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Characterization of potential interactions between transferrin binding proteins in Neisseria gonorrhoeae.

机译：淋病奈瑟氏球菌中转铁蛋白结合蛋白之间潜在相互作用的表征。

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摘要

Neisseria gonorrhoeae requires iron for survival and establishment of infection in the human host. Pathogenic Neisseriae have evolved a repertoire of high-affinity iron acquisition systems to facilitate iron uptake in the human host. This requires specific outer membrane receptors and energy-harnessing cytoplasmic membrane proteins. The transferrin receptor proteins of Neisseria gonorrhoeae are necessary for iron uptake from transferrin in the host. The iron uptake system consists of two transferrin binding proteins, (Tbp) A and B. TbpA is an integral outer membrane, TonB-dependent transporter that forms the pore for iron internalization. TbpB is a surface exposed lipoprotein that makes the iron internalization process more efficient.;TbpA is proposed to consist of two distinct domains: a beta barrel and an N-terminal plug domain. Previous studies have shown that the EIEYE sequence in the TbpA plug domain plays an important role in iron internalization. We undertook a collaborative project to test the hypothesis that the conserved EIEYE sequence in the wild-type TbpA plug binds Fe3+ during the outer membrane iron transport process. CD spectra analysis and fluorescence emission titration spectra of purified recombinant wildtype and mutated plug proteins revealed that Fe3+ is sequestered by the wildtype TbpA plug protein, unlike the mutated plug protein. Modeling data with the wild-type plug predicts the EIEYE sequence is part of a flexible loop structure and acts as an Fe3+ binding site.;Characterization of the Tbps constituting the gonococcal receptor is important to understanding how the gonococcus survives within its host. TbpA and TbpB act together to acquire iron from human transferrin. We hypothesize that the presence of TbpA impacts the exposure or conformation of TbpB. In this study, we have utilized photoactivable cross-linkers to assess the effect of TbpA on TbpB in live gonococcal cells and studied it in presence of ligand and TonB derived energy. We employed insertion mutants, in which TbpA and TbpB contained the hemagglutinin (HA) epitope tag, to probe for impact of TbpA on TbpB. Our results demonstrate that photo-cross-linking altered TbpB size and migration and was dependent on the presence of TbpA. HA epitope insertion mutants in surface exposed loops of TbpA and TbpB did not impact the mobility of cross-linked TbpB. Addition of human transferrin to the de-energized mutant caused a change in TbpB migration after cross-linking. This result indicates that when ligand is bound tightly and irreversibly to de-energized TbpA, the surface accessibility and perhaps conformation of TbpB is altered and TbpA does not interact with TbpB. Our findings were confirmed with recent structural studies of TbpA-TbpB-ligand triple complex, which illustrate that Tbps bind ligand through unique, non-overlapping binding sites such that TbpA and TbpB do not interact.;TbpB is not an essential member of transferrin-iron acquisition pathway. It is surface exposed and tethered to the outer membrane by a lipid moiety. The role of TbpB has not been clearly outlined in the transferrin iron acquisition system. The last objective of this study was to look at the significance of two specific conserved regions of TbpB and its importance in transferrin iron utilization and TbpA-TbpB interaction. Using site-directed mutagenesis we created two mutants, in the first mutant the conserved lipobox of TbpB was replaced with a signal I peptidase cleavage site, and the second mutant contained a deletion of the conserved poly glycine residue stretch, immediately downstream of the lipobox. Our results indicate that lipobox is required for lipidation of TbpB, both the mutants were impaired for transferrin-iron utilization, and neither of the mutations altered TbpA-TbpB interaction.;Overall, these studies help elucidate the functional importance of the specific regions in TbpA and TbpB in Neisseria gonorrhoeae, thereby adding to our understanding of the process of iron acquisition through the transferrin binding proteins.
机译：淋病奈瑟氏球菌需要铁才能在人类宿主中生存和建立感染。致病性奈瑟氏球菌已经进化出一系列高亲和力的铁捕获系统，以促进人类宿主中铁的吸收。这需要特定的外膜受体和能量利用的细胞质膜蛋白。淋病奈瑟氏球菌的转铁蛋白受体蛋白是从宿主体内转铁蛋白摄取铁所必需的。铁吸收系统由两个转铁蛋白结合蛋白（Tbp）A和B组成。TbpA是完整的外膜，依赖TonB的转运蛋白，形成铁内在化的孔。 TbpB是一种表面暴露的脂蛋白，可使铁的内在化过程更有效。TbpA提议由两个不同的域组成：一个β桶和一个N末端的塞域。先前的研究表明，TbpA插入域中的EIEYE序列在铁内在化中起重要作用。我们进行了一项合作项目，以检验外膜铁运输过程中野生型TbpA塞子中保守的EIEYE序列与Fe3 +结合的假说。纯化的重组野生型和突变的栓蛋白的CD光谱分析和荧光发射滴定光谱表明，与突变的栓蛋白不同，Fe3 +被野生型的TbpA栓蛋白螯合。用野生型塞子建模数据预测EIEYE序列是柔性环结构的一部分，并充当Fe3 +结合位点。构成淋球菌受体的Tbps的表征对于理解淋球菌如何在其宿主内生存很重要。 TbpA和TbpB共同作用以从人转铁蛋白中获取铁。我们假设TbpA的存在会影响TbpB的暴露或构象。在这项研究中，我们利用光活化交联剂来评估TbpA对活淋球菌细胞中TbpB的影响，并在存在配体和TonB衍生能量的情况下对其进行了研究。我们使用插入突变体，其中TbpA和TbpB包含血凝素（HA）表位标签，以探查TbpA对TbpB的影响。我们的结果表明，光交联改变了TbpB的大小和迁移，并且取决于TbpA的存在。 TbpA和TbpB的表面暴露环中的HA表位插入突变体不影响交联TbpB的迁移率。将人转铁蛋白添加到断电的突变体中，导致交联后TbpB迁移发生变化。该结果表明，当配体紧密地且不可逆地结合到去电的TbpA上时，TbpB的表面可及性和构象可能会改变，并且TbpA不会与TbpB相互作用。 TbpA-TbpB-配体三重复合物的最新结构研究证实了我们的发现，该研究表明Tbps通过独特的，不重叠的结合位点结合配体，因此TbpA和TbpB不相互作用。TbpB不是运铁蛋白的必需成员。铁获取途径。其表面暴露并通过脂质部分拴在外膜上。在转铁蛋白铁捕获系统中尚未明确概述TbpB的作用。这项研究的最后一个目的是研究两个特定保守区域TbpB的重要性及其在转铁蛋白铁利用和TbpA-TbpB相互作用中的重要性。使用定点诱变，我们创建了两个突变体，在第一个突变体中，保守的TbpB脂质盒被信号I肽酶切割位点取代，第二个突变体在脂质盒的下游立即缺失了保守的聚甘氨酸残基片段。我们的结果表明，脂蛋白盒是TbpB脂质化所必需的，两个突变体均不影响转铁蛋白铁的利用，并且两个突变均未改变TbpA-TbpB的相互作用。总体而言，这些研究有助于阐明TbpA中特定区域的功能重要性。和淋病奈瑟氏球菌中的TbpB，从而加深了我们对通过运铁蛋白结合蛋白获得铁的过程的了解。

著录项

  • 作者

    Mistry, Shreni Dilipkumar.;

  • 作者单位

    Virginia Commonwealth University.;

  • 授予单位 Virginia Commonwealth University.;
  • 学科 Biology Molecular.;Biology Microbiology.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 205 p.
  • 总页数 205
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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