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

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).