The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China.
Res Vet Sci. 2019 Jun;124:70-78. doi: 10.1016/j.rvsc.2019.02.010. Epub 2019 Mar 1.
Mycoplasma bovis is a common pathogenic microorganism of cattle and represents an important hazard on the cattle industry. Adherence to host cells is a significant component of mycoplasma-pathogenesis research. Fibronectin (Fn), an extracellular matrix protein, is a common host cell factor that can interact with the adhesions of pathogens. The aims of this study were to investigate the Fn-binding properties of M. bovis fructose-1,6-bisphosphate aldolase (FBA) and evaluate its role as a cell adhesion factor during mycoplasma colonization. The fba (MBOV_RS00435) gene of M. bovis was cloned and expressed, with the resulting recombinant protein used to prepare rabbit polyclonal antibodies. The purified recombinant FBA (rFBA) was shown to have fructose bisphosphate aldolase activity. Western blot indicated that FBA was an antigenically conserved protein in several M. bovis strains. Western blot combined with immunofluorescent assay (IFA) revealed that FBA was dual-localized to both cytoplasm and membrane in M. bovis. IFA showed that rFBA was able to adhere to embryonic bovine lung (EBL) cells. Meanwhile, an adhesion inhibition assay demonstrated that anti-rFBA antibodies could significantly block the adhesion of M. bovis to EBL cells. Moreover, a dose-dependent binding of rFBA to Fn was found by dot blotting and enzyme-linked immunosorbent assays. Together these results provided evidence that FBA is a surface-localized and antigenic protein of M. bovis, suggesting that it may function as a virulence determinant through interacting with host Fn.
牛支原体是牛的一种常见致病性微生物，是牛养殖业的重要危害因素。黏附宿主细胞是支原体发病机制研究的重要组成部分。纤连蛋白（Fn）是一种细胞外基质蛋白，是一种常见的宿主细胞因子，可以与病原体的黏附物相互作用。本研究旨在研究牛支原体果糖-1,6-二磷酸醛缩酶（FBA）与 Fn 的结合特性，并评估其在支原体定植过程中作为细胞黏附因子的作用。克隆并表达了牛支原体的 fba（MBOV_RS00435）基因，用所得重组蛋白制备兔多克隆抗体。结果表明，纯化的重组 FBA（rFBA）具有果糖双磷酸醛缩酶活性。Western blot 表明 FBA 是几种牛支原体菌株中抗原保守的蛋白。Western blot 结合免疫荧光分析（IFA）表明 FBA 在牛支原体中双重定位于细胞质和膜。IFA 表明 rFBA 能够黏附于牛胚肺（EBL）细胞。同时，黏附抑制试验表明，抗-rFBA 抗体可以显著阻断牛支原体与 EBL 细胞的黏附。此外，斑点印迹和酶联免疫吸附试验发现 rFBA 与 Fn 呈剂量依赖性结合。这些结果表明 FBA 是牛支原体表面定位和抗原性蛋白，提示其可能通过与宿主 Fn 相互作用而作为一种毒力决定因子发挥作用。
