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.
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