Menon S, Pillai D K, Narayanan S
Department of Diagnostic Medicine/ Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, 66506-5606, USA.
Department of Diagnostic Medicine/ Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, 66506-5606, USA.
Anaerobe. 2018 Apr;50:101-105. doi: 10.1016/j.anaerobe.2018.01.015. Epub 2018 Feb 2.
Liver abscesses are of major economic importance to the cattle industry. These are mainly associated with the presence of Fusobacterium necrophorum, a non-spore forming and Gram-negative anaerobe. There are two main subspecies, F. necrophorum subspecies necrophorum and subsp. funduliforme, and they differ molecularly, morphologically, biochemically and in virulence. Previous studies have shown that the outer membrane proteins (OMP) of F. necrophorum subsp. necrophorum are important for its successful binding to immobilized bovine adrenal gland capillary endothelial (EJG) cells. In this study, a 42.4 kDa OMP of F. necrophorum subsp. necrophorum with the highest binding capacity to EJG cells was characterized. The gene was cloned into pFLAG-CTS vector and the proteins were subsequently expressed on the surface of E. coli BL21 DE3 cells. When E. coli carrying the recombinant plasmid (SM 2013) was induced using IPTG, there was significant enhancement in the binding to immobilized EJG cells compared to both uninduced SM 2013 and the E. coli carrying control vector only. When fixed EJG cells were incubated with purified native OMP, SM 2013 showed lowered levels of binding, compared to the uninduced SM 2013 and the E. coli carrying control vector only. Pre-incubation of induced SM 2013 with polyclonal antibodies made against the OMP reduced the binding to immobilized EJG cells to uninduced SM 2013 levels. This gain of function by recombinant E. coli confirms the ability of this protein to act as an adhesion to help binding of F. necrophorum subsp. necrophorum to host cells.
肝脓肿对养牛业具有重大的经济影响。这些脓肿主要与坏死梭杆菌的存在有关，坏死梭杆菌是一种不形成芽孢的革兰氏阴性厌氧菌。有两个主要亚种，即坏死梭杆菌坏死亚种和亚基形亚种，它们在分子、形态、生化和毒力方面存在差异。先前的研究表明，坏死梭杆菌坏死亚种的外膜蛋白（OMP）对于其成功结合固定化的牛肾上腺毛细血管内皮（EJG）细胞很重要。在本研究中，对坏死梭杆菌坏死亚种中与EJG细胞结合能力最强的一种42.4 kDa的OMP进行了表征。该基因被克隆到pFLAG-CTS载体中，随后这些蛋白质在大肠杆菌BL21 DE3细胞表面表达。当使用IPTG诱导携带重组质粒（SM 2013）的大肠杆菌时，与未诱导的SM 2013和仅携带对照载体的大肠杆菌相比，其与固定化EJG细胞的结合显著增强。当将固定的EJG细胞与纯化的天然OMP孵育时，与未诱导的SM 2013和仅携带对照载体的大肠杆菌相比，SM 2013的结合水平降低。用针对该OMP制备的多克隆抗体对诱导的SM 2013进行预孵育，可将其与固定化EJG细胞的结合降低到未诱导的SM 2013水平。重组大肠杆菌的这种功能获得证实了该蛋白作为黏附素帮助坏死梭杆菌坏死亚种与宿主细胞结合的能力。
