Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels
| Autor: | Blacutt, Jacob, Lan, Ziyang, Cosgriff-Hernandez, Elizabeth M., Gordon, Vernita D. |
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| Rok vydání: | 2021 |
| Předmět: |
Cyclic di-GMP
cyclic-di-gmp pily1 confocal microscopy pseudomonas aeruginosa biofilm Green fluorescent protein law.invention 03 medical and health sciences chemistry.chemical_compound Plasmid Confocal microscopy law poly(ethylene glycol) diacrylate lcsh:Science 030304 developmental biology Physics and Biophysics 0303 health sciences Multidisciplinary biology 030306 microbiology Chemistry Biofilm biology.organism_classification Second messenger system Self-healing hydrogels Biophysics lcsh:Q Bacteria Research Article |
| Zdroj: | Royal Society Open Science, Vol 8, Iss 1 (2021) Royal Society Open Science |
| ISSN: | 2054-5703 |
| Popis: | The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure. Biofilm development initiates when bacteria attach to and sense a surface. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, the transition to the biofilm phenotype is controlled by the intracellular signal and second messenger cyclic-di-GMP (c-di-GMP). It is not known how biomedical materials might be adjusted to impede c-di-GMP signalling, and there are few extant methods for conducting such studies. Here, we develop such a method. We allowed P. aeruginosa to attach to the surfaces of poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These bacteria contained a plasmid for a green fluorescent protein (GFP) reporter for c-di-GMP. We used laser-scanning confocal microscopy to measure the dynamics of the GFP reporter for 3 h, beginning 1 h after introducing bacteria to the hydrogel. We controlled for the effects of changes in bacterial metabolism using a promoterless plasmid for GFP, and for the effects of light passing through different hydrogels being differently attenuated by using fluorescent plastic beads as ‘standard candles’ for calibration. We demonstrate that this method can measure statistically significant differences in c-di-GMP signalling associated with different PEGDA gel types and with the surface-exposed protein PilY1. |
| Databáze: | OpenAIRE |
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