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Clarifying the Intracellular and Extracellular Lifestyles of CF Microbes in Three Dimensions

Jorth, P. and Newman, D. K. (2017) Clarifying the Intracellular and Extracellular Lifestyles of CF Microbes in Three Dimensions. In: 31st Annual North American Cystic Fibrosis Conference, 2-4 November, 2017, Indianapolis, IN. https://resolver.caltech.edu/CaltechAUTHORS:20171012-103857991

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Abstract

Imaging of sputum smears and thin sections have suggested that P. aeruginosa persists as extracellular bacterial aggregates, or biofilms, during chronic CF infections. However, emerging evidence indicates that host cells may also provide a protective reservoir for P. aeruginosa during infection. While phagocytes that flood the CF airways normally kill infecting bacteria, CFTR-deficient murine macrophages fail to kill P. aeruginosa, raising the possibility that CF macrophages could house viable bacteria. Epithelial cells may also provide an intracellular home for P. aeruginosa, since P. aeruginosa can invade epithelial cells in tissue culture systems. Together, these results have led to our hypothesis that the intracellular environment is a protective reservoir for P. aeruginosa during chronic CF infections. We are using a state-of-the-art imaging technique termed MiPACT to study how bacteria are spatially organized and functioning in CF sputum in relation to host cells. MiPACT was previously developed for the characterization of growth rates and spatial organization of microbes in millimeter thick three-dimensional CF sputum specimens. A major advantage to MiPACT is that the sputum specimens are embedded in a hydrogel and passively cleared with a detergent, which renders the sputum optically transparent and maintains the original biogeography of the specimens. This represents a substantial advance over traditional techniques like smears and thin sections, which were essentially limited to analyses in two dimensions. In this study, we have further advanced MiPACT in two ways to study how P. aeruginosa interacts with host cells. First, we can now detect bacterial gene expression in situ with fluorescent nucleic acid probes, which will allow us to determine whether specific genes are expressed when bacteria associate with host cells. Second, we have combined MiPACT with immunofluorescence to label bacteria and host cell proteins with fluorescent antibodies. In these preliminary studies, we have developed probes to detect P. aeruginosa alginate gene expression in vitro and successfully used immunofluorescence to label neutrophils, macrophages, and P. aeruginosa in CF sputum in situ. The immunofluorescence revealed that P. aeruginosa can be detected as extracellular and intracellular aggregates in sputum. Ongoing work is being performed to determine which host cells are housing intracellular P. aeruginosa and whether intracellular bacteria are viable or in the process of being killed. These new methods will shed important light on how P. aeruginosa persists in the CF airways.


Item Type:Conference or Workshop Item (UNSPECIFIED)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1002/ppul.23840DOIArticle
http://onlinelibrary.wiley.com/doi/10.1002/ppul.23840/fullPublisherArticle
ORCID:
AuthorORCID
Newman, D. K.0000-0003-1647-1918
Additional Information:© 2017 Wiley Periodicals, Inc. Issue online: 19 September 2017; Version of record online: 19 September 2017.
Issue or Number:S47
Record Number:CaltechAUTHORS:20171012-103857991
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20171012-103857991
Official Citation:(2017), Poster Session Abstracts. Pediatr Pulmonol., 52: S214–S516. doi:10.1002/ppul.23840
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:82310
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:12 Oct 2017 22:18
Last Modified:03 Oct 2019 18:53

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