A Luminescence-Based Coupled Enzyme Assay Enables High-Throughput Quantification of the Bacterial Second Messenger 3'3'-Cyclic-Di-AMP

Cyclic dinucleotide signaling systems, which are found ubiquitously throughout nature, allow organisms to rapidly and dynamically sense and respond to alterations in their environments. In recent years, the second messenger, cyclic di-(3',5')-adenosine monophosphate (c-di-AMP), has been identified as an essential signaling molecule in a diverse array of bacterial genera. We and others have shown that defects in c-di-AMP homeostasis result in severe physiological defects and virulence attenuation in many bacterial species. Despite significant advancements in the field, there is still a major gap in the understanding of the environmental and cellular factors that influence c-di-AMP dynamics due to a lack of tools to sensitively and rapidly monitor changes in c-di-AMP levels. To address this limitation, we describe here the development of a luciferase-based coupled enzyme assay that leverages the cyclic nucleotide phosphodiesterase, CnpB, for the sensitive and high-throughput quantification of 3'3'-c-di-AMP. We also demonstrate the utility of this approach for the quantification of the cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector, 3'3'-cGAMP. These findings establish CDA-Luc as a more affordable and sensitive alternative to conventional c-di-AMP detection tools with broad utility for the study of bacterial cyclic dinucleotide physiology.

Automated summary

Recent research has identified c-di-AMP as an essential signaling molecule in various bacterial genera, with defects leading to severe physiological defects and virulence attenuation. A new method utilizing CnpB for high-throughput and sensitive quantification of c-di-AMP has been developed, providing a more affordable and sensitive alternative for studying bacterial cyclic dinucleotide physiology.