Escherichia coli and Staphylococcus aureus Differentially Regulate Nrf2 Pathway in Bovine Mammary Epithelial Cells: Relation to Distinct Innate Immune Response.

Escherichia coli and Staphylococcus aureus are major mastitis causing pathogens in dairy cattle but elicit distinct immune and an inflammatory response in the udder. However, the host determinants responsible for this difference remains largely unknown. Our initial studies focused on the global transcriptomic response of primary bovine mammary epithelial cells (pbMECs) to heat-killed E. coli and S. aureus. RNA-sequencing transcriptome analysis demonstrates a significant difference in expression profiles induced by E. coli compared with S. aureus. A major differential response was the activation of innate immune response by E. coli, but not by S. aureus. Interestingly, E. coli stimulation increased transcript abundance of several genes downstream of Nrf2 (nuclear factor erythroid 2-related factor 2) that were enriched in gene sets with a focus on metabolism and immune system. However, none of these genes was dysregulated by S. aureus. Western blot analysis confirms that S. aureus impairs Nrf2 activation as compared to E. coli. Using Nrf2-knockdown cells we demonstrate that Nrf2 is necessary for bpMECs to mount an effective innate defensive response. In support of this notion, nuclear Nrf2 overexpression augmented S. aureus-stimulated inflammatory response. We also show that, unlike E. coli, S. aureus disrupts the non-canonical p62/SQSTM1-Keap1 pathway responsible for Nrf2 activation through inhibiting p62/SQSTM1 phosphorylation at S349. Collectively, our findings provide important insights into the contribution of the Nrf2 pathway to the pathogen-species specific immune response in bovine mammary epithelial cells and raise a possibility that impairment of Nrf2 activation contributes to, at least in part, the weak inflammatory response in S. aureus mastitis.

Annexin A2-Mediated Internalization of Staphylococcus aureus into Bovine Mammary Epithelial Cells Requires Its Interaction with Clumping Factor B.

BACKGROUND: Staphylococcus aureus is a leading cause of contagious mastitis in dairy cattle. Internalization of S. aureus by bovine mammary gland epithelial cells is thought to be responsible for persistent and chronic intramammary infection, but the underlying mechanisms are not fully understood. METHODS: In the present study, we evaluated the role of Annexin A2 (AnxA2), a membrane-binding protein, in S. aureus invasion into bovine mammary epithelial cell line (MAC-T). In vitro binding assays were performed to co-immunoprecipitate the binding proteins of AnxA2 in the lysates of S. aureus. RESULTS: AnxA2 mediated the internalization but not adherence of S. aureus. Engagement of AnxA2 stimulated an integrin-linked protein kinase (ILK)/p38 MAPK cascade to induce S. aureus invasion. One of the AnxA2-precipitated proteins was identified as S. aureus clumping factor B (ClfB) through use of mass spectrometry. Direct binding of ClfB to AnxA2 was further confirmed by using a pull-down assay. Pre-incubation with recombinant ClfB protein enhanced S. aureus internalization, an effect that was specially blocked by anti-AnxA2 antibody. CONCLUSION: Our results demonstrate that binding of ClfB to AnxA2 has a function in promoting S. aureus internalization. Targeting the interaction of ClfB and AnxA2 may confer protection against S. aureus mastitis.