Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP

Bacteria colonize
highly diverse and complex environments, from
gastrointestinal tracts to soil and plant surfaces. This colonization
process is controlled in part by the intracellular signal cyclic di-GMP,
which regulates bacterial motility and biofilm formation. To interrogate
cyclic di-GMP signaling networks, a variety of fluorescent biosensors
for live cell imaging of cyclic di-GMP have been developed. However,
the need for external illumination precludes the use of these tools
for imaging bacteria in their natural environments, including in deep
tissues of whole organisms and in samples that are highly autofluorescent
or photosensitive. The need for genetic encoding also complicates
the analysis of clinical isolates and environmental samples. Toward
expanding the study of bacterial signaling to these systems, we have
developed the first chemiluminescent biosensors for cyclic di-GMP.
The biosensor design combines the complementation of split luciferase
(CSL) and bioluminescence resonance energy transfer (BRET) approaches.
Furthermore, we developed a lysate-based assay for biosensor activity
that enabled reliable high-throughput screening of a phylogenetic
library of 92 biosensor variants. The screen identified biosensors
with very large signal changes (∼40- and 90-fold) as well as
biosensors with high affinities for cyclic di-GMP (KD < 50 nM). These chemiluminescent biosensors then
were applied to measure cyclic di-GMP levels in E. coli. The cellular experiments revealed an unexpected challenge for chemiluminescent
imaging in Gram negative bacteria but showed promising application
in lysates. Taken together, this work establishes the first chemiluminescent
biosensors for studying cyclic di-GMP signaling and provides a foundation
for using these biosensors in more complex systems.