Project Summary
Bacteriophages (phages) are viruses that infect and kill bacteria. These viral predators have provided the
selective pressure to drive the evolution of numerous anti-phage immune pathways such as restriction-
modification and CRISPR-Cas systems. A recent surge in the discovery of new anti-phage immune systems has
uncovered an incredible diversity of defensive mechanisms across prokaryotes. Strikingly, many of these
systems appear to be homologous to mammalian anti-viral immune pathways, suggesting that some human
innate immune responses may have originated as anti-phage systems. The immune system of interest in this
proposal first arose in bacteria and is now widely studied in mammals as cGAS-STING. In mammals, the cGAS
enzyme directly binds to cytoplasmic double-stranded DNA (dsDNA) and triggers the production of cyclic GMP-
AMP (cGAMP) molecules that bind to the protein STING to ultimately stimulate interferon genes. cGAS is
normally held in the off state through a wide array of inhibitory post-translational modifications, direct protein
interactions, nucleosome tethering, and phase separation. However, the regulatory mechanisms that inhibit
or activate the homologous system in bacteria, called CBASS, remain unknown. CBASS (cyclic
oligonucleotide-based anti-phage signaling systems) systems are currently thought to drive an abortive infection
outcome, in which the production of one of many potential cyclic oligonucleotides (c-oligos) activates a co-
encoded toxic effector protein and induces cell death. Given this cell death outcome of activated CBASS, we
hypothesize that tight regulatory mechanisms must keep it off, and these mechanisms must be rapidly reversed
during phage infection to turn CBASS on. Biochemical assays have shown that the cGAS-like enzymes in
bacteria, called CD-NTases, constitutively produce c-oligos in vitro and structural work shows that CD-NTases
are in an activated state with the catalytic site permanently competent for substrate nucleotide binding.
Paradoxically, the overexpression of the CD-NTase and effector in bacteria is not toxic in the absence of phage,
confirming that the cell has at least one mechanism to repress or inhibit function. For our studies, we will use the
first described native model system for CBASS anti-phage function, established in Pseudomonas aeruginosa, in
our group. We will first conduct unbiased and targeted genetic screens to identify endogenous CBASS inhibitors
or repressors that allow maintenance and prevent self-toxicity by CBASS. In conjunction, we will identify the
phage component(s) that triggers CBASS by isolating phages that acquire mutations that enable CBASS escape.
Genetics and biochemical experiments will be used to validate the trigger. Surprisingly, preliminary experiments
with this approach revealed the first phage encoded anti-CBASS protein, which will be mechanistically
characterized during this study. Together, these experiments will provide a mechanistic understanding for how
CBASS immune systems interface with, and inhibit, phage replication.
