PSEUDOMONAS AERUGINOSA AND LUNG EPITHELIAL CELL CO-CULTURE METABOLOMICS

铜绿假单胞菌和肺上皮细胞共培养代谢组学

• 批准号: 7959821
• 负责人: Jack Hill
• 金额: $ 12.76万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959821
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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. Tracking the volatile metabolic signature of Pseudomonas aeruginosa (PA14) grown under variable iron concentrations and doses of sub-minimum inhibitory concentrations of ¿-lactam antibiotics The overarching goal of this project is to use novel extractive electrospray ionization mass spectrometry (EESI-MS) to determine the volatile metabolic fingerprint for P. aeruginosa under culture conditions approaching clinical relevance  namely, the growth of P. aeruginosa biofilm on CFTR-mutant lung epithelial cells (with complimentary microarray and immunological measurements). Mucoid biofilms of P. aeruginosa play a dominant role in the morbidity and mortality of those with afflicted with Cystic Fibrosis as well as other illnesses of the respiratory tract. The virulence of P. aeruginosa is significantly impacted by medium iron concentration and it is well known that antibiotics are less effective when P. aeruginosa is in a biofilm. This project initially focuses on the systematic measurement of volatile compounds generated by P. aeruginosa grown aerobically as suspended cultures in minimal media (M9) under iron-limited conditions (via the use of the iron-chelator desiferrisirox; [Fefree]: 0.2-600 ¿M) and is followed by experiments with P. aeruginosa grown in synthetic sputum media with the same limited iron conditions. Our plan is to transition to biofilm-based growth in the summer of 2009 and at that time, start to couple the volatile metabolome measurement to rRNA expression levels using P. aeruginosa microarrays. Thereafter, we will also systematically explore the impact of ¿-lactam antibiotic (e.g., cefalexin) dose on the volatile metabolome signature and gene expression via microarray to determine if indeed there is a unique volatile profile under these conditions. Our funding started in November, 2008. However, the mass spectrometer purchased required the renovation of a space in Votey Hall, which was not finished until mid-March 2009. While the room was being renovated, one graduate student and two undergraduate students mapped out their projects in great detail and built the associated apparatuses required for their project; primarily: the bacterial culture chambers (to collect and direct headspace vapors to the mass spectrometer) and the design and manufacture of the modified mass spectrometer interface for the EESI-MS configuration. In the meantime, I applied for and received small pilot project funding from Dartmouth University's COBRE (Lung Pathobiology) in the amount of $10,000. Most importantly, the methodology assistance for growing CFTR lung cells was offered by the O'Toole group. This knowledge will enhance the probability of success of the next phase of the project, which is to reliably capture the volatile metabolome of the growth of biofilm on CFTR-mutant lung epithelial cells (in conjunction with simultaneous microarray and immunological measurements). A post-doctoral researcher position is currently being advertised and her/his position will be focused on this area. A full-length grant proposal was submitted to the NASA Experimental Program to Stimulate Competitive Research. The title of the project is "The impact of microgravity-grown Pseudomonas aeruginosa and Haemophilus influenzae on human lung cells: integration of virulence, lung cell immune responses, and the volatile metabolome." The grant award would be in the amount of $750,000. I am the PI on the award, however, it was written in collaboration with the major members of the cystic fibrosis research cluster at the University of Vermont (Poynter, Weiss, and Whittaker) and indeed these researchers are the co-Investigators on the proposal submission. My funding goal for 2009/10 is to generate enough data and publications to submit to a R21 proposal in late 2009/early 2010. The R21 proposal will likely focus on the volatile metabolome, but would be informed by an integration of gene expression and immune system function data. Jane Hill  Project 2: Measurement and prediction of E. coli counter current swimming in laminar flow This is a smaller project. Briefly, the objective of this project is to determine the conditions under which E. coli strains will turn and swim against the flow. Our goal is to predict when this phenomenon will occur through an integration of the dominant forces acting on the bacterium and confirmatory experimental measurement. We have initially studied late exponential phase-grown E. coli K12 cells subject to laminar flow in LB media and within a microfluidic channel. We have made some solid progress on this topic. Namely, we have adapted and built several subroutines into an existing MatLab code which enables us to semi-automatically analyze our time-lapse data. The MatLab code is used to import images, track particle motion, and extract out salient features e.g., bacterium aspect ratio and the radius of curvature of bacterium as it turns into the flow. To dramatically enhance the impact of drag on the cell body, we grow motile E. coli K12 on sub-minimum inhibitory concentrations of cefalexin to produce elongated cells (up to 30x normal cell length). Theoretically, this additional surface area enhances the drag force on the bacterium which we hypothesize to be one of the major forces acting on the body under laminar flow conditions. We do see this impact. Additionally, we recently started to model the major forces acting on the bacterial body (i.e., fluid drag, fluid shear, and bacterial motility). Our goal is to link experiment and theory in the form of a manuscript by the end of the summer of 2009. Thereafter, we will focus on the use of our collection of uropathogenic E. coli strains to determine if our model accurately predicts what we measure experimentally and apply this knowledge to enhance our understanding of ascending infections of the urinary tract. Publications: 2008 and 2009 Peccia J., Milton D., Reponen T., and Hill J.E., 2008 J. A Role for Environmental Engineering and Science in Preventing Bioaerosol Related Disease. Environmental Science and Technology (feature) 4631-4637. Low S.Y., Hill J.E., and Peccia P. 2009 DNA aptamers bind specifically and selectively to (1 ¿ 3)-¿-d-glucans Biochemical and Biophysical Research Communications 378 (4): 701-705 Hill J.E. and Cade-Menun B., 2009 Phosphorus-31 nuclear magnetic resonance spectroscopy transect study of poultry operations on the Delmarva Peninsula. Journal of Environmental Quality 38(1): 130-138 Mentoring Summaries: Dr. Elizabeth Bonney Jane Hill, a recent COBRE Junior Faculty, is newly a mentee to me. Jane and I both have training in Chemical Engineering and have similar ways of analysis and thinking about biological problems. As a practicing physician I find her work very exciting and potentially relevant in several areas of clinical infectious disease. My mentoring goals for her are as follows: 1. To help Jane formulate well-focused questions and delineate and expand lines of clinically relevant investigation. 2. To foster connections between Jane and other members of the COBRE and other scientists with relevant expertise. 3. To assist her in the writing of grants. I plan to meet with Jane every 2-3 weeks. Ralph Budd I have met with Dr. Hill monthly since joining the COBRE program. Dr. Hill has been quite active in launching her research program, despite commitments for undergraduate teaching. As this was a concern raised by the EAC, I plan to discuss this with her Dean. Dr. Hill now has her mass spec machine for performing her proposed research on aerosolized components of bacteria. There were some delays in the HVAC system installation system needed, but that is being worked out. She made a visit to the Dartmouth Lung Center group who received her very warmly, particularly her proposed studies on Pseudomonas, which would fit with their regional CF center. They will likely provide some support also for Dr. Hill. Publications: Dr. Hill indicated that two publications are in preparation. Grants: A NASA submission is planned, the Dartmouth co-suppooty, and a potential R21 later in the summer or fall.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 追踪在不同铁浓度和亚最低抑菌浓度的 β-内酰胺抗生素剂量下生长的铜绿假单胞菌 (PA14) 的挥发性代谢特征 该项目的总体目标是使用新型提取电喷雾电离质谱 (EESI-MS) 来确定铜绿假单胞菌在接近临床相关性的培养条件下的挥发性代谢指纹，即铜绿假单胞菌生物膜在 CFTR 突变肺上皮细胞上的生长（具有互补的微阵列和免疫学测量）。铜绿假单胞菌的粘液生物膜在囊性纤维化以及其他呼吸道疾病患者的发病率和死亡率中起主导作用。铜绿假单胞菌的毒力受到中等铁浓度的显着影响，众所周知，当铜绿假单胞菌处于生物膜中时，抗生素的效果较差。该项目最初侧重于系统测量铜绿假单胞菌在铁限制条件下（通过使用铁螯合剂 desiferrisirox；[Fefree]：0.2-600 µM）在基本培养基 (M9) 中需氧生长为悬浮培养物时产生的挥发性化合物，随后对在相同铁限制条件下的合成痰培养基中生长的铜绿假单胞菌进行实验。我们的计划是在 2009 年夏天过渡到基于生物膜的生长，届时，开始使用铜绿假单胞菌微阵列将挥发性代谢组测量与 rRNA 表达水平结合起来。此后，我们还将通过微阵列系统地探讨β-内酰胺抗生素（例如头孢氨苄）剂量对挥发性代谢组特征和基因表达的影响，以确定在这些条件下是否确实存在独特的挥发性特征。 我们的资助从2008年11月开始。然而，购买的质谱仪需要对Votey Hall的一个空间进行翻修，直到2009年3月中旬才完成。在翻修房间的过程中，一名研究生和两名本科生详细规划了他们的项目，并建造了项目所需的相关设备；主要是：细菌培养室（收集顶空蒸汽并将其引导至质谱仪）以及针对 EESI-MS 配置改进的质谱仪接口的设计和制造。与此同时，我向达特茅斯大学 COBRE（肺病理学）申请并获得了 10,000 美元的小型试点项目资助。最重要的是，O'Toole 小组提供了生长 CFTR 肺细胞的方法学帮助。这些知识将提高该项目下一阶段成功的可能性，即可靠地捕获 CFTR 突变肺上皮细胞上生物膜生长的挥发性代谢组（结合同步微阵列和免疫学测量）。 目前正在招聘博士后研究员职位，她/他的职位将集中在该领域。 一份完整的拨款提案已提交给美国宇航局刺激竞争研究实验计划。 该项目的标题是“微重力生长的铜绿假单胞菌和流感嗜血杆菌对人类肺细胞的影响：毒力、肺细胞免疫反应和挥发性代谢组的整合”。 补助金金额为 750,000 美元。 我是该奖项的 PI，然而，它是与佛蒙特大学囊性纤维化研究集群的主要成员（Poynter、Weiss 和 Whittaker）合作撰写的，实际上这些研究人员是提交提案的共同研究者。我 2009/10 年度的资助目标是产生足够的数据和出版物，以便在 2009 年底/2010 年初提交给 R21 提案。R21 提案可能会关注不稳定的代谢组，但会通过基因表达和免疫系统功能数据的整合来提供信息。 Jane Hill 项目2：层流中大肠杆菌逆流游动的测量和预测 这是一个较小的项目。简而言之，该项目的目标是确定大肠杆菌菌株会转向并逆流游动的条件。我们的目标是通过整合作用于细菌的主导力和验证性实验测量来预测这种现象何时发生。我们最初研究了在 LB 介质和微流体通道内经受层流的晚期指数生长期大肠杆菌 K12 细胞。我们在这个主题上取得了一些扎实的进展。也就是说，我们在现有的 MatLab 代码中调整并构建了几个子程序，这使我们能够半自动分析延时数据。 MatLab 代码用于导入图像、跟踪粒子运动并提取显着特征，例如细菌长宽比和细菌进入流动时的曲率半径。为了显着增强阻力对细胞体的影响，我们在亚最低抑制浓度的头孢氨苄下培养能动的大肠杆菌 K12，以产生细长的细胞（高达正常细胞长度的 30 倍）。从理论上讲，这种额外的表面积增强了对细菌的阻力，我们假设这是层流条件下作用于身体的主要力量之一。我们确实看到了这种影响。此外，我们最近开始模拟作用于细菌体的主要力（即流体阻力、流体剪切和细菌运动）。我们的目标是在 2009 年夏末之前以手稿的形式将实验和理论联系起来。此后，我们将专注于使用我们收集的尿路致病性大肠杆菌菌株来确定我们的模型是否准确预测我们实验测量的结果，并应用这些知识来增强我们对尿路上行感染的理解。 出版物：2008 年和 2009 年 Peccia J.、Milton D.、Reponen T. 和 Hill J.E.，2008 J. 环境工程和科学在预防生物气溶胶相关疾病中的作用。 环境科学与技术（专题）4631-4637。 Low S.Y.、Hill J.E. 和 Peccia P. 2009 DNA 适体特异性且选择性地结合 (1 ¿ 3)-¿-d-葡聚糖 生物化学和生物物理研究通讯 378 (4): 701-705 Hill J.E. 和 Cade-Menun B.，2009 年德尔马瓦半岛家禽养殖的 Phosphorus-31 核磁共振波谱横断面研究。环境质量杂志38(1): 130-138 辅导总结： 伊丽莎白·邦尼博士 Jane Hill，新近加入 COBRE 初级教员，也是我的新学员。简和我都接受过化学工程方面的培训，对生物问题的分析和思考方式也很相似。 作为一名执业医师，我发现她的工作非常令人兴奋，并且可能与临床传染病的多个领域相关。 我对她的指导目标如下： 1. 帮助 Jane 提出重点突出的问题，并描绘和扩展临床相关调查的范围。 2. 促进 Jane 与 COBRE 其他成员以及其他具有相关专业知识的科学家之间的联系。 3. 协助她撰写补助金。 我计划每 2-3 周与 Jane 会面一次。 拉尔夫·巴德 自从加入 COBRE 计划以来，我每月都会与 Hill 博士会面。 尽管致力于本科教学，希尔博士一直非常积极地启动她的研究项目。 由于这是 EAC 提出的问题，我计划与她的院长讨论这个问题。 希尔博士现在拥有她的质谱仪，用于执行她提议的细菌雾化成分研究。 所需的暖通空调系统安装系统存在一些延迟，但正在解决中。 她拜访了达特茅斯肺中心小组，该小组非常热情地接待了她，特别是她提出的关于假单胞菌的研究，这将适合他们的地区 CF 中心。 他们也可能会为希尔博士提供一些支持。 出版物：希尔博士表示，两份出版物正在准备中。 赠款：计划向 NASA 提交申请，达特茅斯联合支持，以及可能在夏季或秋季晚些时候推出的 R21。