PSEUDOMONAS AERUGINOSA AND LUNG EPITHELIAL CELL CO-CULTURE METABOLOMICS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 子项目说明 在此资助期间，我们开发了二次电喷雾电离质谱（SESI-MS）技术，以提交我们的第一篇出版物（见"正在审查的论文"，#4），提交并获得了NASA-EPSCoR的资助和囊性纤维化基金会（通过达特茅斯学院）的试点项目资助，以支持相关工作，并有两篇论文被接受出版。 有人明智地建议我把研究目标集中在小组内，以便发展一个独特的研究利基。 因此，我们将重点放在两个领域：使用SESI-MS确定铜绿假单胞菌（Pa）的代谢状态，并建立在我们的磷酸肌醇（IP）跟踪记录的基础上，我们还关注Pa通过IP配体在肺环境中对Fe的利用。 项目领域1：Pa Mucoidy 来自囊性纤维化患者的85%的Pa临床分离株由于mucA突变而表现出粘液样表型。 因此，第一个项目的重点是快速确定Pa的粘体表型，因为它与mucA/MucA在摇瓶、生物膜和小鼠肺环境中的突变或降解有关。我们的目标是连接氨基酸代谢和挥发性侧链（如Tyr和Trp）的产生以及HCN产生的途径。 项目领域2：通过磷酸肌醇利用铁 第二个项目的重点是确定受体，进口，和顺序的酶促步骤，导致PA利用主机铁和肌醇磷酸盐。 我们在这里建立在我们对受体-配体相互作用的理解以及我们在理解原核生物中的氧化还原化学和磷酸酶生产生物学方面的专业知识。 赠款提交计划 在过去的六个月里，在肌醇磷酸化学和微生物转化领域已经提交了两项赠款。 三个拨款目标即将到来：NSF CAREER，USDA-NIFA和2011年2月的NIH RO1目标。 美国国家科学基金会和美国农业部的资助将集中在SESI-MS应用于快速检测大肠杆菌。大肠杆菌O157：H7的碎牛肉，在努力支持SESI-MS病原体挥发性数据库建设企业和利基。 R01提案将可能关注ENaC小鼠模型中Pa非粘液-粘液转变的代谢特征。 2009/10年度出版物 1.低S.Y.，希尔J.E.，和Peccia P.2009 A DNA Aptamer Recognizes the Aspergillus fumigatus Allergen Biochemical and Biophysical Research Communications 386（3）544 - 548 2.Johnson，N.R.和Hill，J.E.通过酶解的家禽粪便改良土壤的磷组成：高通量方法的证明和对酶不稳定P SSSAJ的提示 2009/10年度审查中的出版物 1.贾尔斯，C.D.，Cade-Menun，B.，和Hill，J.E.（正在审查）随时间跟踪的家禽粪便改良土壤中的磷迁移和转化 [2]贾尔斯，C.D.，Cade-Menun，B.，和Hill，J.E.土壤和粪肥中的肌醇磷酸：吸收、循环和测定 3. Haznedaroglu B.Z.，佐鲁·O希尔J.E.，步行者S.L.（正在审查中）确定鞭毛在运动和非运动肠道沙门氏菌血清型转运中的作用 4. Zhu J，Bean H.，郭耀，Hill J.E.细菌培养物中挥发性有机物的SESI-MS快速检测

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. SUBPROJECT DESCRIPTION During this funding period we have developed the secondary electrospray ionization mass spectrometry (SESI-MS) technology to the point of submitting our first publication (see 'papers under review', #4), submitted and received funding from NASA-EPSCoR and pilot project funding from the Cystic Fibrosis Foundation (via Dartmouth College) to support related work, and had two papers accepted for publication. It was wisely suggested that I focus the research goals within the group so as to develop a distinct research niche. Therefore, we have focuses ourselves in two areas: P. aeruginosa (Pa) metabolic state determination using SESI-MS and to build on our inositol phosphate (IP) track record, we also focus on Pa utilization of Fe via IP ligands in the lung environment. Project area 1: Pa Mucoidy 85% of Pa clinical isolates from patients with cystic fibrosis exhibit the mucoid phenotype as a consequence of mutations in mucA. Therefore, the first project focuses on the rapid determination of the mucoidy phenotype for Pa as it relates to mutation or degradation of mucA/MucA in the: shake flask, biofilm, and mouse lung environment. We target pathways that link amino acid metabolism and the production of side chains that are volatile, such as Tyr and Trp, as well as, the production of HCN. Project area 2: Pa utilization of Fe via inositol phosphates The second project focuses on the determination of the receptor, import, and sequence of enzymatic steps that lead to Pa utilization of host Fe and inositol phosphates. We build here on our understanding of receptor-ligand interactions as well as our expertise in understanding redox chemistry and the biology of phosphatase production in prokaryotes. Grant submission plans Two grants in the area of inositol phosphate chemistry and transformation via microorganisms have been submitted in the past six months. Three grant targets are on the horizon: NSF CAREER, USDA-NIFA, and an NIH RO1 target of February 2011. The NSF and USDA grants will focus on SESI-MS application to rapid detection of E. coli O157:H7 on ground beef, in an effort to support the SESI-MS pathogen volatile database-building enterprise and niche. The RO1 proposal will likely focus on the metabolic signature of the Pa non-mucoid-mucoid transition in the ENaC mouse model. Publications 2009/10 Accepted/Published 1.Low S.Y., Hill J.E., and Peccia P. 2009 A DNA Aptamer Recognizes the Asp f 1 Allergen of Aspergillus fumigatus Biochemical and Biophysical Research Communications 386 (3) 544-548 2.Johnson, N.R. and Hill, J.E. Phosphorus composition of a poultry manure-amended soil via enzymatic hydrolysis: demonstration of a high-throughput method and hints on enzyme-labile P SSSAJ Publications 2009/10 under review 1.Giles, C.D., Cade-Menun, B., and Hill, J.E. (under review) Phosphorus mobility and transformation in a poultry manure-amended soil tracked over time 2.Giles, C.D., Cade-Menun, B., and Hill, J.E. (under review) The Inositol Phosphates in Soils and Manures: Abundance, Cycling, and Measurement 3.Haznedaroglu B.Z., Zorlu O., Hill J.E., Walker S.L. (under review) Identifying the role of flagella in the transport of motile and nonmotile Salmonella enterica serovars 4.Zhu J, Bean H., Kuo Y., Hill J.E. Fast detection of headspace volatile organic compounds from bacterial cultures by SESI-MS