Effective treatment of many infectious diseases is limited by ignorance of the infected environment and how pathogens adapt to it and alter it. Unfortunately, methods to conduct in situ studies that would provide such insight are largely absent from biomedicine, and there is an urgent need to develop new technical approaches. The existing, dominant paradigm in infectious disease research is to use (necessarily imperfect) reductionist experiments with model organisms to study their physiology and thereby identify new drug targets. Yet ecosystems are much more than the sum of their parts, and interactions (both competitive and cooperative) between species can significantly affect the behavior of individuals. Because of this, traditional approaches likely reveal only part of the bigger picture. Growing awareness of the importance of the human microbiome in determining human health and disease has resulted in an increased appreciation for microbial ecology, yet studies in this area still have focused almost entirely on surveying the microbial communities present within various parts of the body (e.g. the NIH-funded Human Microbiome Project). Here we proposed to develop and apply a number of new approaches to answer the following questions about opportunistic pathogens in the lungs of cystic fibrosis (CF) patients: i) who is there, and how are they spatially associated with each other and with the host? ii) how active are they, as a function of both space and time? and iii) what metabolic pathways are they utilizing? In doing so we will draw on our experience in the field of geobiology, using tools originally developed to track and understand microbes in remote habitats like deep-sea sediments. We will employ state- of-the-art microelectrodes to characterize the chemical environment of the host lung at micrometer length scales, looking for chemical gradients that shape the behavior of the microbial ecosystem and/or are generated by it. Incorporation of 2H from water into lipids will be developed as a novel proxy for in situ growth rates, and spatial mapping of 15N incorporation into proteins using nanoSIMS will be used to discern sub-micrometer patterns of metabolic activity. Fluorescence in situ hybridization (FISH) using 16S rRNA-directed probes will enable the spatial organization between different bacterial species within biofilms to be determined at the level of single cells. Finally, we will develop new FISH probes targeting particular mRNA transcripts using a novel hybridization chain-reaction technique to map metabolic gene expression. These methods will be refined and validated using planktonic and biofilm cultures of Pseudomonas aeruginosa and subsequently applied to CF patient samples in collaboration with clinicians from Children's Hospital L.A. and the USC Adult CF Clinic. Expectorated sputum will be used to gain insight into the temporal evolution of the lung microbiome, whereas explanted lungs will be used to study its spatiometabolic organization. While the CF microbiome will serve as our starting point for the development of these new methods, they ultimately have the potential to transform the study of diverse types of infectious diseases ranging from tuberculosis to malaria.

Public Health Relevance

The design of effective therapeutics to combat infectious diseases is profoundly limited by our ignorance of how pathogens survive in the human host at different stages of infection. Traditionally, studies attempting to gain insight into this problem have been performed in the laboratory using model systems and conditions that imperfectly mimic the human host. Here, we propose to directly measure the chemistry, structure and metabolic activity of pathogens in situ using a novel suite of approaches.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL117328-03
Application #
8682825
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Caler, Elisabet V
Project Start
2012-09-24
Project End
2017-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Babin, Brett M; Bergkessel, Megan; Sweredoski, Michael J et al. (2016) SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 113:E597-605
DePas, William H; Starwalt-Lee, Ruth; Van Sambeek, Lindsey et al. (2016) Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling. MBio 7:
Kopf, Sebastian H; Sessions, Alex L; Cowley, Elise S et al. (2016) Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum. Proc Natl Acad Sci U S A 113:E110-6
Kreamer, Naomi N; Phillips, Rob; Newman, Dianne K et al. (2015) Predicting the impact of promoter variability on regulatory outputs. Sci Rep 5:18238
Kreamer, Naomi N; Costa, Flavia; Newman, Dianne K (2015) The ferrous iron-responsive BqsRS two-component system activates genes that promote cationic stress tolerance. MBio 6:e02549
Van Sambeek, Lindsey; Cowley, Elise S; Newman, Dianne K et al. (2015) Sputum glucose and glycemic control in cystic fibrosis-related diabetes: a cross-sectional study. PLoS One 10:e0119938
Cowley, Elise S; Kopf, Sebastian H; LaRiviere, Alejandro et al. (2015) Pediatric Cystic Fibrosis Sputum Can Be Chemically Dynamic, Anoxic, and Extremely Reduced Due to Hydrogen Sulfide Formation. MBio 6:e00767
Kopf, Sebastian H; McGlynn, Shawn E; Green-Saxena, Abigail et al. (2015) Heavy water and (15) N labelling with NanoSIMS analysis reveals growth rate-dependent metabolic heterogeneity in chemostats. Environ Microbiol 17:2542-56
Glasser, Nathaniel R; Kern, Suzanne E; Newman, Dianne K (2014) Phenazine redox cycling enhances anaerobic survival in Pseudomonas aeruginosa by facilitating generation of ATP and a proton-motive force. Mol Microbiol 92:399-412
Fox, Emily P; Cowley, Elise S; Nobile, Clarissa J et al. (2014) Anaerobic bacteria grow within Candida albicans biofilms and induce biofilm formation in suspension cultures. Curr Biol 24:2411-6

Showing the most recent 10 out of 11 publications