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Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels.

ABSTRACT: 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.

SUBMITTER: Blacutt J

PROVIDER: S-EPMC7890475 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels.

Blacutt Jacob J Lan Ziyang Z Cosgriff-Hernandez Elizabeth M EM Gordon Vernita D VD

Royal Society open science 20210106 1

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 <i>Pseudomonas aeruginosa</i> 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 fe ...[more]

PMID: 33614081

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Project description:The synthesis of signaling molecules is one strategy bacteria employ to sense alterations in their environment and rapidly adjust to those changes. In Gram-negative bacteria, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) regulates the transition from a unicellular motile state to a multicellular sessile state. However, c-di-GMP signaling has been less intensively studied in Gram-positive organisms. To that end, we constructed a fluorescent yfp reporter based on a c-di-GMP-responsive riboswitch to visualize the relative abundance of c-di-GMP for single cells of the Gram-positive model organism Bacillus subtilis Coupled with cell-type-specific fluorescent reporters, this riboswitch reporter revealed that c-di-GMP levels are markedly different among B. subtilis cellular subpopulations. For example, cells that have made the decision to become matrix producers maintain higher intracellular c-di-GMP concentrations than motile cells. Similarly, we find that c-di-GMP levels differ between sporulating and competent cell types. These results suggest that biochemical measurements of c-di-GMP abundance are likely to be inaccurate for a bulk ensemble of B. subtilis cells, as such measurements will average c-di-GMP levels across the population. Moreover, the significant variation in c-di-GMP levels between cell types hints that c-di-GMP might play an important role during B. subtilis biofilm formation. This study therefore emphasizes the importance of using single-cell approaches for analyzing metabolic trends within ensemble bacterial populations.IMPORTANCE Many bacteria have been shown to differentiate into genetically identical yet morphologically distinct cell types. Such population heterogeneity is especially prevalent among biofilms, where multicellular communities are primed for unexpected environmental conditions and can efficiently distribute metabolic responsibilities. Bacillus subtilis is a model system for studying population heterogeneity; however, a role for c-di-GMP in these processes has not been thoroughly investigated. Herein, we introduce a fluorescent reporter, based on a c-di-GMP-responsive riboswitch, to visualize the relative abundance of c-di-GMP for single B. subtilis cells. Our analysis shows that c-di-GMP levels are conspicuously different among B. subtilis cellular subtypes, suggesting a role for c-di-GMP during biofilm formation. These data highlight the utility of riboswitches as tools for imaging metabolic changes within individual bacterial cells. Analyses such as these offer new insight into c-di-GMP-regulated phenotypes, especially given that other biofilms also consist of multicellular communities.

Dataset Information

Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels.

Publications

Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels.

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