All experimental procedures with animals were conducted according to the guidelines for the care and use of experimental animals that have been established by the Inner Mongolia University Animal Care and Use Committee.

Primary BMECs were isolated and identified as described (52). Briefly, mammary tissue was obtained from Chinese Holstein cows after being slaughtered on a commercial cattle slaughter farm. After surgical removal of mammary tissue from the slaughtered cow, it was placed in sterile, ice-cold phosphate-buffered saline (PBS) that was supplemented with 300 U/mL penicillin G and 100 mg/mL streptomycin (Sigma-Aldrich, Inc., USA) and transported immediately to the laboratory. Purified primary BMECs were isolated and maintained in DMEM/F12 medium (Hyclone Laboratories, Inc., Logan, UT, USA) that contained 10% fetal bovine serum. Cells were cultured in 25 cm² tissue culture flasks at 37°C in humidified air with 5% CO₂. P2 to P4 BMECs that were in the logarithmic growth phase were used for all experimental assays.

Glu (Cat# G8415), Asp (Cat# A7219), and Leu (Cat# L8912) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). β-casein (Cat# EIA06975Bo) was purchased from Wuhan Xinqidi (Wuhan Xinqidi Biological Technology Co. Ltd. Wuhan, China). Rapamycin (Cat# 53123-88-9) was purchased from Gene Operation (Gene Operation, Ann Arbor, MI, USA). Rapamycin was dissolved in ethanol (Sigma-Aldrich, Inc., USA) to a stock concentration of 50 mg/mL, stored at −20°C, and diluted to the appropriate final concentration with culture medium before use. The concentration of ethanol in the final solution did not exceed 0.5% (v/v) in any experiment. β-mercaptoethanol (Cat# M8211) was obtained from Solarbio (Solarbio Science & Technology, Co., Ltd. Beijing, China). Dil (Cat# KGMP002) was purchased from KeyGEN (KeyGEN BioTECH, Co., Ltd. Jiangsu, China). DAPI (Cat# C1005) and Hoechst 33342 Staining Kit (Cat# C1022) were acquired from Beyotime (Beyotime Biotechnology, Co., Ltd. Shanghai, China). Annexin V-FITC/PI Cell Apoptosis Detection Kit (Cat# FA101) was purchased from TransGen (TransGen Biotech Co. Ltd. Beijing, China). Alexa Fluor® 594 Phalloidin (Cat# A12381) was purchased from Invitrogen (Invitrogen, Carlsbad, New Mexico, USA). 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE) were purchased from Sigma (Sigma-Aldrich, Inc. St. Louis, MO, USA).

Anti-4EBP1 (Cat# ab2606), anti-p-mTOR (Ser2448) (Cat# ab109268), anti-mTOR (Cat# ab32028), goat anti-rabbit (Cat# ab136817), and goat anti-mouse (Cat# ab205719) were purchased from Abcam (Abcam plc 330 Cambridge Science Park, Cambridge, UK). Anti-p-S6 (Ser240/244) (Cat# 5364s), anti-p-4EBP1 (Thr37/46) (Cat# 2855s), anti-p-NF-κB p65 (Ser536) (Cat# 3033), anti-NF-κB p65 (Cat# 8242), and anti-p-STAT5 (Tyr694) (Cat# 4322) were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., Beverley, MA, USA). Anti-S6 (Cat# sc-74459) was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., 10410 Finnell Street Dallas, Texas 75220 USA). Anti-STAT5A (Cat# 13179-1-AP), anti-SLC1A3 (Cat# 20785-1-AP), anti-SLC7A5 (Cat# 13752-1-AP) and anti-Caspase 3 (Cat# 66470-2-Ig) were purchased from Proteintech (Proteintech Group, Inc., 5500 Pearl Street, Suite 400 Rosemont, IL 60018, USA). Goat FITC-conjugated anti-rabbit IgG (Cat# 115-095-003) and FITC-conjugated anti-mouse IgG (Cat# 115-095-146) were purchased from Jackson (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Anti-β-actin (Cat# A5441) was purchased from Sigma (Sigma-Aldrich, Inc., St. Louis, MO, USA).

BMECs were infected with S. aureus (ATCC 27543) for 2 h at an MOI of 30, and the extracellular bacteria were killed and lysed with antibiotics and lysozyme. Intracellularly infected cell cultures were continued and maintained in medium for 2, 4, and 8 h. The cells were lysed, and the number of intracellular bacteria was determined by spread plate method.

BMECs were seeded on a slide and incubated overnight. Bacteria (S. aureus) were washed with PBS and then incubated with CFSE [5 (6)-carboxyfluorescein diacetate N-succinimidyl ester] at 4°C for 15 min. The stained bacteria were centrifuged for 10 min at 3,000 × g at 4°C 3 times. Cells were infected by the stained bacteria at an MOI of 30 for 2 h, washed three times with PBS, and fixed with 4% paraformaldehyde for 20 min. After being treated with 1% Triton X-100 for 5 min, the cells were stained with Alexa Fluor® 594 Phalloidin for 1 h in the dark, washed three times with PBS, and counterstained with 100 μl DAPI for 3 min to assess the nuclear morphology. Finally, the slide was mounted with glycerin for examination under a laser scanning confocal microscope (NIKON A1R, Nikon Corp., Tokyo, Japan).

BMECs were infected with bacteria (S. aureus) for 2 h at an MOI of 30, and the extracellular bacteria were killed and lysed with antibiotics and lysozyme for 2 h. The infected cells were washed three times with PBS, centrifuged for 10 min at 3,000 × g at 4°C, and fixed with 2.5% glutaraldehyde overnight, the precipitation was wrapped in the 1% agarose. Agarose blocks with samples avoid light post fixed with 1% OsO₄ in 0.1 M PB (pH 7.4) for 2 h at room temperature. The tissues were sequentially fed with 30%-50%-70%-80%-95%-100%-100% alcohol and dehydrated for 20 min each time, 100% acetone twice, 15 min each time. Resin penetration and embedding as followed: Acetone, EMBed 812 = 1:1 for 2–4 h at 37°C; Acetone, EMBed 812 = 1:2 overnight at 37°C; pure EMBed 812 for 5–8 h at 37°C; Pouring the pure EMBed 812 into the embedding models and insert the tissues into the pure EMBed 812, and then keep in 37°C overnight. The embedding models with resin and samples were moved into 65°C to polymerize for more than 48 h. The resin blocks were cut to 60–80 nm thin on the ultra microtome, and the tissues were fished out onto the 150 meshes cuprum grids with formvar film, and the 2% uranium acetate saturated alcohol solution avoid light staining for 8 min and then rinsed in 70% ethanol for three times. 2.6% Lead citrate avoid CO₂ staining for 8 min. After dried by the filer paper, the cuprum grids were put into the grids board and dried overnight at room temperature. Finally, the samples were examined by TEM (Hitachi HT7700, Hitachi, Ltd., Tokyo, Japan) to detect intracellular bacteria.

BMECs were seeded into 6-well plates, incubated until 80% confluence, and treated with the indicated conditions, including infection with S. aureus; serum and amino acid starvation, followed by amino acid stimulation and rapamycin.

To examine how S. aureus invasion suppresses milk protein synthesis, BMECs were infected with S. aureus for 2 h, and the extracellular bacteria were killed and lysed with antibiotics and lysozyme. The intracellularly infected cells were continued in culture and maintained in medium for 8 h. Cell culture supernatants were collected to measure extracellular β-casein, α-lactalbumin, and β-lactoglobulin using ELISA kits (Wuhan Xinqidi Biological Technology Co. Ltd. Wuhan, China) per the manufacturer's instructions. Intracellularly infected cells were harvested with trypsin and centrifuged to remove the supernatant, and cell lysates were prepared through five freeze-thaw cycles. The total protein concentration of the control and treatment groups was standardized by adjusting the volume of the protein lysate. An equal volume of each total protein lysate was analyzed for β-casein, α-lactalbumin, and β-lactoglobulin by ELISA.

To determine how rapamycin treatment decreases milk protein synthesis, BMECs were treated with 100 nM rapamycin for 8 h, the cell culture supernatants were collected, the cells were harvested, and cell lysates were prepared and standardized. The levels of extracellular and intracellular β-casein, α-lactalbumin, and β-lactoglobulin were analyzed by ELISA.

To examine how exogenous amino acids induce casein synthesis, BMECs were serum-starved for 16 h, amino acid-starved for 1 h, and then stimulated with amino acids for 1 h. Control and treated cells were harvested, and cell lysates were prepared and standardized. The level of intracellular β-casein was analyzed by ELISA.

To study the suppression of amino acid uptake by S. aureus, BMECs were serum-starved for 16 h, amino acid-starved for 1 h, stimulated with amino acids for 1 h, and infected intracellularly with S. aureus for 8 h. Cell culture supernatants were collected to measure Glu, Asp, and Leu using ELISA kits (Wuhan Xinqidi Biological Technology Co. Ltd. Wuhan, China).

To examine how S. aureus invasion suppresses amino acid induced-casein synthesis, four groups of BMECs were compared: control, amino acid induction (Glu, Asp, Leu), S. aureus invasion (8 h), and amino acid stimulation with bacterial infection. BMECs were serum starved for 16 h, amino acid-starved for 1 h, stimulated with amino acids for 1 h, and infected intracellularly with S. aureus for 8 h. Control and treated cells were harvested, and cell lysates were prepared and standardized. Intracellular β-casein was analyzed by ELISA.

To determine the contents of alpha-hemolysin (Hlα) and Plasmin, BMECs were infected with S. aureus for 2 h, and the extracellular bacteria were killed and lysed with antibiotics and lysozyme. The contents of α-hemolysin (Hla), and Plasmin in cell medium and in cells were measured after infection 8 h using ELISA kits (Wuhan Xinqidi Biological Technology Co. Ltd. Wuhan, China). To determine whether β-casein was directly degraded by S. aureus in culture medium, 1.5 × 10³ CFU/mL S. aureus were inoculated into DMEM/F12 medium which β-casein was dissolved to the final concentration of 1 μg/mL, and maintained in 37°C. The content of β-casein was determined after 8 h.

Absorbance at 450 and 630 nm was read on a Varioskan Flash Multimode Reader (Thermo Fisher Scientific, Pittsburgh, PA, USA). All measurements were performed in triplicate, and the mean value of the 3 independent measurements was used for statistical analysis.

Western blot was used to measure the indicated proteins and phosphorylated proteins as described (53). Briefly, BMECs were managed as four groups, i.e., control cells (uninfected cells), cells were infected by S. aureus 2, 4, and 8 h, respectively. Four groups of cells were culture in medium simultaneously, and then three infected groups were inoculated with S. aureus at different time points and continued in co-culture. Finally, the cells were harvested with trypsin at the same time. The harvested BMECs were washed with cold PBS, and lysed in cell lysis buffer. The lysis buffer comprised 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, PMSF, and phosphatase inhibitors. Equal amounts (40 μg) of protein were electrophoresed on 10% (w/v) sodium dodecyl sulfate-polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and incubated with the primary antibody. Peroxidase-conjugated secondary antibody and enhanced chemiluminescence (ECL) reagent were used to detect the signals with the Western Blotting System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). The bands were quantified on a Gel-Pro Analyzer 4.0 (Media Cybernetics, USA).

RT-qPCR was performed to measure EAAT1/GLAST/SLC1A3 and LAT1/SLC7A5 in BMECs in the treatment and control groups. Cells were infected with S. aureus for 2, 4, and 8 h or treated with 100 nM rapamycin for 8 h, and total RNA was extracted from untreated and treated cells. Total RNA was prepared with RNAiso Plus per the manufacturer's instructions (9109, TaKaRa Co. Ltd., Dalian, China). Briefly, the cells were washed with PBS and lysed in RNAiso Plus, and chloroform was added to the cell lysates for homogenization; the top aqueous layer was transferred to a new tube after centrifugation, and isopropanol was added to the supernatant and mixed well. Total RNA was precipitated by centrifugation, and the pellet was dissolved in RNase-free water.

mRNA was reverse-transcribed with oligo (dT)₁₂₋₁₈ primer using the AMV first Strand cDNA Synthesis Kit (Takara Co. Ltd., China). cDNA sequences were amplified with the primers in Supplementary Table S1. The KAPA SYBP FAST qPCR Kit Optimized for LightCycler 480 (KAPA, Inc., Boston, MA, USA) was used for the PCR with the primers (Supplementary Table S1), according to the manufacturer's instructions. The program comprised an initial denaturation step at 95°C for 5 min; 40 cycles of 95°C for 5 s, 54°C for 30 s, and 72°C for 20 s; and a final extension of 72°C for 10 min. Three technical replicates were run in each experiment. 2-ΔΔCT values were calculated to determine expression levels, and the qPCR results were compared by student's t-test between untreated and treated groups. Three independent experiments were performed.

Cells were seeded onto a slide, incubated overnight, and infected with S. aureus for 2, 4, and 8 h. After being washed with PBS and fixed with 4% paraformaldehyde for 15 min, the cells were blocked with 1% BSA for 1 h. Then, the cells were incubated with primary antibodies against SLC1A3 and SLC7A5, p-STAT5 and p-NF-κB p65 at 4°C overnight and FITC-labeled goat anti-rabbit IgG for 1 h at room temperature. DAPI was used to stain the nucleus. Finally, the slide was mounted with glycerin and examined under a laser scanning confocal microscope (NIKON A1R, Nikon Corp., Tokyo, Japan).

Adhesion assay of S. aureus was achieved in two phases. First, BMECs were infected with S. aureus at MOI 30 for 30, 60, and 90 min, respectively. End of infection, BMECs were continued and maintained for 8 h in medium with antibiotics and lysozyme. After incubation, BMECs were harvested with trypsin and washed softly three times with PBS to remove extracellular dead bacteria, and then lysed using lysis buffer. The number of intracellular bacteria was determined by bacterial colony count. Second, BMECs were infected with S. aureus for 30 min at MOI 30, and then the cells were cultured for 8 h in a medium with antibiotics and lysozyme. After incubation, BMECs were harvested with trypsin and washed softly three times with PBS to remove non-adherent bacteria, remaining bacteria considered to be adherent but not internalized in cells, and then lysed using lysis buffer. The bacteria were evaluated by bacterial colony count, which were considered as adherent bacteria.

BMECs were infected with S. aureus at MOI 30 for 2 h, then the antibiotics and lysozyme were used to kill and lyse the extracellular bacteria. The intracellularly infected cells were continued in culture and maintained in medium for 8 h, and then the apoptosis were assessed with the Hoechst 33342 Staining Kit and FITC Annexin V Apoptosis Detection Kit according to the manufacturer's instructions, respectively. Following treatment, cells were stained with Hoechst for 5 min and washed with PBS, followed by observation under a fluorescence microscope (Observer A1, Zeiss, Oberkochen, Germany). For the flow cytometry assay, cells were collected after treatment, and washed with PBS, and then stained with FITC-Annexin V and PI. Cells were subsequently analyzed using flow cytometry (Cytoflex, Beckman, CA, USA).

Statistical analyses were conducted using SPSS PASW Statistics for Windows, v18.0 (SPSS Inc.: Chicago, IL, USA). Data were analyzed using standard parametric statistics and one-way ANOVA, followed by Tukey's method. Data are expressed as mean ± SD. The results are presented as the average of at least 3 independent experiments. Western blot results were quantified on a Gel-Pro Analyzer 4.0 (Media Cybernetics, USA). Statistical significance was accepted when p ≤ 0.05.

In order to confirm S. aureus can be internalized by BMECs, we infected BMECs with S. aureus for 2 h at MOI 30 and then killed and lysed the extracellular bacteria with antibiotics and lysozyme. The infected cells were maintained in medium for 2-8 h, and the bacteria were evaluated intracellularly and extracellularly by bacterial colony count. The results showed that 1.5 × 10³ CFU/mL − 5.1 × 10³ CFU/mL were counted in whole BMEC lysates, whereas extracellular bacteria were not found in the culture medium (Supplementary Table S2), indicating that intracellular S. aureus proliferated. Further, to confirm S. aureus invasion of BMECs, we stained S. aureus with fluorescent dye and observed the bacteria under a laser scanning confocal microscope (LSCM) (Supplementary Figure S1A). S. aureus was also found in cytosolic vacuoles in BMECs by TEM (Supplementary Figure S1B). These results indicate that S. aureus was internalized by BMECs in vitro.

To examine whether S. aureus invasion suppresses the synthesis of milk protein in BMECs, cells were infected with S. aureus at 30 MOI for 2 h, and extracellular bacteria were then killed and lysed with antibiotics and lysozyme. The infected cells were maintained in medium for 8 h, and β-casein, α-lactalbumin, and β-lactoglobulin were determined by ELISA. The levels of β-casein, α-lactalbumin and β-lactoglobulin decreased intracellularly (Figures 1A–C) and in medium (Figures 1D–F), indicating that S. aureus invasion inhibits the synthesis of milk protein in BMECs.

Considering milk protein can be degraded by endogenous protease or bacterial enzymes, to eliminate the possibility of milk proteins were degraded by these enzymes, we first determined the level of endogenous protease Plasmin by ELISA in S. aureus infected cells, and found that the level of Plasmin was not increased in the culture medium and in cells of the S. aureus-infected cells, compared to control (Supplementary Figure S2A). Next, to determine whether β-casein was directly degraded by S. aureus during infection of 8 h, we simulated the conditions under which S. aureus infected BMECs, i.e., 1.5 × 10³ CFU/mL S. aureus (Supplementary Table S2) were inoculated into DMEM/F12 medium which β-casein, and the content of β-casein was determined by ELISA after 8 h. Comparing to the control, the level of β-casein did not show significant decline in infected group (Supplementary Figure S2B). These data indicate that the decrease in milk protein content was caused by intracellular infection of S. aureus, rather than by both endogenous and bacterial enzymes. Then, to eliminate the possibility of apoptosis induced by S. aureus leading to the decrease of milk protein, we examined apoptosis 8 h after S. aureus infection, and the results showed that no apoptosis was found in BMECs (Supplementary Figures S3A–C), suggesting that the decrease of milk protein was not caused by apoptosis.

Epithelial cells are the central component of bovine mammary alveoli, which produce milk during lactation. Mammary epithelial cells are considered to derive amino acids from blood to synthesize milk proteins. Thus, we tested whether S. aureus invasion prevents cells from absorbing amino acids from the culture medium. Control and S. aureus-infected BMECs were subjected to serum and amino acid starvation, after which Glu, Asp, and Leu were added to the medium and measured by ELISA. The levels of Glu, Asp, and Leu in the medium of S. aureus-infected cells was significantly higher than that in the control (Figures 2A–C), indicating that S. aureus prevents BMECs from taking up amino acids from the culture medium.

Amino acids initiate mTORC1 signaling to promote protein synthesis. Thus, we speculated that mTORC1 activation and milk protein synthesis are stimulated by exogenous amino acids in BMECs. We treated serum- and amino acid-starved cells with Glu, Asp, and Leu and measured mTORC1 activation and β-casein (Csn 2) in BMECs. The results showed that mTORC1 activation (Figures 3A–C) and β-casein (Figure 3D) were increased in BMECs, demonstrating that exogenous amino acids initiate mTORC1 activation and induce β-casein (Csn 2) synthesis.

To characterize the suppression of amino acid-induced casein synthesis by S. aureus, β-casein was measured in four groups of BMECs: control, amino acid-treated (Glu, Asp, Leu), S. aureus invasion, and S. aureus invasion with amino acids (Glu, Asp, Leu). Exogenous amino acids significantly increased β-casein concentrations, an effect that S. aureus infection mitigated (Figures 4A–C). These data indicate that S. aureus invasion suppresses amino acid-induced casein synthesis.

To validate the underlying mechanism by which S. aureus invasion prevents amino acid uptake and amino acid-induced casein synthesis in BMECs, we examined the expression of SLC1A3(EAAT1/GLAST) and SLC7A5(LAT1) by RT-qPCR. The mRNA levels of SLC1A3 and SLC7A5 increased at 2 and 4 h and declined at 8 h in S. aureus-invaded BMECs (Figures 5A,B). SLC1A3 and SLC7A5 were detected by western blot and immunofluorescence, following the same trend as the mRNA levels in S. aureus-infected BMECs (Figures 5C–E). These results suggest that S. aureus invasion impairs the expression of amino acid transporter genes at the mRNA and protein levels in BMECs.

Based on the findings, we speculated that certain transcription factors that are related to these genes are also impaired in S. aureus-invaded BMECs. To identify transcription factors for SLC1A3 and SLC7A5, we performed a bioinformatic analysis, which predicted NF-κB and STAT5 (Supplementary Figure S4, S5). Further, we examined the phosphorylation of NF-κB and STAT5 and nuclear localization by western blot and immunofluorescence. We found that the phosphorylation of NF-κB and STAT5 was reduced, and the nuclear translocation of phosphorylated NF-κB p65 and STAT5 was also attenuated at 8 h after bacterial infection (Figures 6A–C). These data suggest that SLC1A3 and SLC7A5 expression is directed by NF-κB and STAT5 in BMECs. The experiments above demonstrate that exogenous amino acids induce mTORC1 signaling and that S. aureus invasion prevents the uptake of exogenous amino acids. Thus, we speculated that S. aureus invasion decreases the activity of mTORC1 in BMECs. In S. aureus-infected BMECs, the phosphorylation of mTOR and S6 fell 8 h after invasion (Figure 6D), indicating that mTORC1 is involved in the expression of SLC1A3 and SLC7A5.

Considering non-invasive S. aureus is also very important in mastitis, to eliminate the possibility of S. aureus adhesion to affect the activity of mTORC1, and NF-κB and STAT5, and the expression of SLC1A3 and SLC7A5, we first examined adhesion of S. aureus to BMECs, and found that the bacteria only adhered to BMECs but failed to internalize within 30 min. Next, BMECs were infected with S. aureus for 30 min, and then the cells were cultured for 8 h in a medium with antibiotics and lysozyme. The content of β-casein, α-lactalbumin, and β-lactoglobulin in cell culture medium was determined by ELISA, and the expression of the targeting proteins by Western blot. The results showed that there was no significant difference between the bacterial adhesion group and the control group (Supplementary Figures S6A–C), indicating that S. aureus adhesion has no effect on the milk protein synthesis, mTORC1 signaling, the activity of NF-κB p65 and STAT5, and the expression of SLC1A3 and SLC7A5. Further, to eliminate the possibility of S. aureus toxin effect on milk protein synthesis, alpha-hemolysin (Hlα), which is the most abundant toxin in S. aureus, was determined by ELISA after infection 8 h. The results showed that α-hemolysin was not detectable both in cell medium and cells (Supplementary Figure S7), suggesting that toxins were not produced by S. aureus within 8 h.

To verify the mTORC1 pathway regulates the expression of SLC1A3 and SLC7A5 through NF-κB and STAT5 is being affected during S. aureus infection, and demonstrate the function of mTORC1 signaling in the expression of SLC1A3 and SLC7A5 in BMECs, cells were managed as four groups, i.e., control cells, cells were infected by S. aureus 8 h, cells were treated by 100 nM rapamycin 8 h and cells were both infected by S. aureus and treated by 100 nM rapamycin 8 h, respectively. The phosphorylation of NF-κB and STAT5 and SLC1A3 and SLC7A5 expression were examined by western bolt and RT-qPCR. The results showed that the phosphorylation of mTOR, S6, NF-κB, and STAT5 was inhibited by S. aureus and rapamycin in BMECs (Figure 7A), and SLC1A3 and SLC7A5 mRNA and protein were downregulated by S. aureus and rapamycin (Figures 7A,B), indicating that mTORC1 pathway was inhibited during the S. aureus infection, and mTORC1 regulates the expression of SLC1A3 and SLC7A5 via NF-κB and STAT5.

Next, to confirm the function of mTORC1 in milk protein synthesis, we measured β-casein, α-lactalbumin, and β-lactoglobulin intracellularly and in the culture medium. Milk proteins synthesis (Figures 8A–C) and secretion (Figures 8D–F) were lower, indicating that mTORC1 signaling controls milk protein synthesis in BMECs. These data demonstrate that mTORC1 governs milk protein synthesis by regulating the expression of SLC1A3 and SLC7A5 in BMECs.