'Royal College of Obstetricians & Gynaecologists (RCOG)'
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