Defining the role of cyclic trinucleotide signaling in bacteriophage immunity through the activation and regulation of HORMA-associated cGAS/DncV-related nucleotidyltransferases In their natural environment, bacterial cells are in a constant conflict with the surrounding cells and bacteriophages. Therefore, they have developed a variety of signaling pathways which are responsible for detecting and adapting to changes both their internal and external environment. These signals can be propagated through the synthesis of small molecules, also called second messengers, which amplify the signal and can bind to downstream effectors. It has recently been discovered that one class of enzymes, cGAS/DncV- related nucleotidyltransferases, are responsible for synthesizing a wide variety of cyclic di- and tri-nucleotides, which have been shown to act as second messengers. In bacteria, dinucleotides regulate a plethora of behaviors ranging from biofilm formation, to intracellular ion concentration, to defense against bacteriophage infection. With the rise in bacterial resistance to small molecule drugs and increased interest in alternative approaches, including phage therapy, it is imperative that we understand the responses to bacteriophage infection. Furthermore, there is growing evidence that cyclic dinucleotides are sensed by hosts upon infection, leading to high interest in bacterial cyclic oligonucleotide signaling. In this study, we propose to study the molecular mechanisms and biological roles of a novel bacteriophage defense pathway recently discovered in a diverse set of environmental and pathogenic bacterial strains. This pathway, whose components are contained in an operon, has an enzyme, CdnC, which synthesizes cyclic tri-AMP (cAAA) and co-exists with the first-identified bacterial proteins containing the HORMA domain, a peptide-binding domain found in many critical signaling pathways in eukaryotes. The operons also encode an ortholog of the AAA+ ATPase Pch2, an important regulator of eukaryotic HORMA domain proteins. Our extensive preliminary data shows that CdnC is inactive on its own, but synthesizes cAAA when bound to the HORMA in a closed conformation as well as DNA. cAAA in turn binds and activates a DNA endonuclease, NucC, also found in the operon. I have found that this pathway confers immunity to bacteriophage ?, and that immunity requires the CdnC, HORMA, and NucC proteins. In this project, I will identify the bacteriophage protein(s) recognized by HORMA to initiate signaling, and define the spectrum of bacteriophage immunity imparted by the operon. I will further define the activities of NucC and determine whether it specifically targets phage DNA, or rather degrades the bacterium?s own genome. Finally, I will determine how the CdnC operon is regulated transcriptionally, through the activity of a DNA binding protein/metallopeptidase pair with homology to transcriptional control proteins in Deinococcus. Overall, this work will determine the regulation of a novel trinucleotide signaling pathway in bacteria, and further the understanding of the mechanisms through which it provides bacteriophage immunity.
Here, I propose to investigate the mechanisms of a novel bacteriophage immunity pathway that likely senses foreign proteins, activates synthesis of a cyclic trinucleotide signaling molecule, and activates a nuclease to provide immunity against bacteriophage infection. The proposed project will identify the phage protein(s) sensed by this pathway?s HORMA domain proteins, determine mechanism of immunity and the spectrum of bacteriophages that the pathway senses, and reveal whether and how the pathway is feedback-regulated. Overall, the work will reveal the fundamental mechanisms of a new class of bacterial defense pathway that stands alongside restriction-modification and CRISPR/Cas systems, with implications for human therapeutic strategies and for the understanding of complex microbial communities.
