Centre de Recerca en Sanitat Animal, (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain.
Vet Res. 2012 Jul 27;43(1):57. doi: 10.1186/1297-9716-43-57.
Haemophilus parasuis, a member of the family Pasteurellaceae, is a common inhabitant of the upper respiratory tract of healthy pigs and the etiological agent of Glässer's disease. As other virulent Pasteurellaceae, H. parasuis can prevent phagocytosis, but the bacterial factors involved in this virulence mechanism are not known. In order to identify genes involved in phagocytosis resistance, we constructed a genomic library of the highly virulent reference strain Nagasaki and clones were selected by increased survival after incubation with porcine alveolar macrophages (PAM). Two clones containing two virulent-associated trimeric autotransporter (VtaA) genes, vtaA8 and vtaA9, respectively, were selected by this method. A reduction in the interaction of the two clones with the macrophages was detected by flow cytometry. Monoclonal antibodies were produced and used to demonstrate the presence of these proteins on the bacterial surface of the corresponding clone, and on the H. parasuis phagocytosis-resistant strain PC4-6P. The effect of VtaA8 and VtaA9 in the trafficking of the bacteria through the endocytic pathway was examined by fluorescence microscopy and a delay was detected in the localization of the vtaA8 and vtaA9 clones in acidic compartments. These results are compatible with a partial inhibition of the routing of the bacteria via the degradative phagosome. Finally, antibodies against a common epitope in VtaA8 and VtaA9 were opsonic and promoted phagocytosis of the phagocytosis-resistant strain PC4-6P by PAM. Taken together, these results indicate that VtaA8 and VtaA9 are surface proteins that play a role in phagocytosis resistance of H. parasuis.
副猪嗜血杆菌，巴斯德氏菌科的一个成员，是健康猪上呼吸道的常见居民，也是 Glässer 病的病原体。与其他毒力巴斯德氏菌一样，副猪嗜血杆菌可以阻止吞噬作用，但参与这种毒力机制的细菌因素尚不清楚。为了鉴定参与吞噬作用抗性的基因，我们构建了高毒力参考菌株 Nagasaki 的基因组文库，并且通过在猪肺泡巨噬细胞（PAM）孵育后存活能力增加来选择克隆。通过这种方法选择了两个分别含有两个毒力相关三聚体自转运蛋白（VtaA）基因 vtaA8 和 vtaA9 的克隆。通过流式细胞术检测到这两个克隆与巨噬细胞的相互作用减少。产生了单克隆抗体并用于证明这些蛋白存在于相应克隆和副猪嗜血杆菌吞噬抗性菌株 PC4-6P 的细菌表面。通过荧光显微镜检查了 VtaA8 和 VtaA9 在细菌通过内吞途径运输中的作用，并检测到 vtaA8 和 vtaA9 克隆在酸性隔室中的定位延迟。这些结果与细菌通过降解吞噬体的路由的部分抑制是一致的。最后，针对 VtaA8 和 VtaA9 中的共同表位的抗体具有调理作用，并促进 PAM 对吞噬抗性菌株 PC4-6P 的吞噬作用。总之，这些结果表明 VtaA8 和 VtaA9 是副猪嗜血杆菌吞噬作用抗性中的表面蛋白。
