Structural characterization of outer membrane proteins from Yersinia pestis

鼠疫耶尔森氏菌外膜蛋白的结构表征

• 批准号: 7733943
• 负责人: Susan Buchanan
• 金额: $ 34.02万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733943
• 关键词: Adverse effects; Air; Amendment; Animals; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Antigens; Bacteria; Binding; Biological; Biological Assay; Biology; Bite; Bubonic Plague; Cells; Cellular biology; Cessation of life; Citrate; Citrates; Collaborations; Communicable Diseases; Condition; Crystallization; Crystallography; DNA Microarray Chip; DNA Microarray format; Development; Disease; Disease Outbreaks; Drug Design; Drug resistance; Economics; Effectiveness; Environment; Epidemic; Epitopes; Escherichia coli; European; Fleas; Future; Human; Hygiene; Immune system; Individual; Infection; Iron; Knowledge; Ligands; Lung; Manuscripts; Mediating; Membrane; Membrane Proteins; Membrane Transport Proteins; Methods; Molecular Profiling; Monitor; Multi-Drug Resistance; Mus; Names; National Institute of Allergy and Infectious Disease; Natural Immunity; Nature; Nuclear Magnetic Resonance; Pathway interactions; Patients; Pharmaceutical Preparations; Plague; Plague Vaccine; Plasmids; Pneumonic Plague; Preparation; Preventive; Process; Production; Prohibit; Proteins; Rattus; Recording of previous events; Recurrence; Reporting; Research; Resistance; Rodent; Siderophores; Site-Directed Mutagenesis; Speed; Structure; System; Thinking; Time; Toxin; Travel; Update; Vaccinated; Vaccination; Vaccines; Virulence; Virulent; Whole Cell Vaccine; World Health Organization; X-Ray Crystallography; Yersinia pestis; analytical ultracentrifugation; base; capsule; colicin; contagion; design; experience; extracellular; ferric citrate; flasks; improved; innovation; interest; middle age; novel vaccines; pathogenic bacteria; polypeptide; programs; receptor; respiratory; response; solid state; structural biology; vaccine development

2. Current research Our collaborator at NIAID, Dr. B. Joseph Hinnebusch, has used DNA microarrays to compare expression profiles of Y. pestis recovered from plague infected rats as opposed to Y. pestis that are grown in flask cultures6. Using these methods they have identified eight putative outer membrane proteins that are found in higher amounts in infected animals. Almost all of these are proteins found in the outer envelope of Y. pestis and for this reason it is thought that they might make good vaccine targets. This is because portions of these proteins protrude into the surrounding environment of the bacteria where they can be readily detected by the immune system of an infected but vaccinated human being. Additionally almost all are involved in the import of iron into the cell. The ability to acquire iron is essential for the survival of most bacteria including pathogenic bacteria. In Y. pestis the ability to obtain iron from the iron poor environment of the infected host is correlated to virulence and lethality of infection in mice. Our lab has a lot of expertise on the basic and structural biology of iron import pathways E. coli4,5. The experiences gained on the handling of similar proteins in E. coli have already been valuable in the study of Y. pestis proteins. We have proceeded to clone, express, purify and crystallize these 8 proteins with two general aims: first, to generate protein for vaccine development studies and second, to make use of the excess material for structural studies. So far we have expressed and successfully purified 5 out of the 8 proteins. We have successfully used X-ray crystallography to solve the crystal structure of a Yersinia pestis membrane TonB type receptor and a second outer membrane protein not involved in iron transport (two manuscripts in preparation). It is hoped that these structures will reveal structural epitopes useful for vaccine development and/or antibiotic development. 3. Future research In the immediate future we would like to use X-ray crystallography to look at the molecular interactions of the TonB receptor with its natural ligands a microbiocidal colicin and an iron siderophore. In addition to crystallography ligand-receptor interactions may have to be studied by analytical ultracentrifugation and/or solid state Nuclear Magnetic Resonance (NMR). This information might be useful in the rational design of new antibiotic compounds. We would like to also look at iron import processes that are downstream of the binding interaction of the TonB receptor and its ligands. For this we hope to use site directed mutagenesis in combination with a cell biology assays. Additionally the project described in section 2 is still ongoing. More proteins will have to be expressed and purified and thousands more crystallization conditions will have to be screened in order to obtain diffraction quality crystals so that more structures of the remaining proteins can be solved. Further collaborations will have to be established to use the information gained from this project for vaccine development or rational drug design. 1. http://www.who.int/mediacentre/factsheets/fs267/en/ 2. Galimand, M., Guiyoule, A., Gerbaud, G., Rasoamanana, B., Chanteau, S., Carniel, E., Courvalin P. (1997) Multidrug resistance in Yersinia pestis mediated by a transferable plasmid. N Engl J Med. 337(10), 702-4. 3. http://disarmament2.un.org/wmd/bwc/index.html 4. Buchanan, S. K., Smith, B. S., Venkatramani, L., Xia, D., Palnitkar, M., Chakraborty, R., van der Helm, D. & Deisenhofer, J. (1999). Crystal structure of the outer membrane active transporter FepA from Escherichia coli. Nature Struc. Biol. 6, 56-63. 5. Yue, W.W., Grizot, S. & Buchanan, S.K. (2003). Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA. J. Mol. Biol. 332, 353-368. 6. Sebbane, F., Lemaitre, N., Sturdevant, D.E., Rebeil, R., Virtaneva, K., Porcella, S.F. & Hinnebusch, B.J. (2006). Adaptive response of Yersina pestis to extracellular effectorsof innate immunity during bubonic plague. Proc. Natl. Acad. Sci. USA 103, 11766-11771.

2. 目前的研究 我们在 NIAID 的合作者 B. Joseph Hinnebusch 博士使用 DNA 微阵列比较了从感染鼠疫的大鼠中回收的鼠疫耶尔森氏菌与培养瓶中培养的鼠疫耶尔森氏菌的表达谱6。利用这些方法，他们鉴定了八种假定的外膜蛋白，这些蛋白在受感染的动物中含量较高。几乎所有这些都是在鼠疫耶尔森氏菌外膜中发现的蛋白质，因此人们认为它们可能成为良好的疫苗靶点。这是因为这些蛋白质的一部分突出到细菌的周围环境中，在那里它们可以很容易地被受感染但已接种疫苗的人类的免疫系统检测到。此外，几乎所有这些都参与铁向细胞的输入。获取铁的能力对于大多数细菌（包括病原菌）的生存至关重要。在鼠疫耶尔森氏菌中，从受感染宿主的贫铁环境中获取铁的能力与小鼠感染的毒力和致死率相关。我们的实验室在铁输入途径大肠杆菌4,5 的基础和结构生物学方面拥有大量专业知识。在大肠杆菌中处理类似蛋白质所获得的经验对于鼠疫耶尔森氏菌蛋白质的研究已经很有价值。 我们已经开始克隆、表达、纯化和结晶这 8 种蛋白质，有两个总体目标：第一，产生用于疫苗开发研究的蛋白质；第二，利用多余的材料进行结构研究。到目前为止，我们已经表达并成功纯化了 8 种蛋白质中的 5 种。我们已经成功地利用X射线晶体学解析了鼠疫耶尔森氏菌膜TonB型受体和不参与铁转运的第二种外膜蛋白的晶体结构（两份手稿正在准备中）。希望这些结构将揭示可用于疫苗开发和/或抗生素开发的结构表位。 3. 未来的研究 在不久的将来，我们希望使用 X 射线晶体学来观察 TonB 受体与其天然配体（杀菌大肠杆菌素和铁铁载体）的分子相互作用。除了晶体学之外，配体-受体相互作用可能还需要通过分析超速离心和/或固态核磁共振（NMR）来研究。这些信息可能有助于合理设计新的抗生素化合物。 我们还想研究 TonB 受体及其配体结合相互作用下游的铁输入过程。为此，我们希望将定点诱变与细胞生物学测定相结合。 此外，第 2 节中描述的项目仍在进行中。必须表达和纯化更多的蛋白质，并且必须筛选数千种结晶条件，以获得衍射质量的晶体，从而可以解析剩余蛋白质的更多结构。 必须建立进一步的合作，以利用从该项目中获得的信息来开发疫苗或合理的药物设计。 1. http://www.who.int/mediacentre/factsheets/fs267/en/ 2. Galimand, M., Guiyoule, A., Gerbaud, G., Rasoamanana, B., Chanteau, S., Carniel, E., Courvalin P. (1997) 可转移质粒介导的鼠疫耶尔森氏菌的多药耐药性。 N 英格兰医学杂志。 337(10)、702-4。 3. http://disarmament2.un.org/wmd/bwc/index.html 4. Buchanan, S.K.、Smith, B.S.、Venkatramani, L.、Xia, D.、Palnitkar, M.、Chakraborty, R.、van der Helm, D. 和 Deisenhofer, J. (1999)。大肠杆菌外膜主动转运蛋白 FepA ​​的晶体结构。自然结构。生物。 6、56-63。 5. Yue, W.W.、Grizot, S. 和 Buchanan, S.K. （2003）。无铁柠檬酸盐和柠檬酸铁与 TonB 依赖性外膜转运蛋白 FecA 结合的结构证据。 J.莫尔。生物。 332、353-368。 6. Sebbane, F.、Lemaitre, N.、Sturdevant, D.E.、Rebeil, R.、Virtaneva, K.、Porcella, S.F. ＆Hinnebusch，B.J.（2006）。鼠疫耶尔森氏菌对先天免疫细胞外效应器的适应性反应。过程。国家。阿卡德。科学。美国 103，11766-11771。