Deep Sequencing-Based Transcriptional Analysis of Bovine Mammary Epithelial Cells Gene Expression in Response to In Vitro Infection with Staphylococcus aureus Stains

Abstract

Staphylococcus aureus (S. aureus) is an important etiological organism in chronic and subclinical mastitis in lactating cows. Given the fundamental role the primary bovine mammary epithelial cells (pBMECs) play as a major first line of defense against invading pathogens, their interactions with S. aureus was hypothesized to be crucial to the establishment of the latter’s infection process. This hypothesis was tested by investigating the global transcriptional responses of pBMECs to three S. aureus strains (S56,S178 and S36) with different virulent factors, using a tag-based high-throughput transcriptome sequencing technique. Approximately 4.9 million total sequence tags were obtained from each of the three S. aureus-infected libraries and the control library. Referenced to the control, 1720, 219, and 427 differentially expressed unique genes were identified in the pBMECs infected with S56, S178 and S36 S. aureus strains respectively. Gene ontology (GO) and pathway analysis of the S56-infected pBMECs referenced to those of the control revealed that the differentially expressed genes in S56-infected pBMECs were significantly involved in inflammatory response, cell signalling pathways and apoptosis. In the same vein, the clustered GO terms of the differentially expressed genes of the S178-infected pBMECs were found to comprise immune responses, metabolism transformation, and apoptosis, while those of the S36-infected pBMECs were primarily involved in cell cycle progression and immune responses. Furthermore, fundamental differences were observed in the levels of expression of immune-related genes in response to treatments with the three S. aureus strains. These differences were especially noted for the expression of important pro-inflammatory molecules, including IL-1α, TNF, EFNB1, IL-8, and EGR1. The transcriptional changes associated with cellular signaling and the inflammatory response in this study may reflect different immunomodulatory mechanisms that underlie the interaction between pBMECs and S. aureus strains during infection by the latter.

Genome-Wide Transcriptional and Post-transcriptional Regulation of Innate Immune and Defense Responses of Bovine Mammary Gland to Staphylococcus aureus

“…Chemokine signaling pathway plays important roles in regulating and mobilizing immune-relevant cells, and is involved in providing directional cues for leukocytes to the site of inflammation (Mellado et al, 2001), indicating the importance of recruitment and activation of immune cells to sites of infection during acute S. aureus mastitis. Noticeably, several other pathways primarily involved in pro-inflammatory and innate immune system were uncovered in the present study but not in Wang et al (2013), including cytokine-cytokine receptor interaction, Jak-STAT signaling pathway (FDR = 0.05; Figure S5) and Toll-like receptor signaling pathway, all of which were also demonstrated to be associated with S. aureus mastitis in both dairy cows and goats by previous studies (Pisoni et al, 2010; He et al, 2016). However, several human neurological disease-related pathways (e.g., Huntingson's, Alzheimer's, and Parkinson's diseases) were only observed in Wang et al (2013).…”

“…Noticeably, several other pathways primarily involved in pro-inflammatory and innate immune system were uncovered in the present study but not in Wang et al (2013), including cytokine-cytokine receptor interaction, Jak-STAT signaling pathway (FDR = 0.05; Figure S5) and Toll-like receptor signaling pathway, all of which were also demonstrated to be associated with S. aureus mastitis in both dairy cows and goats by previous studies (Pisoni et al, 2010; He et al, 2016). However, several human neurological disease-related pathways (e.g., Huntingson's, Alzheimer's, and Parkinson's diseases) were only observed in Wang et al (2013). The findings here and in Wang et al (2013) indicate that the transcriptional responses of the mammary gland to IMI with S. aureus in vivo could be different from responses of Mac-T cells in vitro due to the complex in vivo environment and the interplays among various cell types.…”

“…However, several human neurological disease-related pathways (e.g., Huntingson's, Alzheimer's, and Parkinson's diseases) were only observed in Wang et al (2013). The findings here and in Wang et al (2013) indicate that the transcriptional responses of the mammary gland to IMI with S. aureus in vivo could be different from responses of Mac-T cells in vitro due to the complex in vivo environment and the interplays among various cell types.…”

“…More recently, another study exploited the transcriptional responses of Mac-T cells to the infection with S. aureus in vitro using RNA-Seq (Wang et al, 2013). Two significantly enriched KEGG pathways were shared by their study and the present study (FDR < 0.05), NOD-like receptor signaling pathway and chemokine signaling pathway.…”

Rapamycin Inhibits Expression of Elongation of Very-long-chain Fatty Acids 1 and Synthesis of Docosahexaenoic Acid in Bovine Mammary Epithelial Cells

“…Primary BMECs were cultured and purified according to previously reported methods (Wang et al, 2013). Purified BMECs were cultured in DMEM/F12 media (DMEM/F12, Gibco, Paisley, PA49RF, Scotland, UK) containing 10% fetal bovine serum (Hyclone Laboratories, Inc. Logan, UT, USA), bovine insulin (5 μg/mL, Sigma-Aldrich, Inc. St. Louis, MO, USA), hydrocortisone (1 μg/mL, Sigma-Aldrich, Inc., USA), progesterone (1 μg/mL, Sigma-Aldrich, Inc., USA), transferrin (5 μg/mL, Sigma-Aldrich, Inc., USA), glutamine (200 mM, Sigma-Aldrich, Inc., USA), EGF (10 ng/mL, Sigma-Aldrich, Inc., USA), 100 U/mL penicillin G, and 100 mg/mL streptomycin (Sigma-Aldrich, Inc., USA).…”