VERMONT COBRE PROJECT 7: BETAINE REGULATION OF PSEUDOMONAS VIRULENCE

佛蒙特州 COBRE 项目 7：甜菜碱对假单胞菌毒力的调节

• 批准号: 8167737
• 负责人: MATTHEW J WARGO
• 金额: $ 20.63万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167737
• 关键词: Allergens; Allergic; Animal Experiments; Applications Grants; Appointment; Area; Award; Betaine; Binding; Binding Proteins; Biological; Breathing; Carbon; Carnitine; Catabolism; Cell Culture Techniques; Cells; Choline; Computer Retrieval of Information on Scientific Projects Database; Cystic Fibrosis; Data; Dissection; Enzymes; Epithelial Cells; European; Faculty; Foundations; Funding; Fungal Antigens; Future; Genes; Genetic Transcription; Goals; Grant; Human Resources; Immune response; Immunity; Immunology; Infection; Institution; Interleukin-6; Investigation; Journals; Knowledge; Laboratories; Learning; Lung; Lysophosphatidylcholines; Manuscripts; Metabolism; Microbiology; Miltefosine; Modeling; Molecular; Mucous body substance; Paper; Pathway interactions; Phenotype; Phospholipase; Phospholipase C; Phospholipases A; Physiology; Post-Transcriptional Regulation; Postdoctoral Fellow; Production; Proteins; Protons; Pseudomonas; Pseudomonas aeruginosa; Publications; Publishing; Pulmonary Surfactants; Recruitment Activity; Regulation; Regulon; Reporter; Reporting; Research; Research Personnel; Resources; Respiratory physiology; Role; Source; Specificity; System; Therapeutic; Therapeutic Agents; Time; Training; United States National Institutes of Health; Vermont; Virulence; Virulence Factors; Work; Writing; design; dimethylglycine; graduate student; in vivo; inhibitor/antagonist; mutant; professor; programs; quaternary ammonium compound; research study; response; small molecule; tool; Allergens; Allergic; Animal Experiments; Applications Grants; Appointment; Area; Award; Betaine; Binding; Binding Proteins; Biological; Breathing; Carbon; Carnitine; Catabolism; Cell Culture Techniques; Cells; Choline; Computer Retrieval of Information on Scientific Projects Database; Cystic Fibrosis; Data; Dissection; Enzymes; Epithelial Cells; European; Faculty; Foundations; Funding; Fungal Antigens; Future; Genes; Genetic Transcription; Goals; Grant; Human Resources; Immune response; Immunity; Immunology; Infection; Institution; Interleukin-6; Investigation; Journals; Knowledge; Laboratories; Learning; Lung; Lysophosphatidylcholines; Manuscripts; Metabolism; Microbiology; Miltefosine; Modeling; Molecular; Mucous body substance; Paper; Pathway interactions; Phenotype; Phospholipase; Phospholipase C; Phospholipases A; Physiology; Post-Transcriptional Regulation; Postdoctoral Fellow; Production; Proteins; Protons; Pseudomonas; Pseudomonas aeruginosa; Publications; Publishing; Pulmonary Surfactants; Recruitment Activity; Regulation; Regulon; Reporter; Reporting; Research; Research Personnel; Resources; Respiratory physiology; Role; Source; Specificity; System; Therapeutic; Therapeutic Agents; Time; Training; United States National Institutes of Health; Vermont; Virulence; Virulence Factors; Work; Writing; design; dimethylglycine; graduate student; in vivo; inhibitor/antagonist; mutant; professor; programs; quaternary ammonium compound; research study; response; small molecule; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. I joined the UVM faculty in September of 2009, and have spent the time between then and April 2010 setting up my laboratory, staffing and training, writing grant proposals, and advancing my independent research program. All of these points are briefly discussed below. My research is focused on the interaction between Pseudomonas aeruginosa and the mammalian lung, particularly as relates to sensing and metabolism of pulmonary surfactant compounds by P.a. The research effort over this past year has been greatly aided by the new personnel in the laboratory, and those areas under current study are delineated in the summaries below: The first research topic is the role of P.a. phospholipases in alteration of lung function during infection and the general role of secreted phospholipases in P.a. virulence. I have shown in experiments continued from my post-doctoral research that a potential therapeutic agent, miltefosine, was capable of inhibiting the decline in lung function during infection caused by the secreted hemolytic phospholipase C, PlcH. These observations were combined with our general infection model and are being submitted in late April or early May to JCI. This manuscript is the final paper from my postdoctoral work. Any further experiments regarding the regulation of PlcH and its production in vivo will be reported solely from my laboratory, while any future miltefosine work will continue to be collaborative with Dr. Deborah Hogan (Dartmouth). Further investigation of the potential therapeutic value of miltefosine has been funded by the Cystic Fibrosis Foundation Pilot funding program awarded to Dr. Hogan and myself, the funds of which will be split evenly between UVM and Dartmouth. During microarray analyses, I discovered an uncharacterized predicted secreted phospholipase A that was induced in response to pulmonary surfactant. I have created the deletion strain, transcriptional reporters, and other tools necessary for its characterization. In October 2009, Annette LaBauve (graduate student) joined the lab and has been working to understand the regulation of this phospholipase, as well as express and purify the protein to verify the functional prediction. We are planning on assessing any phenotype of the deletion mutant both in cell culture and animal experiments. This secreted phospholipase A has the potential to tie in well with Dr. Jon Boyson's work with NKT cells, as a product of these phospholipase A enzymes, lysophosphatidylcholine, has been shown to be sufficient to function as an activator of NKT cells. Pilot research investigating the interplay between phospholipases and activation of NKT and epithelial cells is supported by a pilot grant via the VCIID CoBRE to Drs. Boyson, Poynter, and myself. The goal of this pilot proposal is to gather sufficient preliminary data to generate one or more publications and submit a multi-PI R01 in the first half of 2011. The second research topic covers the catabolism of choline by P.a. and the regulation of the catabolic genes and virulence factors related to this pathway. These studies have been greatly advanced by two lab personnel, Dr. Ken Hampel (Research Assistant Professor) and Liam Fitzsimmons (Technician). Liam joined the lab in October 2009 and has been spearheading the effort to rationally design, synthesize, and characterize small molecule mimics of choline to aid in dissection of the entire pathway, particularly focused on identifying inhibitors and non-catalizable inducers. We have succeeded in both of these avenues. Propargylcholine is a potent inhibitor of the dimethylglycine catabolism enzyme system, and further characterization of the inhibitory mechanism is underway. Ethylcholine is a non-inhibitory, non-catablizable inducer of the choline and betaine transcriptional regulons. Liam has been trained on proton and carbon NMR and has verified these syntheses, and has also synthesized and validated control compounds that are similar to the active ones but without biological effect, to provide appropriate control compounds. We hope to use the knowledge gained from these compounds to inform the design of therapeutic compounds targeting this pathway. Dr. Hampel joined the lab in March 2010, and is studying the role of transcriptional and post-transcriptional regulation within the choline catabolism pathway, following up on findings that I had made approximately a year ago. Ken is also leading the effort to use microarray studies to characterize the choline and betaine transcriptional regulons using mutants and the small molecules generated by Liam. In addition to further dissection of the role of choline catabolism in vivo, this second research topic has been the primary focus of my start-up funding. The interplay of P.a. choline catabolism and lung physiology during infection is the topic of my K22 proposal which scored a 27 impact/priority score, and is awaiting the final adjustments to the payline. Other funding: I learned recently that I was unsuccessful in my Pew Scholars application. My future funding plans, regardless of the K22 being funded, include submission of an R01 in October 2010 and submission of a Hood Foundation Young Investigator proposal in September 2010. Publications (2009-2010): Note: only publication #5 was published after appointment to UVM. 1. Allard, J.B., Rinaldi, L., Wargo, M.J., Allen, G.A., Akira, S., Uematsu, S., Poynter, M.E., Hogan, D.A., Rincon, M., and Whittaker, L.A. 2009. Th2 allergic immune response to inhaled fungal antigens is modulated by TLR-4-independent bacterial products. European Journal of Immunology 39(3):776-788. PMCID: PMC2750775 2. Wargo, M.J., Ho, T., Gross, M., Whittaker, L.A., and Hogan, D.A. 2009. GbdR regulates P. aeruginosa plcH and pchP transcription in response to choline catabolites. Infection and Immunity. 77(3):1103-1111. PMCID: PMC2643652 3. Wargo, M.J. and Hogan, D.A. 2009. Identification of genes required for Pseudomonas aeruginosa carnitine catabolism. Microbiology (SGM). 155(7):2411-2419. (NIHMS 191666). 4. Neveu, W.A., Allard, J.B., Dienz, O., Wargo, M.J., Ciliberto, G., Whittaker, L.A., and Rincon, M. 2009. IL-6 is required for airway mucus production induced by inhaled fungal allergens. Journal of Immunology 183(3):1732-1738. (PMC in progress by corresponding author) 5. Chen, C., Malek, A.A., Wargo, M.J., Hogan, D.A., and Beattie, G.A. 2010. The ATP-binding cassette Cbc (choline/betaine/carnitine) transporter recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds. Molecular Microbiology 75(1):29-45. (PMC in progress by publisher).

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我于2009年9月加入了UVM教职员工，并在2010年4月之间花费了时间，设立了我的实验室，人员配备和培训，撰写赠款建议以及推进我的独立研究计划。所有这些观点将在下面简要讨论。 我的研究重点是铜绿假单胞菌与哺乳动物肺之间的相互作用，特别是与P.A.肺表面活性剂化合物的传感和代谢有关。过去一年中的研究工作得到了实验室的新人员的极大帮助，并且在以下摘要中划定了当前研究的那些领域： 第一个研究主题是P.A.的角色磷脂酶在感染过程中肺功能改变以及分泌磷脂酶在P.A.中的一般作用中的一般作用。毒力。我在实验中的研究中继续进行了研究后的研究，潜在的治疗剂Miltefosine能够抑制由分泌的溶血性磷脂酶C引起的感染期间肺功能下降。这些观察结果与我们的一般感染模型相结合，并于4月下旬或5月初提交给JCI。该手稿是我博士后工作的最后一篇论文。关于PLCH调节及其在体内生产的任何进一步的实验都将仅从我的实验室报告，而将来的任何米尔特富氨酸工作都将继续与Deborah Hogan博士（Dartmouth）合作。囊性纤维化基金会试点资金计划授予了霍根博士和我本人的囊性纤维化基金会试点资金计划，进一步研究了米尔特福修的潜在治疗价值，其资金将在UVM和达特茅斯之间平均分配。 在微阵列分析期间，我发现了一个未表征的预测的分泌的磷脂酶A，该磷脂酶A响应于肺表面活性剂。我创建了删除菌株，转录记者以及其表征所需的其他工具。 2009年10月，Annette Labauve（研究生）加入了实验室，并一直在努力了解该磷脂酶的调节，并表达和净化蛋白质以验证功能性预测。我们计划在细胞培养和动物实验中评估缺失突变体的任何表型。该分泌的磷脂酶A有可能与Jon Boyson博士与NKT细胞的工作相吻合，作为这些磷脂酶A酶的产物，溶血磷脂酰胆碱已被证明足以充当NKT细胞的激活剂。试点研究调查了磷脂酶之间的相互作用以及NKT和上皮细胞的激活，通过vCIID COBRE支持DRS。博伊森，poynter和我自己。该试点提案的目的是收集足够的初步数据来生成一个或多个出版物，并在2011年上半年提交多PI R01。 第二个研究主题涵盖了P.A.胆碱的分解代谢。以及与该途径相关的分解代谢基因和毒力因子的调节。两名实验室人员Ken Hampel（研究助理教授）和Liam Fitzsimmons（技术人员）大大提出了这些研究。利亚姆（Liam）于2009年10月加入了实验室，并一直率先努力设计，合成和表征胆碱的小分子模仿，以帮助解剖整个途径，尤其是专注于识别抑制剂和不可诱导的诱导剂。我们在这两个途径中都取得了成功。丙酰胆碱是二甲基甘氨酸分解代谢酶系统的有效抑制剂，抑制机制的进一步表征正在进行中。乙基胆碱是胆碱和甜菜碱转录调节子的不抑制性，不可抑制的诱导剂。利亚姆（Liam）接受了质子和碳NMR的培训，并已验证了这些合成，并且还合成和经过验证的对照化合物，与活性相似但没有生物学效应，以提供适当的控制化合物。我们希望利用从这些化合物中获得的知识来告知针对该途径的治疗化合物的设计。 Hampel博士于2010年3月加入实验室，并正在研究胆碱可分解代谢途径中转录和转录后调节的作用，跟进了我大约一年前所做的发现。 Ken还领导着使用微阵列研究的努力来使用突变体以及Liam产生的小分子来表征胆碱和甜菜碱转录调节。 除了进一步解剖胆碱分解代谢在体内的作用外，第二个研究主题是我启动资金的主要重点。 P.A.的相互作用感染过程中的胆碱分解代谢和肺部生理学是我的K22提案的主题，该提案得分为27个影响/优先级得分，并且正在等待对支付线的最终调整。 其他资金：我最近得知我在皮尤学者的申请中没有成功。我未来的资金计划，无论是由K22提供资金，都包括2010年10月提交R01，以及2010年9月提交的Hood Foundation Foundation年轻调查员提案。 出版物（2009-2010）：注意：仅在任命UVM后出版了5号出版物。 1。Allard，J.B.，Rinaldi，L.，Wargo，M.J.，Allen，G.A.，Akira，S.，Uematsu，S.欧洲免疫学杂志39（3）：776-788。 PMCID：PMC2750775 2。Wargo，M.J.，Ho，T.，Gross，M.，Whittaker，L.A。和Hogan，D.A。 2009。GBDR调节铜绿假单胞菌PLCH和PCHP转录，以响应胆碱分解代谢物。感染和免疫力。 77（3）：1103-1111。 PMCID：PMC2643652 3。Wargo，M.J。和Hogan，D.A。 2009。鉴定铜绿假单胞菌肉碱分解代谢所需的基因。微生物学（SGM）。 155（7）：2411-2419。 （NIHMS 191666）。 4。Neveu，W.A.，Allard，J.B.，Dienz，O.，Wargo，M.J.，Ciliberto，G.，Whittaker，L.A。和Rincon，M.2009。IL-6是由吸入真菌过敏原引起的气道粘液生产所必需的。免疫学杂志183（3）：1732-1738。 （通讯作者正在进行的PMC） 5。Chen，C.，Malek，A.A.，Wargo，M.J.，Hogan，D.A。和Beattie，G.A。 2010。ATP结合Cassette CBC（胆碱/甜菜碱/carnitine）转运蛋白募集多种底物结合蛋白具有很强的特异性，这些蛋白对于不同的季铵化合物。分子微生物学75（1）：29-45。 （发布者正在进行的PMC）。