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: 8360778
• 负责人: MATTHEW J WARGO
• 金额: $ 21.67万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-20 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360778
• 关键词: Animal Experiments; Applications Grants; Area; Award; Betaine; Biological; Carbon; Catabolism; Cell Culture Techniques; Cells; Centers of Research Excellence; Choline; Communicable Diseases; Cystic Fibrosis; Data; Dissection; Enzymes; Epithelial Cells; Faculty; Foundations; Funding; Future; Genes; Goals; Grant; Human Resources; Infection; Investigation; Knowledge; Laboratories; Lung; Lysophosphatidylcholines; Manuscripts; Metabolism; Miltefosine; Modeling; National Center for Research Resources; Paper; Pathway interactions; Phenotype; Phospholipase; Phospholipase C; Phospholipases A; Physiology; Post-Transcriptional Regulation; Postdoctoral Fellow; Principal Investigator; Production; Proteins; Protons; Pseudomonas; Pseudomonas aeruginosa; Publications; Pulmonary Surfactants; Regulation; Regulon; Reporter; Reporting; Research; Research Infrastructure; Resources; Respiratory physiology; Role; Source; System; Therapeutic; Therapeutic Agents; Time; Training; United States National Institutes of Health; Vermont; Virulence; Virulence Factors; Work; Writing; cost; design; dimethylglycine; graduate student; in vivo; inhibitor/antagonist; mutant; professor; programs; 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我于2009年9月加入了UVM教师，并在2010年4月之前建立了我的实验室，人员配备和培训，撰写拨款申请，并推进了我的独立研究计划。下文将简要讨论所有这些问题。 我的研究主要集中在铜绿假单胞菌和哺乳动物肺之间的相互作用，特别是与肺表面活性物质化合物的传感和代谢有关。过去一年的研究工作得到了实验室新人员的极大帮助，目前研究的领域概述如下： 第一个研究主题是P.A.的作用。磷脂酶在感染期间肺功能改变中的作用以及分泌型磷脂酶在P. a.毒性。我在博士后研究中继续进行的实验表明，一种潜在的治疗药物米替福新能够抑制分泌型溶血性磷脂酶C（PlcH）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月底或5月初提交给JCI。这是我博士后工作的最后一篇论文。关于PlcH调节及其体内生产的任何进一步实验将仅由我的实验室报告，而任何未来的米替福新工作将继续与Deborah Hogan博士（达特茅斯）合作。对米替福新潜在治疗价值的进一步研究得到了授予霍根博士和我本人的囊性纤维化基金会试点资助计划的资助，该基金将在UVM和达特茅斯之间平均分配。 在微阵列分析过程中，我发现了一种未表征的预测分泌型磷脂酶A，它是在对肺表面活性物质的反应中诱导的。我已经创建了缺失菌株，转录报告，和其他必要的工具，其特征。2009年10月，Annette LaBauve（研究生）加入实验室，并一直致力于了解这种磷脂酶的调控，以及表达和纯化蛋白质以验证功能预测。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。这种分泌的磷脂酶A有可能与Jon Boyson博士对NKT细胞的研究密切相关，因为这些磷脂酶A酶的产物溶血磷脂酰胆碱已被证明足以作为NKT细胞的激活剂。调查磷脂酶与NKT和上皮细胞活化之间相互作用的试点研究得到了VCIIID CoBRE向Boyson博士，Poynter博士和我本人提供的试点资助的支持。本试点提案的目标是收集足够的初步数据以生成一份或多份出版物，并在2011年上半年提交多PI R 01。 第二个研究课题涉及P. a.以及与该途径相关的分解代谢基因和毒力因子的调控。两名实验室人员Ken Hampel博士（研究助理教授）和Liam Fitzsimmons（技术员）大大推进了这些研究。Liam于2009年10月加入该实验室，并一直致力于合理设计，合成和表征胆碱的小分子模拟物，以帮助解剖整个途径，特别是专注于识别抑制剂和非催化诱导剂。我们在这两个方面都取得了成功。炔丙基胆碱是二甲基甘氨酸催化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非抑制性、不可催化诱导剂。Liam接受过质子和碳NMR的培训，并验证了这些合成，还合成并验证了与活性化合物相似但无生物学效应的对照化合物，以提供适当的对照化合物。我们希望利用从这些化合物中获得的知识来设计针对这一途径的治疗化合物。Hampel博士于2010年3月加入该实验室，正在研究胆碱催化剂通路中转录和转录后调控的作用，跟进我大约一年前的发现。Ken还领导了使用微阵列研究来表征胆碱和甜菜碱转录调节子的工作，这些研究使用了Liam产生的突变体和小分子。 除了进一步剖析胆碱催化剂在体内的作用外，这第二个研究课题一直是我启动资金的主要重点。P.A.的相互作用感染期间的胆碱催化剂和肺生理学是我的K22提案的主题，该提案获得了27分的影响/优先级分数，正在等待对付款线的最终调整。