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Autophagy of bovine mammary epithelial cell induced by intracellular Staphylococcus aureus

  • Microbial Pathogenesis and Host-Microbe Interaction
  • Published:
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Abstract

Bovine mastitis is a common disease in the dairy industry that causes great economic losses. As the primary pathogen of contagious mastitis, Staphylococcus aureus (S. aureus) can invade bovine mammary epithelial cells, thus evading immune defenses and resulting in persistent infection. Recently, autophagy has been considered an important mechanism for host cells to clear intracellular pathogens. In the current study, autophagy caused by S. aureus was detected, and the correlation between autophagy and intracellular S. aureus survival was assessed. First, a model of intracellular S. aureus infection was established. Then, the autophagy of MAC-T cells was evaluated by confocal microscopy and western blot. Moreover, the activation of the PI3K-Akt-mTOR and ERK1/2 signaling pathways was determined by western blot. Finally, the relationship between intracellular bacteria and autophagy was analyzed by using autophagy regulators (3-methyladenine [3-MA], rapamycin [Rapa] and chloroquine [CQ]). The results showed that S. aureus caused obvious induction of autophagosome formation, transformation of LC3I/II, and degradation of p62/SQSTM1 in MAC-T cells; furthermore, the PI3K-Akt-mTOR and ERK1/2 signaling pathways were activated. The number of intracellular S. aureus increased significantly with autophagy activation by rapamycin, whereas the number decreased when the autophagy flux was inhibited by chloroquine. Therefore, this study indicated that intracellular S. aureus can induce autophagy and utilize it to survive in bovine mammary epithelial cells.

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References

  • Bah, A. and Vergne, I. 2017. Macrophage autophagy and bacterial infections. Front. Immunol.8, 1483.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bardiau, M., Detilleux, J., Farnir, F., Mainil, J.G., and Ote, I. 2014. Associations between properties linked with persistence in a collection of Staphylococcus aureus isolates from bovine mastitis. Vet. Microbiol.169, 74–79.

    Article  PubMed  Google Scholar 

  • Batavani, R.A., Asri, S., and Naebzadeh, H. 2007. The effect of subclinical mastitis on milk composition in dairy cows. Iran. J. Vet. Res.8, 205–211.

    Google Scholar 

  • BenYounès, A., Tajeddine, N., Tailler, M., Malik, S.A., Shen, S., Métivier, D., Kepp, O., Vitale, I., Maiuri, M.C., and Kroemer, G. 2011. A fluorescence-microscopic and cytofluorometric system for monitoring the turnover of the autophagic substrate p62/SQSTM1. Autophagy7, 883–891.

    Article  PubMed  CAS  Google Scholar 

  • Chen, N. and Karantza-Wadsworth, V. 2009. Role and regulation of autophagy in cancer. Biochim. Biophys. Acta1793, 1516–1523.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cheng, P., Ni, Z., Dai, X., Wang, B., Ding, W., Rae Smith, A., Xu, L., Wu, D., He, F., and Lian, J. 2013. The novel BH-3 mimetic apogossypolone induces Beclin-1- and ROS-mediated autophagy in human hepatocellular carcinoma [corrected] cells. Cell Death Dis.4, e489.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Choi, J., Jo, M., Lee, E., and Choi, D. 2014. ERK1/2 is involved in luteal cell autophagy regulation during corpus luteum regression via an mTOR-independent pathway. Mol. Hum. Reprod.20, 972–980.

    Article  CAS  PubMed  Google Scholar 

  • Fortunato, F. and Kroemer, G. 2009. Impaired autophagosome-lysosome fusion in the pathogenesis of pancreatitis. Autophagy5, 850–853.

    Article  CAS  PubMed  Google Scholar 

  • Francoz, D., Bergeron, L., Nadeau, M., and Beauchamp, G. 2012. Prevalence of contagious mastitis pathogens in bulk tank milk in Québec. Can. Vet. J.53, 1071–1078.

    PubMed  PubMed Central  Google Scholar 

  • Haugaard, K., Tusell, L., Perez, P., Gianola, D., Whist, A.C., and Heringstad, B. 2013. Prediction of clinical mastitis outcomes within and between environments using whole-genome markers. J. Dairy Sci.96, 3986–3993.

    Article  CAS  PubMed  Google Scholar 

  • Jiang, P. and Mizushima, N. 2015. LC3- and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells. Methods75, 13–18.

    Article  CAS  PubMed  Google Scholar 

  • Jung, C.H., Ro, S.H., Cao, J., Otto, N.M., and Kim, D.H. 2010. mTOR regulation of autophagy. FEBS Lett.584, 1287–1295.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y., and Yoshimori, T. 2000. LC3/ a mammalian homolog of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J.19, 5720–5728.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kabeya, Y., Mizushima, N., Yamamoto, A., Oshitani-Okamoto, S., Ohsumi, Y., and Yoshimori, T. 2004. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J. Cell Sci.117, 2805–2812.

    Article  CAS  PubMed  Google Scholar 

  • Kim, J., Choi, S., Kim, J.O., and Kim, K.K. 2018. Autophagy-mediated upregulation of cytoplasmic claudin 1 stimulates the degradation of SQSTM1/p62 under starvation. Biochem. Biophys. Res. Commun.496, 159–166.

    Article  CAS  PubMed  Google Scholar 

  • Kimura, T., Takabatake, Y., Takahashi, A., and Isaka, Y. 2013. Chloroquine in cancer therapy: a double-edged sword of autophagy. Cancer Res.73, 3–7.

    Article  CAS  PubMed  Google Scholar 

  • Kobayashi, S., Kojidani, T., Osakada, H., Yamamoto, A., Yoshimori, T., Hiraoka, Y., and Haraguchi, T. 2010. Artificial induction of autophagy around polystyrene beads in nonphagocytic cells. Autophagy6, 36–45.

    Article  CAS  PubMed  Google Scholar 

  • Ktistakis, N.T. and Tooze, S.A. 2016. Digesting the expanding mechanisms of autophagy. Trends Cell Biol.26, 624–635.

    Article  CAS  PubMed  Google Scholar 

  • Lee, W.K., Probst, S., Santoyo-Sánchez, M.P., Al-Hamdani, W., Diebels, I., von Sivers, J.K., Kerek, E., Prenner, E.J., and Thévenod, F. 2017. Initial autophagic protection switches to disruption of autophagic flux by lysosomal instability during cadmium stress accrual in renal NRK-52E cells. Arch. Toxicol.91, 3225–3245.

    Article  CAS  PubMed  Google Scholar 

  • LoPiccolo, J., Blumenthal, G.M., Bernstein, W.B., and Dennis, P.A. 2008. Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations. Drug Resist. Updat.11, 32–50.

    Article  CAS  PubMed  Google Scholar 

  • Lundberg, Å., Nyman, A.K., Aspán, A., Börjesson, S., Unnerstad, H.E., and Waller, K.P. 2016. Udder infections with Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis at calving in dairy herds with suboptimal udder health. J. Dairy Sci.99, 2102–2117.

    Article  CAS  PubMed  Google Scholar 

  • Mestre, M.B., Fader, C.M., Sola, C., and Colombo, M.I. 2010. Alphahemolysin is required for the activation of the autophagic pathway in Staphylococcus aureus-infected cells. Autophagy6, 110–125.

    Article  CAS  PubMed  Google Scholar 

  • Mizushima, N., Levine, B., Cuervo, A.M., and Klionsky, D.J. 2008. Autophagy fights disease through cellular self-digestion. Nature451, 1069–1075.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mohamed, W., Sommer, U., Sethi, S., Domann, E., Thormann, U., Schütz, I., Lips, K.S., Chakraborty, T., Schnettler, R., and Alt, V. 2014. Intracellular proliferation of S. aureus in osteoblasts and effects of rifampicin and gentamicin on S. aureus intracellular proliferation and survival. Eur. Cell Mater.28, 258–268.

    Article  CAS  PubMed  Google Scholar 

  • Morissette, G., Ammoury, A., Rusu, D., Marguery, M.C., Lodge, R., Poubelle, P.E., and Marceau, F. 2009. Intracellular sequestration of amiodarone: role of vacuolar ATPase and macroautophagic transition of the resulting vacuolar cytopathology. Br. J. Pharmacol.157, 1531–1540.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Neumann, Y., Bruns, S.A., Rohde, M., Prajsnar, T.K., Foster, S.J., and Schmitz, I. 2016. Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase. Autophagy12, 2069–2084.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ogawa, M. and Sasakawa, C. 2006. Bacterial evasion of the autophagic defense system. Curr. Opin. Microbiol.9, 62–68.

    Article  CAS  PubMed  Google Scholar 

  • Pankiv, S., Clausen, T.H., Lamark, T., Brech, A., Bruun, J.A., Outzen, H., Øvervatn, A., Bjørkøy, G., and Johansen, T. 2007. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J. Biol. Chem.282, 24131–24145.

    Article  CAS  PubMed  Google Scholar 

  • Ravikumar, B., Sarkar, S., Davies, J.E., Futter, M., Garcia-Arencibia, M., Green-Thompson, Z.W., Jimenez-Sanchez, M., Korolchuk, V.I., Lichtenberg, M., Luo, S., et al. 2010. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol. Rev.90, 1383–1435.

    Article  CAS  PubMed  Google Scholar 

  • Ravikumar, B., Vacher, C., Berger, Z., Davies, J.E., Luo, S., Oroz, L.G., Scaravilli, F., Easton, D.F., Duden, R., O’Kane, C.J., et al. 2004. Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet.36, 585–595.

    Article  CAS  PubMed  Google Scholar 

  • Rodrigues, P.H., Bélanger, M., Dunn, W. Jr., Progulske-Fox, A. 2008. Porphyromonas gingivalis and the autophagic pathway: an innate immune interaction? Front. Biosci.13, 178–187.

    Article  CAS  PubMed  Google Scholar 

  • Sato, M., Seki, T., Konno, A., Hirai, H., Kurauchi, Y., Hisatsune, A., and Katsuki, H. 2016. Fluorescent-based evaluation of chaper-one-mediated autophagy and microautophagy activities in cultured cells. Genes Cells21, 861–873.

    Article  CAS  PubMed  Google Scholar 

  • Scaccabarozzi, L., Locatelli, C., Pisoni, G., Manarolla, G., Casula, A., Bronzo, V., and Moroni, P. 2011. Short communication: Epidemiology and genotyping of Candida rugosa strains responsible for persistent intramammary infections in dairy cows. J. Dairy Sci.94, 4574–4577.

    Article  CAS  PubMed  Google Scholar 

  • Schnaith, A., Kashkar, H., Leggio, S.A., Addicks, K., Krönke, M., and Krut, O. 2007. Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death. J. Biol. Chem.282, 2695–2706.

    Article  CAS  PubMed  Google Scholar 

  • Shinojima, N., Yokoyama, T., Kondo, Y., and Kondo, S. 2007. Roles of the Akt/mTOR/p70S6K and ERK1/2 signaling pathways in curcumin-induced autophagy. Autophagy3, 635–637.

    Article  CAS  PubMed  Google Scholar 

  • Sinha, B. and Fraunholz, M. 2010. Staphylococcus aureus host cell invasion and post-invasion events. Int. J. Med. Microbiol.300, 170–175.

    Article  PubMed  CAS  Google Scholar 

  • Stuhr, T. and Aulrich, K. 2010. Intramammary infections in dairy goats: recent knowledge and indicators for detection of subclinical mastitis. Forestry4, 267–279.

    Google Scholar 

  • Tanida, I., Ueno, T., and Kominami, E. 2008. LC3 and autophagy. Methods Mol. Biol.445, 77–88.

    Article  CAS  PubMed  Google Scholar 

  • Tollersrud, T., Kenny, K., Reitz, A.R. Jr., and Lee, J.C. 2000. Genetic and serologic evaluation of capsule production by bovine mammary isolates of Staphylococcus aureus and other Staphylococcus spp. from Europe and the United States. J. Clin. Microbiol.38, 2998–3003.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Van Loenen, H.J., Dijkmans, B.A., and De Vries, E. 1990. Concentration dependency of cyclosporin and chloroquine as inhibitors of cell proliferation and immunoglobulin production upon mitogen stimulation of mononuclear cells. Clin. Exp. Rheumatol.8, 59–61.

    CAS  PubMed  Google Scholar 

  • Wu, Y.T., Tan, H.L., Shui, G., Bauvy, C., Huang, Q., Wenk, M.R., Ong, C.N., Codogno, P., and Shen, H.M. 2010. Dual role of 3-methyladenine in modulation of autophagy via different temporal patterns of inhibition on class I and III phosphoinositide 3-kinase. J. Biol. Chem.285, 10850–10861.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xie, S., Chen, M., Yan, B., He, X., Chen, X., and Li, D. 2014. Identification of a role for the PI3K/AKT/mTOR signaling pathway in innate immune cells. PLoS One9, e94496.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Xie, J., Pang, Y., Wang, C., Wei, S., Wang, P., and Tang, J. 2015. Effects of autophagy inhibitor 3-methyladenine on growth and Notch1 protein expression in colorectal cancer cells. Chin. J. General Surgery24, 527–531.

    CAS  Google Scholar 

  • Zhu, J., Wu, J., Frizell, E., Liu, S.L., Bashey, R., Rubin, R., Norton, P., and Zern, M.A. 1999. Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis. Gastroenterology117, 1198–1204.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The project was supported by the National Science Foundation of China (31802259, 31872535), Shandong Key R&D Program (GG201809160113), Shandong Natural Science Foundation of China (ZR2018MC027, ZR2016CQ25), China Postdoctoral Science Foundation (2018M632704, 2019T-120601), and Funds of Shandong “Double Tops” Program.

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Authors and Affiliations

  1. College of Veterinary Medicine, Shandong Agricultural University, Tai’an, 271018, P. R. China

    Na Geng, Jianwei Lu, Yuliang Xu, Xiaozhou Wang, Run Wang & Yongxia Liu

  2. Research Center for Animal Disease Control Engineering, Shandong Agricultural University, Tai’an, 271018, P. R. China

    Kangping Liu & Jianzhu Liu

  3. College of Veterinary Medicine, China Agricultural University, Beijing, 100193, P. R. China

    Bo Han

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  1. Na Geng
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  2. Kangping Liu
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Correspondence to Jianzhu Liu or Yongxia Liu.

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Geng, N., Liu, K., Lu, J. et al. Autophagy of bovine mammary epithelial cell induced by intracellular Staphylococcus aureus. J Microbiol. 58, 320–329 (2020). https://doi.org/10.1007/s12275-020-9182-8

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  • DOI: https://doi.org/10.1007/s12275-020-9182-8

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