VtaA8 and VtaA9 from Haemophilus parasuis delay phagocytosis by alveolar macrophages.
VtaA8 and VtaA9 from Haemophilus parasuis delay phagocytosis by alveolar macrophages.
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DOI：
10.1186/1297-9716-43-57
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发表时间：
2012-07-27
影响因子：
4.4
通讯作者：
Aragon V
中科院分区：
文献类型：
--
作者：
Costa-Hurtado M;Ballester M;Galofré-Milà N;Darji A;Aragon V
副猪嗜血杆菌（Haemophilus parasuis）是巴氏杆菌科（Pasteurellaceae）的成员，是健康猪上呼吸道的常见寄生菌，也是格莱瑟氏病的病原体。与其它致病性巴氏杆菌科（Pasteurellaceae）一样，H.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。为了鉴定与吞噬抗性相关的基因，我们构建了高毒力参考株长崎的基因组文库，并通过与猪肺泡巨噬细胞（PAM）孵育后存活率增加来选择克隆。通过这种方法，选择了两个克隆含有两个毒性相关的三聚体自身转运蛋白（VtaA）基因，vtaA 8和vtaA 9，分别。通过流式细胞术检测到两个克隆与巨噬细胞的相互作用减少。制备单克隆抗体，并用于证明这些蛋白质在相应克隆的细菌表面和H.副猪嗜血杆菌吞噬抗性菌株PC 4 -6P。通过荧光显微镜检查VtaA 8和VtaA 9在细菌通过内吞途径运输中的作用，并且在酸性隔室中检测到vtaA 8和vtaA 9克隆的定位延迟。这些结果与通过降解性吞噬体的细菌路由的部分抑制是相容的。最后，针对VtaA 8和VtaA 9中的共同表位的抗体是调理剂，并促进PAM对吞噬抗性菌株PC 4 -6P的吞噬作用。综上所述，这些结果表明VtaA 8和VtaA 9是在H.副猪。
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.
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