Anti-Staphylococcus aureus Single-Chain Fragment Variables Play a Protective Anti-Inflammatory Role In Vitro and In Vivo.

Lei Zhang,Fengqing Wang,Jianguo Zhu,Yan Jia,Kalbinur Tohti,Manling Cheng,Dangjin Wu,Yan Zhang,Fanqing Zhang,Xin Ye

doi:10.3390/vaccines9111300

Abstract

Staphylococcus aureus is a causative agent of bovine mastitis, capable of causing significant economic losses to the dairy industry worldwide. This study focuses on obtaining single-chain fragment variables (scFvs) against the virulence factors of S. aureus and evaluates the protective effect of scFvs on bovine mammary epithelial (MAC-T) cells and mice mammary gland tissues infected by S. aureus. After five rounds of bio-panning, four scFvs targeting four virulence factors of S. aureus were obtained. The complementarity-determining regions (CDRs) of these scFvs exhibited significant diversities, especially CDR3 of the VH domain. In vitro, each of scFvs was capable of inhibiting S. aureus growth and reducing the damage of MAC-T cells infected by S. aureus. Preincubation of MAC-T cells with scFvs could significantly attenuate the effect of apoptosis and necrosis compared with the negative control group. In vivo, the qPCR and ELISA results demonstrated that scFvs reduced the transcription and expression of Tumor Necrosis Factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8, and IL-18. Histopathology and myeloperoxidase (MPO) results showed that scFvs ameliorated the histopathological damages and reduced the inflammatory cells infiltration. The overall results demonstrated the positive anti-inflammatory effect of scFvs, revealing the potential role of scFvs in the prevention and treatment of S. aureus infections.

Highlights

Staphylococcus aureus refers to a natural inhabitant of mammalian skin and mucous epithelia bacterium, acting as the leading causative agent of various diseases in man and domestic animals [1]
Five S. aureus strains applied here were termed as USA300, ATCC25923 and ATCC29213, XD69 and XD79
The results showed that compared with the C group, the transcription level and expression level of TNF- α, IL-18, IL-6, IL-1β and IL-8 increased significantly in mammary gland tissues infected by S. aureus, especially IL-1β and IL-8 (p < 0.05)

Summary

Introduction

Staphylococcus aureus refers to a natural inhabitant of mammalian skin and mucous epithelia bacterium, acting as the leading causative agent of various diseases in man and domestic animals [1]. After invading the udder via the nipple duct, S. aureus can recognize mammary epithelial and inflammatory cells by secreting a series of related toxins and adhesion factors, destroying the integrity of the cell membrane through the joint action of other virulence factors; the microbes invade into the cells, causing damage of mammary gland tissues and inflammation [4,5]. The following four proteins (fibronectin binding protein A (FnBPA), coagulase (Coa), β-hemolysin (Hlb), glyceraldehyde-3-phosphate dehydrogenase (GapC)), as important virulence factors, play a vital role in the pathogenicity and inflammation of S. aureus. FnBPA can mediate the binding of S. aureus to fibrinogen and fibronectin on the surface of host cells, which contributes to the invasion of S. aureus to the host cells [6]. These research of virulence factors contribute to the clarification of disease pathogenesis [12]

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Results

Conclusion