Fluorescent Detection of the Ubiquitous Bacterial Messenger 3',5' Cyclic Diguanylic Acid by Using a Small Aromatic Molecule.

Teng-Fei Xuan,Zi-Qiang Wang,Jing Lin,Jun Liu,Wei-Min Chen

doi:10.3389/fmicb.2019.03163

Teng-Fei Xuan, Zi-Qiang Wang + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2019.03163

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Journal: Frontiers in microbiology	Publication Date: Jan 14, 2020
Citations: 6	License type: CC BY 4.0

Affiliation: Jinan University

Abstract

3′,5′ Cyclic diguanylic acid (c-di-GMP) has been shown to play a central role in the regulation of bacterial physiological processes such as biofilm formation and virulence production, and is regarded as a potential target for the development of anti-infective drugs. A method for the facile detection of the bacterial level of cellular c-di-GMP is required to explore the details of c-di-GMP signaling and design drugs on the basis of this pathway. Current methods of c-di-GMP detection have limited sensitivity or difficultly in probe preparation. Herein a new fluorescent probe is reported for the detection of c-di-GMP at concentrations as low as 500 nM. The probe was developed on the basis of the G-quadruplex formation of c-di-GMP induced by aromatic molecules. When used on crude bacterial cell lysates, it can effectively distinguish between the low c-di-GMP levels of bacteria in plankton and the high c-di-GMP levels in biofilm. The method described here is simple, inexpensive, sensitive, and suitable for practical applications involving the rapid detection of cellular c-di-GMP levels in vitro after simple bacterial lysis and filtration.

Highlights

First discovered in 1987 (Ross et al, 1987), 3′,5′-cyclic diguanylic acid (c-di-GMP) is a ubiquitous second messenger that has been shown to play a central role in the regulation of bacterial biofilm formation (Tsiry et al, 2015)
Nakayama et al demonstrated that the fluorescence of the c-di-GMP G-quadruplex inducer thiazole orange (TO) is enhanced in a c-di-GMP/TO complex (Zhang et al, 2006; Nakayama et al, 2011a,b), which can be used to establish a simple method for c-di-GMP detection in vitro
Inducing c-di-GMP to form G-quadruplex in the presence of monovalent cations and enhancing the fluorescence of the aromatic inducer is a promising method for the detection of cellular c-di-GMP

Summary

INTRODUCTION

First discovered in 1987 (Ross et al, 1987), 3′,5′-cyclic diguanylic acid (c-di-GMP) is a ubiquitous second messenger that has been shown to play a central role in the regulation of bacterial biofilm formation (Tsiry et al, 2015). Nakayama et al demonstrated that the fluorescence of the c-di-GMP G-quadruplex inducer thiazole orange (TO) is enhanced in a c-di-GMP/TO complex (Zhang et al, 2006; Nakayama et al, 2011a,b), which can be used to establish a simple method for c-di-GMP detection in vitro. A small aromatic molecule, A18 ((E)-2-(2-(1H-indol-3-yl) vinyl) -3-methyl-3λ4-benzo [d] thiazole iodide), has been designed and synthesized It is useful for c-di-GMP detection because of its G-quadruplex formation property and offers effective c-di-GMP G-quadruplex induction (Nakayama et al, 2011a,b; Kelsey et al, 2012), enabling the detection limit for c-di-GMP to be reduced to roughly 500 nM. The improvement of sensitivity and selectivity makes the immediate detection and quantification of cellular c-di-GMP levels possible through simple lysis and filtration of bacterial samples, without the need for prior HPLC purification

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSIONS