PROJECT SUMMARY This is a proposed supplement to Dr. Aaron Whiteley’s NIH Director’s New Innovator Award (DP2AT012346) titled “Deciphering the crosstalk between bacteria and their mammalian hosts”. Bacteria must defend themselves against the constant threat of bacteriophages (phages) and mobile genetic elements that seek to parasitize bacterial hosts for their selfish replication and spread. The conflict between bacteria and their threats has driven the evolution of genes that cooperate to form sophisticated signaling pathways to protect bacteria; these pathways collectively form the bacterial immune system. Interestingly, a subset of proteins found in the bacterial immune system are homologous to proteins in the human immune system, which provides a basis for cross-kingdom signaling, from bacteria to humans. The focus of this supplement is on understanding the bacterial immune system to improve our understanding and design of probiotic bacteria that might better survive in the human gut or influence human immune signaling pathways. The proposed goals for this supplement are to investigate the prevalence and function of known bacterial immune pathways in the microbiome and to uncover novel immune pathways that restrict horizontal gene transfer. In the first Aim, we will investigate the antiphage system CBASS (Cyclic Oligonucleotide Based Antiphage Signaling Systems) in the context of the human microbiome. CBASS is a potent antiphage system that is evolutionarily related to the human cGAS-STING antiviral pathway. A defining feature of CBASS systems is a CD-NTase (cGAS/DncV-like nucleotidyltransferase) protein, which is homologous to cGAS. Both cGAS and CD-NTases synthesize cyclic nucleotides that elicit potent immune signaling. In the parent grant, we investigate how cyclic nucleotides produced by bacteria in the gut may impact mammalian host immune signaling. This aim synergizes with that work by investigating the abundance of CBASS systems in metagenomes derived from healthy and disease-related mammalian microbiomes and interrogating their function in heterologous systems. In the second Aim, we will discover novel aspects of the bacterial immune system that restrict horizontal gene transfer by screening diverse bacteria using a functional assay. We will then assess the role of these systems in bacteria from the mammalian microbiome, including estimating their prevalence and disease association in metagenomes. The supplemental aims proposed will illuminate cryptic aspects of antiphage systems, interdomain signaling, and how bacteria control mobile genetic elements. Further, findings from this work will contribute to the ultimate development of novel pre- and probiotic complementary therapies that can durably remain within the human gut microbiome and even promote appropriate immune responses.