An Immunogenic, Surface-Exposed Domain of Haemophilus ducreyi Outer Membrane Protein HgbA Is Involved in Hemoglobin Binding
An Immunogenic, Surface-Exposed Domain of Haemophilus ducreyi Outer Membrane Protein HgbA Is Involved in Hemoglobin Binding
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DOI：
10.1128/iai.00034-09
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发表时间：
2009-07-01
影响因子：
3.1
通讯作者：
Elkins, Christopher
中科院分区：
文献类型：
--
作者：
Nepluev, Igor;Afonina, Galyna;Elkins, Christopher
HgbA是杜克雷嗜血杆菌获得血红蛋白（Hb）的唯一TonB依赖性受体。Hb与HgbA的结合是从Hb获得血红素的初始步骤。为了更好地理解这一步骤，我们通过删除11个假定的表面暴露环中的每一个来诱变hgbA，并在宿主菌株H中反式表达每一种突变蛋白。鸭嘴兽FX547 hgbA.所有突变蛋白均被表达、输出，并通过抗HgbA免疫球蛋白G（IgG）在表面进行检测。在环5和7的HgbA的序列删除废除血红蛋白结合在两种不同的格式。与此相反，HgbA蛋白在其他九个环中含有缺失保留的能力，结合血红蛋白。表达突变蛋白的克隆都不能在含有低浓度Hb的平板上生长。以前，我们证明了在一个猪模型的软下疳感染，HgbA疫苗赋予完全的保护，从挑战感染。使用抗HgbA IgG从这项研究和上述缺失突变体，我们表明，环4，5，和7的HgbA的免疫原性和表面暴露和IgG针对环4和5阻断Hb结合。此外，环6被蛋白酶在完整的H上切割。ducreyi，表明表面暴露。这些数据暗示了HgbA的中心结构域（相对于一级氨基酸序列）在Hb结合中的重要性，并表明该分子的该区域可能具有作为亚单位疫苗的潜力。
HgbA is the sole TonB-dependent receptor for hemoglobin (Hb) acquisition of Haemophilus ducreyi. Binding of Hb to HgbA is the initial step in heme acquisition from Hb. To better understand this step, we mutagenized hgbA by deletion of each of the 11 putative surface-exposed loops and expressed each of the mutant proteins in trans in host strain H. ducreyi FX547 hgbA. All mutant proteins were expressed, exported, and detected on the surface by anti-HgbA immunoglobulin G (IgG). Deletion of sequences in loops 5 and 7 of HgbA abolished Hb binding in two different formats. In contrast, HgbA proteins containing deletions in the other nine loops retained the ability to bind Hb. None of the clones expressing mutant proteins were able to grow on plates containing low concentrations of Hb. Previously we demonstrated in a swine model of chancroid infection that an HgbA vaccine conferred complete protection from a challenge infection. Using anti-HgbA IgG from this study and the above deletion mutants, we show that loops 4, 5, and 7 of HgbA were immunogenic and surface exposed and that IgG directed against loops 4 and 5 blocked Hb binding. Furthermore, loop 6 was cleaved by protease on intact H. ducreyi, suggesting surface exposure. These data implicate a central domain of HgbA (in respect to the primary amino acid sequence) as important in Hb binding and suggest that this region of the molecule might have potential as a subunit vaccine.