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

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.

Single-Chain Fragment Variables Targeting Leukocidin ED Can Alleviate the Inflammation of Staphylococcus aureus-Induced Mastitis in Mice

“…At present, there is no efficient treatment for bovine mastitis. Previous studies in our lab have shown that scFvs targeting different antigens had a significant protective effect on mastitis in mice [ 8 , 25 , 40 ]. In brief, we obtained four scFvs (ZL8 and ZL42 aimed at LukE, ZL22 and ZL23 aimed at LukD) using the phage display technique.…”

“…Staphylococcus aureus ( S. aureus ) is a major opportunistic pathogen of humans and animals and one of the most pathogenic microorganisms in bovine mastitis [ 5 , 6 , 7 ]. S. aureus can secrete large amounts of virulence factors, such as fibronectin-binding protein A (FnBPA), coagulase (Coa), β-hemolysin (Hlb), glyceraldehyde-3-phosphate dehydrogenase (GapC) and leukotoxin [ 8 , 9 , 10 ]. The interaction between virulence factors is the main reason for the pathogenicity of S. aureus [ 1 , 11 ].…”

“…Studies have shown that genetically engineered antibodies provide an important means for the prevention and treatment of dairy cow mastitis. As one of the most common genetic engineering antibodies, scFv has been demonstrated to provide protection for bovine mastitis caused by S. aureus [ 8 , 25 ]. ScFv consists of a variable region of light chain (VL) and heavy chain (VH), which retains the complete antigen-binding sites of the full-length antibody [ 24 ].…”

“…Now phage display technology has been increasingly used in livestock research (e.g., phage display scFv libraries generated from camel, rabbit, pig, sheep and chicken) [ 31 , 32 , 33 , 34 , 35 ]. Studies have shown that scFv targeting S. aureus can effectively inhibit the growth of S. aureus in vitro and has a good protective effect on mastitis in mice [ 8 ].…”

“…The secretion level of the pro-inflammatory cytokines is one of the prominent indicators of inflammatory response and plays a key role in the host’s defense against invasive pathogenic microorganisms [ 36 ]. Previous studies in our lab have also proven that the detection of the transcription and expression of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-8 and IL-18 can reflect the pathogenicity of S. aureus and the anti-inflammatory role of scFv in anti-bacterial immune response [ 8 , 25 ]. Appropriate levels of pro-inflammatory cytokines are important for the immune response against pathogens, but excessive production of cytokines may lead to severe tissue damage [ 37 ].…”