Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, 74078-2007, USA.
Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, 74078-2007, USA.
Vet Microbiol. 2019 Dec;239:108487. doi: 10.1016/j.vetmic.2019.108487. Epub 2019 Nov 4.
Mannheimia haemolytica colonizes the nasopharynx of cattle and can cause severe fibrinous pleuropneumonia. IgA proteases are metalloendopeptidases released by bacteria that cleave IgA, enhancing colonization of mucosa. The objectives of these studies were to characterize M. haemolytica IgA1 and IgA2 proteases in vitro and in silico, to clone and sequence the genes for these proteases, and to demonstrate immunogenicity of components of the entire IgA protease molecule. Both IgA protease genes were cloned, expressed, and sequenced. Sequences were compared to other published sequences. Components were used to immunize mice to determine immunogenicity. Sera from healthy cattle and cattle that recovered from respiratory disease were examined for antibodies to IgA proteases. In order to assay the cleavage of bovine IgA with IgA1 protease, M. haemolytica culture supernatant was incubated with bovine IgA. Culture supernatant cleaved purified bovine IgA in the presence of ZnCl. Both IgA proteases contain three domains, 1) IgA peptidase, 2) PL1_Passenger_AT and 3) autotransporter. IgA1 and IgA2 peptidases have molecular weights of 96.5 and 87 kDa, respectively. Convalescent bovine sera with naturally high anti-M. haemolytica antibody titers had high antibodies against all IgA1 & IgA2 protease components. Mouse immunizations indicated high antibodies to the IgA peptidases and autotransporters but not to PL1_Passenger_AT. These data indicate that M. haemolytica produces two IgA proteases that are immunogenic, can cleave bovine IgA, and are produced in vivo, as evidenced by antibodies in convalescent bovine sera. Further studies could focus on IgA protease importance in pathogenesis and immunity.
溶血曼海姆菌定植于牛的鼻咽部，可引起严重的纤维素性化脓性胸膜肺炎。分泌型 IgA 蛋白酶是由细菌释放的金属内肽酶，可裂解 IgA，从而增强黏膜定植。本研究旨在对溶血曼海姆菌分泌型 IgA1 和 IgA2 蛋白酶进行体外和计算机模拟分析，克隆和测序这些蛋白酶的基因，并证明整个 IgA 蛋白酶分子的成分具有免疫原性。两种 IgA 蛋白酶基因均被克隆、表达和测序。对序列进行了比较分析，以确定与其他已发表序列的同源性。将这些成分用于免疫小鼠，以确定其免疫原性。对来自健康牛和呼吸道疾病康复牛的血清进行检测，以确定其对 IgA 蛋白酶的抗体。为了检测 IgA1 蛋白酶对牛 IgA 的裂解作用，将溶血曼海姆菌的培养上清液与牛 IgA 孵育。在 ZnCl2 的存在下，培养上清液可裂解纯化的牛 IgA。两种 IgA 蛋白酶均包含三个结构域：1）IgA 肽酶，2）PL1_Passenger_AT 和 3）自转运蛋白。IgA1 和 IgA2 肽酶的分子量分别为 96.5 和 87 kDa。具有天然高抗溶血曼海姆菌抗体滴度的康复牛血清对所有 IgA1 和 IgA2 蛋白酶成分均具有高抗体。小鼠免疫表明，对 IgA 肽酶和自转运蛋白的抗体水平较高，但对 PL1_Passenger_AT 的抗体水平较低。这些数据表明，溶血曼海姆菌产生两种具有免疫原性、可裂解牛 IgA 的 IgA 蛋白酶，并且在体内产生，这一点可通过康复牛血清中的抗体得到证明。进一步的研究可以集中在 IgA 蛋白酶在发病机制和免疫中的重要性上。
