<i>Streptococcus lutetiensis</i> Induces Autophagy via Oxidative Stress in Bovine Mammary Epithelial Cells

Abstract

<i>Streptococcus lutetiensis</i>, an emerging pathogen causing bovine mastitis, has not been well characterized. We reported that <i>S. lutetiensis</i> was pathogenic both <i>in vivo</i> and <i>in vitro</i> and caused inflammatory reactions in the mammary gland. However, roles of autophagy and oxidative stress in the pathogenesis of <i>S. lutetiensis</i>-induced mastitis are unclear. In this study, an autophagy model of <i>S. lutetiensis</i>-infected bovine mammary epithelial cells (bMECs) was used to assess oxidative stress and autophagy flux. Expressions of Beclin1, light chain 3II, and Sequestosome 1/p62 were elevated in bMECs after <i>S. lutetiensis</i> infection. In addition, autophagosome and lysosome formation confirmed autophagy occurred. Based on LysoTracker Red and acridine orange, lysosome degradation was blocked, and lower expressions of lysosomal-associated membrane protein 2, cathepsins D, and cathepsins L confirmed lysosomal damage. Concurrently, the nuclear factor erythroid 2-related factor 2 (Nrf2), kelch-like ECH-associated protein 1 (Keap1), heme oxygenase 1 (HO1), and NAD (P)H: quinone oxidoreductase 1 (NQO1), and basilic proteins associated with the Nrf2/Keap1 signaling pathway, were detected. Decreased keap1 and increased Nrf2, HO1, NQO1, and reactive oxygen species (ROS) indicated increased oxidative stress. Treatment with N-Acetyl-L-cysteine (NAC), an ROS inhibitor, decreased both oxidative stress and autophagy. Therefore, we concluded that <i>S. lutetiensis</i> caused intracellular oxidative stress and autophagy in bMECs. In addition, crosstalk between autophagy and oxidative stress affected the autophagic flux and blocked downstream autophagy. The Nrf2-keap1-p62 pathway participated in this process, with ROS acting upstream of these effects, interfering with normal cell functions.