Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, Sir Graeme Davies Building, 120 University Place, Glasgow G12 8TA, UK.
Infect Immun. 2011 Nov;79(11):4332-41. doi: 10.1128/IAI.05469-11. Epub 2011 Sep 6.
Mannheimia haemolytica is the etiological agent of pneumonic pasteurellosis of cattle and sheep; two different OmpA subclasses, OmpA1 and OmpA2, are associated with bovine and ovine isolates, respectively. These proteins differ at the distal ends of four external loops, are involved in adherence, and are likely to play important roles in host adaptation. M. haemolytica is surrounded by a polysaccharide capsule, and the degree of OmpA surface exposure is unknown. To investigate surface exposure and immune specificity of OmpA among bovine and ovine M. haemolytica isolates, recombinant proteins representing the transmembrane domain of OmpA from a bovine serotype A1 isolate (rOmpA1) and an ovine serotype A2 isolate (rOmpA2) were overexpressed, purified, and used to generate anti-rOmpA1 and anti-rOmpA2 antibodies, respectively. Immunogold electron microscopy and immunofluorescence techniques demonstrated that OmpA1 and OmpA2 are surface exposed, and are not masked by the polysaccharide capsule, in a selection of M. haemolytica isolates of various serotypes and grown under different growth conditions. To explore epitope specificity, anti-rOmpA1 and anti-rOmpA2 antibodies were cross-absorbed with the heterologous isolate to remove cross-reacting antibodies. These cross-absorbed antibodies were highly specific and recognized only the OmpA protein of the homologous isolate in Western blot assays. A wider examination of the binding specificities of these antibodies for M. haemolytica isolates representing different OmpA subclasses revealed that cross-absorbed anti-rOmpA1 antibodies recognized OmpA1-type proteins but not OmpA2-type proteins; conversely, cross-absorbed anti-rOmpA2 antibodies recognized OmpA2-type proteins but not OmpA1-type proteins. Our results demonstrate that OmpA1 and OmpA2 are surface exposed and could potentially bind to different receptors in cattle and sheep.
溶血曼海姆菌是牛和羊肺炎巴氏杆菌病的病原体;两种不同的 OmpA 亚类，OmpA1 和 OmpA2，分别与牛和羊分离株相关。这些蛋白在四个外环的远端不同，参与粘附，并且可能在宿主适应中发挥重要作用。溶血曼海姆菌被多糖荚膜包围，OmpA 的表面暴露程度未知。为了研究牛和羊溶血曼海姆菌分离株中 OmpA 的表面暴露和免疫特异性，表达了来自牛血清型 A1 分离株（rOmpA1）和羊血清型 A2 分离株（rOmpA2）的 OmpA 跨膜结构域的重组蛋白，分别进行了过表达、纯化，并用于产生抗 rOmpA1 和抗 rOmpA2 抗体。免疫金电子显微镜和免疫荧光技术表明，OmpA1 和 OmpA2 在不同血清型和不同生长条件下生长的多种溶血曼海姆菌分离株中表面暴露，且不受多糖荚膜的掩盖。为了探索表位特异性，用异源分离株交叉吸收抗 rOmpA1 和抗 rOmpA2 抗体以去除交叉反应抗体。这些交叉吸收的抗体具有高度特异性，仅在 Western blot 分析中识别同源分离株的 OmpA 蛋白。对代表不同 OmpA 亚类的溶血曼海姆菌分离株的这些抗体结合特异性的更广泛检查表明，交叉吸收的抗 rOmpA1 抗体识别 OmpA1 型蛋白而不识别 OmpA2 型蛋白；相反，交叉吸收的抗 rOmpA2 抗体识别 OmpA2 型蛋白而不识别 OmpA1 型蛋白。我们的结果表明，OmpA1 和 OmpA2 是表面暴露的，并且可能与牛和羊中的不同受体结合。
