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