Elsevier

Microbial Pathogenesis

Volume 106, May 2017, Pages 25-29
Microbial Pathogenesis

TGF-β1 promotes Staphylococcus aureus adhesion to and invasion into bovine mammary fibroblasts via the ERK pathway

https://doi.org/10.1016/j.micpath.2017.01.044Get rights and content

Highlights

  • BMFB model was established susceptible to S. aureus infection after TGF-β1 treatment.

  • TGF-β1-promoted fibrosis is linked to the ability of S. aureus to infect BMFBs.

  • ERK increases BMFBs susceptibility via promoting Collagen I and α-SMA expression.

Abstract

Fibroblasts are the structural base of mammary breast tissues. TGF-β1 can regulate the fibrotic process; however, it remains unclear whether TGF-β1 influences the susceptibility of fibroblasts to bacteria. Staphylococcus aureus (S. aureus) is a major bacterium in both chronic and subclinical mastitis in lactating cows that acts by invading host cells. To better understand the function of TGF-β1 in bovine mammary fibroblasts' (BMFBs) susceptibility to bacteria as well as the mechanisms involved, a primary BMFB model was established by treating cells with TGF-β1 followed by infection with S. aureus. The results revealed that the adhesion and invasion of S. aureus into BMFBs was significantly increased after cells were treated with 5 ng/ml TGF-β1 for 12 h. Moreover, TGF-β1 can increase Collagen I and α-SMA expression via activation of ERK signaling. However, the increased adhesion and invasion of S. aureus can be blocked by specific antibodies against either Collagen I or α-SMA, indicating that the increased adhesion and invasion are dependent on TGF-β1-induced upregulation of both Collagen I and α-SMA. Using PD98059, an ERK inhibitor, could also decrease the adhesion and invasion of S. aureus. These results indicate that TGF-β1 could promote S. aureus adhesion to and invasion into BMFBs by increasing Collagen I and α-SMA expression and may provide a novel target for controlling bovine mastitis.

Introduction

Fibroblasts are the structural base of many tissues and play an important role in their functions. In mammary tissue, bovine mammary fibroblasts (BMFBs) primarily support epithelial cells, influence the growth of epithelial cells and affect acinar formation [1]. BMFBs can also secrete cytokines, chemokines and growth factors to regulate udder inflammation [2]. Mastitis in cows is one of the most common infectious diseases encountered in the dairy industry. S. aureus is the most important and prevalent mastitis pathogen and can cause clinical and subclinical mammary infections [3]; in some cases, the cows can develop chronic mastitis and breast fibrosis, which results in a loss of lactation and can cause serious economic losses [4].

At present, TGF-β1 has gained more attention as a pro-fibrotic factor in the scientific field. TGF-β1 treatment can convert the normal mouse fibroblast cell line NRK-49F into a fibrosis model in vitro [5]. TGF-β1 promotes fibrosis associated with extracellular matrix (ECM) deposition [6], [7]. In addition, some studies found that the surface of S. aureus contains class-specific adhesin proteins (adhesins), also known as the microbial surface adhesion molecule matrix components (microbial surface components recognizing adhesive matrix molecules, MSCRAMMs). These components include fibronectin-binding protein A (FnBPA), collagen binding protein (Cna) and fibrinogen binding protein coagulation factor (ClfA), and these virulence factors have a high affinity for host ECM components [8], [9]. However, understanding these proteins is very important for revealing the mechanism of cow mammary gland bacterial infections.

Therefore, this research established a model of S. aureus-infected BMFBs to observe the susceptibility of BMFBs to S. aureus and its main mechanism upon induction of BMFBs fibrosis by TGF-β1. It was confirmed that TGF-β1 significantly promotes S. aureus adhesion to and invasion into BMFBs during fibrosis, and this process has some relevance with activation of the ERK signaling pathway which promotes Collagen I and α-SMA expression. These results suggest that TGF-β1 might be one cause of exacerbated dairy cow mastitis. Therefore, this study sheds light on the mechanism of the pathogenesis of S. aureus-based dairy cow mastitis and provides a new target for the prevention and treatment of S. aureus-based dairy cow mastitis.

Section snippets

Bacterial strains and growth conditions

The S. aureus strain ATCC35556 (a gift from the laboratory of Naisheng Zhang) was used for all experiments. The bacteria for infection experiments were cultured in 37 °C MH medium (Hopebio, China) in a shaker overnight for approximately 10 h until OD600 = 1.6, at which point the bacteria concentration in the culture medium is approximately 5.3 × 106 cfu/ml. Then, the bacteria were washed with DMEM/F12 (Gibco, USA) three times prior to the adhesion and invasion assays.

Cell culture

A primary culture

TGF-β1- induced S. aureus adhere to and invade into bovine mammary fibroblasts

Previous work has demonstrated that TGF-β1 levels in the cows' peripheral blood is significantly enhanced when the cows are fed straw with lower nutrition value. Furthermore, the lower nutritional value of the crude diet was accompanied the higher incidence of clinical and subclinical mastitis. Therefore, we studied whether TGF-β1 could influence S. aureus adhesion to and invasion into BMFBs. BMFBs were stimulated with TGF-β1 for 3 h, 6 h, 12 h, 24 h, 36 h, 48 h and 72 h and then infected with

Discussion

In this study, we found that TGF-β1 can significantly promote S. aureus adhesion to and invasion into BMFBs and that this process is closely related to ERK-induced Collagen I and α-SMA deposition. Long-term expression as well as high levels of TGF-β1 may be important potential causes of S. aureus-induced dairy cow mastitis. As is commonly known, the cause of mastitis is mainly due to the presence of a pathogenic infection, which emphasizes the need for a clean environment, drug treatment, and

Conflict of interest

None to declare.

Acknowledgments

This work was supported by a grant from the National Key Basic Research Program of China (No. 2011CB100800).

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    1

    Junling Yang and Liancheng Lei make contribution equally to the experiments.

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