Development of a 3’3’‐Cyclic GMP‐AMP Enzyme Linked Immunoassay Reveals Phage Infection Reduces DncV Activity

Abstract

AbstractEast Lansing, MI 48824 Cyclic di‐nucleotides (CDNs) are central signaling molecules in organisms spanning the tree of life. In bacteria, CDNs mediate many important physiological functions such as biofilm formation, motility, and virulence. CDNs are also implicated in activation of cellular biological defense systems in both bacteria and eukaryotes. In bacteria, the CDN 3’3’‐cyclic GMP‐AMP (3’3’‐cGAMP) activates a putative phage defense system in Vibrio cholerae and controls central physiological processes in Geobacter sulfurreducens and Bdellovibrio bacteriovorus. Therefore, access to a rapid, accurate 3’3’‐cGAMP quantification assay would enable further studies of this signaling molecule. Here, we describe validation of a novel 3’3’‐cGAMP enzyme linked immunoassay (ELISA) developed by Cayman Chemicals. We demonstrate that the concentrations of 3’3’‐cGAMP determined by this ELISA strongly correlate with those obtained using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Furthermore, during these studies we show that the V. cholerae 3’3’‐cGAMP synthase, DncV, when expressed by itself in Escherichia coli, is not activated by phage infection.