Abstract
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.
Highlights
Introduction iationsMastitis is the most prevalent disease in dairy cows and is a major cause of economic losses in dairy farms worldwide [1].A wide spectrum of pathogenic agents has been implicated in the etiology of mastitis, of which Escherichia coli and Staphylococcus aureus are the most commonly involved gramnegative and gram-positive bacteria, respectively [2,3,4,5]
To characterize the modulatory effect of E. coli and S. aureus on the gene expression profiles, primary bovine mammary epithelial cells (pbMECs) were stimulated with heat-killed E. coli or S. aureus for 24 h
We identified 1478 (8.5% of 17297 genes) differentially expressed genes (DEGs) from the transcriptome profiles of cells stimulated by heat-inactivated E. coli compared to unstimulated cells (Figure 1A), comprising 933 up- (Additional file 1) and 545 downregulated (Additional file 2) genes
Summary
Introduction iationsMastitis (inflammation of the mammary gland and udder tissue) is the most prevalent disease in dairy cows and is a major cause of economic losses in dairy farms worldwide [1].A wide spectrum of pathogenic agents has been implicated in the etiology of mastitis, of which Escherichia coli and Staphylococcus aureus are the most commonly involved gramnegative and gram-positive bacteria, respectively [2,3,4,5]. Mastitis (inflammation of the mammary gland and udder tissue) is the most prevalent disease in dairy cows and is a major cause of economic losses in dairy farms worldwide [1]. It is well known that these two pathogens elicit a distinct inflammatory response in the bovine udder. Intramammary infections with S. aureus typically cause a much weaker innate immune response, resulting in lifelong pathogen persistence with severe tissue damage [8,9]. Small interfering RNAs (siRNAs) were designed and synthesized by GenePharma (Shanghai, China). Cells were transfected with target-specific or nontargeting negative control siRNAs using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions. The sequence of Nrf siRNA is 50 -GCAAUUCAACGAGGCUC. The sequence of negative control siRNA is 5’- UUCUCCGAACGUGUCACGUTT -3’ (sense) and 5’ACGUGACACGUUCGGAGAATT-3’ (antisense). Knockdown efficiencies were determined by qPCR and Western blot analysis
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