[F32] Revealing the scope of antiviral ADP-ribosylation through the phage defensesystem DarHTG3

Description

Project summary/Abstract Prokaryotes encode a highly diverse set of innate immune systems to protect themselves from bacteriophage infection. These antiviral genes are valuable research targets, as the competition between bacteria and phage drives the rapid, modular evolution of defenses and counter-defenses. Delving into this naturally-selected pool of antiviral systems has revealed ancient origins of mammalian immune defenses, and bacterial immune systems have been repurposed for molecular biology and gene editing. However, this resource remains largely untapped. One understudied link between human and bacterial antiviral defense is a modification called adenosine diphosphate (ADP)-ribosylation. ADP-ribosyltransferases (ARTs) reversibly conjugate ADP-ribose (ADPr) to substrates such as proteins and nucleic acids. Mammalian ARTs target viral proteins and sites of DNA damage. Many bacteria carry two-gene operons termed darTG which consist of a DNA ART toxin which targets DNA, and a cognate DNAADPr-glycohydrolase (ARG) antitoxin that reverses this activity. Dr. LeRoux discovered that these elements are a phage defense system. The goal of this proposal is to leverage a newly discovered, distinct branch of DarTG systems to illuminate the modular evolution of ADP-ribose-based antiviral defenses, potentially revealing conserved principles of immunity across domains of life. DarHTG3 consists of three components: a predicted helicase (DarH3), an ART toxin (DarT3) and an ARG antitoxin (DarG3). DarT3 resembles DarT1, but DarH3 and DarG3 are not conserved in other DarTG systems, and their functions had not been investigated. Preliminary experiments revealed that DarT3 ADP-ribosylates an unknown base of DNA, potentiated by DarH3. DarG3 rescues bacterial cells from DarHT3 toxicity, and shares a catalytic domain with the human enzymes ARH1-3, but these human proteins do not act on DNA. Structural predictions of DarG3 reveal a distinct C-terminal region which may mediate DNA binding. These data inform the hypothesis that the C-terminal domain of DarG3 allows for DNA binding and ADPr removal, until phage infection triggers the formation of the DarHT3 complex, which nonspecifically and lethally ADP-ribosylates phage and host DNA to block phage spread. Aim 1 will identify the target of DarT3, and map phage-induced ADP-ribosylation in vivo to clarify how DarHTG3 restricts infection. Aim 2 will test whether DarH3 is a bona fide helicase which recruits DarT3 to DNA, and define the protein-protein interactions which regulate this activity. Aim 3 will determine the role of the C-terminus of DarG3 in the reversal of DNA ADP-ribosylation. Insight into the molecular details of this system will reveal how modular evolution diversifies antiviral defenses. This work will be carried out under Dr. LeRoux, a highly supportive mentor experienced in toxin-antitoxin phage defenses. It will be co-sponsored by Dr. Feldman, an expert in bacterial physiology. A panel of secondary men Istituzione: WASHINGTON UNIVERSITY PI: Trevor Griesman Progetto: 1F32AI202943-01

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Source: reporter.nih.gov