Structure-function studies of the virulence associated proteins of Rhodococcus equi

马红球菌毒力相关蛋白的结构-功能研究

• 批准号: BB/J007900/1
• 负责人: Anthony Wilkinson
• 金额: $ 48.25万
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ007900%2F1
• 关键词: Structure; function; studies; virulence; associated

The bacterium Rhodococcus equi (R. equi) infects a range of grazing animals and poses a threat to animal health especially to that of horses. In foals aged 3-5 months, R. equi infection causes a lung disease, which if untreated, can lead to death. The bacteria, which are transmitted through the faeces, are inhaled into the lungs in the form of aerosolised dusts. In common with type of bacterium which causes tuberculosis in humans, R. equi cells are surrounded by a complex protective cell wall rich in compounds called mycolic acids, moreover these bacteria share the ability to survive within macrophages, a type of cell of the human immune system. Macrophages normally sweep up and destroy infecting bacteria by first engulfing them and then exposing them to lethal chemical stresses. Many pathogenic bacteria evade macrophage engulfment altogether, while others such as R. equi have developed survival strategies that prevent their chemical destruction once inside the macrophage. Survival in this 'lion's den' is a critical first step in establishing infection and is essential for the later development of virulence. In this study, we are concerned with virulence-associated proteins (Vaps) of R. equi and with one particular member of this set of proteins, VapA. Serum taken from foals with R. equi pneumonia, are rich in antibodies that react with VapA consistent with this protein being exposed on the surface of the bacterium where it is 'visible' to the host. It therefore has potential as a vaccine against R. equi infection. VapA has also been shown to be indispensible for R. equi virulence making it a potential target for the design of new drugs to cure R. equi infections. Interestingly, VapA production by the bacteria is turned on by elevated temperature, increased acidity and by oxidative stress. These are the conditions that the bacteria encounter upon entry into the macrophage. It has therefore been proposed that VapA plays an important role in enabling R. equi to survive and then persist in this hostile environment inside the macrophage.In view of its important role in R. equi virulence, we wish to extend the understanding of VapA to include its molecular structure and function. A first question concerns how VapA is exposed on the surface of the bacterium. In many other bacterial species, cell surface proteins possess a tagging sequence that leads to the attachment of a hydrocarbon chain which facilities tethering of the protein to the cell membrane. VapA does possess the requisite tagging sequence suggesting either that VapA is not tagged or that it is tagged by a novel mechanism. We propose to extract and isolate VapA from R. equi cells and determine whether it is tagged by analysing the protein by state of the art techniques which measure molecular mass. A further goal is to determine the three dimensional structure of VapA using X-ray diffraction methods. Sometimes it is possible to model the structure of a protein if its sequence has sufficient similarity to the sequence of another protein of known structure. This is not possible for VapA. The only proteins with sequence similarity to VapA are a set of nine other Vap proteins from R. equi. For structure determination we will prepare crystals of VapA, and/or its Vap relatives and use these crystals to collect X-ray scattering data. The structure of the protein once determined will be scrutinised for its functional implications for cell surface localisation and virulence. Finally there is evidence to suggest that the Vap proteins interact with one another and this may be important for virulence. We propose to purify the individual proteins and in mixing experiments explore the strength of these interactions and the composition of the complexes that are formed.

马红球菌（Rhodococcus equi，R. Equi）感染一系列放牧动物并对动物健康尤其是马的健康构成威胁。在3-5个月大的马驹中，R.马感染引起肺部疾病，如果不治疗，可能导致死亡。通过粪便传播的细菌以雾化粉尘的形式被吸入肺部。与引起人类结核病的细菌类型一样，R。马细胞被富含称为分枝菌酸的化合物的复杂保护性细胞壁包围，此外，这些细菌具有在巨噬细胞（人类免疫系统的一种细胞）内存活的能力。巨噬细胞通常会先吞噬感染细菌，然后将它们暴露在致命的化学胁迫下，从而清除并摧毁它们。许多致病菌完全逃避巨噬细胞的吞噬，而其他如R。马已经发展出了生存策略，一旦进入巨噬细胞，就可以防止它们的化学破坏。在这个“狮子窝”中生存是建立感染的关键第一步，对后来的毒力发展至关重要。在这项研究中，我们关注的是R的毒性相关蛋白（Vaps）。马和一个特定的成员，这组蛋白质，VapA。从R.马肺炎的病原体富含与VapA反应的抗体，这与这种蛋白质暴露在细菌表面上对宿主“可见”一致。因此，它具有作为抗R.马传染病VapA也被证明是R.等毒力，使其成为设计治疗R.马传染病有趣的是，细菌产生的VapA是由升高的温度，增加的酸度和氧化应激开启的。这些是细菌进入巨噬细胞时遇到的条件。因此，有人提出，VapA在使R。equi在巨噬细胞内的这种恶劣环境中生存并持续存在。为了研究VapA的等毒力，我们希望将对VapA的理解扩展到包括其分子结构和功能。第一个问题涉及VapA如何暴露在细菌表面。在许多其他细菌物种中，细胞表面蛋白质具有标记序列，其导致烃链的附着，这促进蛋白质与细胞膜的束缚。VapA确实具有必要的标记序列，这表明VapA未被标记或被新机制标记。我们建议从R. Equi细胞，并通过测量分子量的现有技术分析蛋白质来确定其是否被标记。另一个目标是使用X射线衍射方法确定VapA的三维结构。有时，如果一个蛋白质的序列与另一个已知结构的蛋白质的序列有足够的相似性，就有可能对该蛋白质的结构建模。这对于VapA来说是不可能的。与VapA具有序列相似性的唯一蛋白质是来自R.等。对于结构测定，我们将制备VapA和/或其Vap相关物的晶体，并使用这些晶体收集X射线散射数据。一旦确定蛋白质的结构，将仔细检查其对细胞表面定位和毒力的功能影响。最后，有证据表明Vap蛋白相互作用，这可能对毒力很重要。我们建议纯化单个蛋白质，并在混合实验中探索这些相互作用的强度和形成的复合物的组成。

项目成果

Crystal structures of the GH18 domain of the bifunctional peroxiredoxin-chitinase CotE from Clostridium difficile.

来自艰难梭菌的双功能过氧化还原蛋白-几丁质酶 CotE 的 GH18 结构域的晶体结构。

• DOI: 10.1107/s2053230x20006147
• 发表时间: 2020
• 期刊: Acta crystallographica. Section F, Structural biology communications
• 作者: Whittingham JL

Commonality of Virulence-Promoting Function in Rhodococcus equi Virulence Associated Proteins (Vaps)

• DOI: 10.1155/2023/9141112
• 发表时间: 2023-10-10
• 期刊: CELLULAR MICROBIOLOGY
• 影响因子: 3.4
• 作者: Ganderton，Timothy R.;Ghete，Daniel;Pryor，Paul R.
• 通讯作者: Pryor，Paul R.

Structural characterisation of the virulence-associated protein VapG from the horse pathogen Rhodococcus equi.

• DOI: 10.1016/j.vetmic.2015.01.027
• 发表时间: 2015-08-31
• 期刊: Veterinary microbiology
• 影响因子: 3.3
• 作者: Okoko T;Blagova EV;Whittingham JL;Dover LG;Wilkinson AJ
• 通讯作者: Wilkinson AJ

Structure of the virulence-associated protein VapD from the intracellular pathogen Rhodococcus equi.

• DOI: 10.1107/s1399004714012632
• 发表时间: 2014-08
• 期刊: Acta crystallographica. Section D, Biological crystallography
• 作者: Whittingham JL;Blagova EV;Finn CE;Luo H;Miranda-CasoLuengo R;Turkenburg JP;Leech AP;Walton PH;Murzin AG;Meijer WG;Wilkinson AJ
• 通讯作者: Wilkinson AJ