde for at least 2 h, then fixed in 1% osmium tetroxide for 2 h at 4 °C. After dehydration in a graded ethanol series, samples were embedded in epoxy resin-acetone mixtures for 2 h, followed by immersion in a pure resin solution overnight at 37 °C. After polymerization, ultrathin sections were cut, stained with saturated uranyl acetate in 50% ethanol and lead citrate, and examined with a transmission electron microscope (JEM-1400, JEOL, Tokyo, Japan).
Immunofluorescence staining and fluorescence microscopy
To assess co-localization of autophagy-associated genes with intracellular M. bovis, LC3B, Lamp-2a or M. bovis were detected by immunofluorescence staining and confocal laser microscopy . Cells on coverslips were fixed with 4% paraformaldehyde (PFA) for 15 min at RT, then washed twice with PBS. Fixed cells were blocked with 3% bovine serum albumin (BSA) in PBS for 15 min, followed by permeabilization with 0.2% Triton X-100 in PBS for 45 min at room temperature. Afterwards, cells were incubated with an appropriate antibody for 1 h at room temperature, washed 3 times with PBS, then incubated with Alexa Fluor-conjugated secondary antibodies for 0.5 h at RT. Finally, coverslips were stained with fluorescence mounting medium containing DAPI and mounted on glass slides. Images were captured with a Nikon A1 LFOV confocal microscope at laser wavelengths of 405, 561 and 488 nm and analyzed with Image J software with the JaCoP plugin. Representative cells were selected and photographed. Twenty cells for each sample and at least 60 cells in each group were used for statistical analyses.
All assays were repeated 3 times independently, unless otherwise stated. There were 3 biological replicates in every treatment, unless otherwise stated. All data were analyzed by 1-way ANOVA using SPSS 22.0 (IBM Corp., Armonk, NY, USA) and data reported as mean ± standard deviation of 3 independent experiments.
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