The role of a toxin-antitoxin system in the arms race between bacteria and phage

Abstract

All domains of life face the fundamental challenge of responding to infection by pathogens. In bacteria, the need to survive predation by phages, or bacterial viruses, has driven the evolution of numerous highly sophisticated anti-phage elements, including restriction modification and CRISPR-Cas systems. These defense mechanisms, in turn, have influenced the emergence of phage anti-defense systems, highlighting the intense, ongoing evolutionary arms race between phages and their bacterial hosts. Toxin-antitoxin (TA) systems are widespread phage defense systems in bacteria comprising a bacteriostatic or bactericidal toxin and an antitoxin that normally keeps the toxin inactive. Following phage infection, the toxin is activated, or liberated from its cognate antitoxin, to block viral recognition. However, the precise activating signals of phage-defensive TA systems, the targets of toxins following activation, and the mechanisms that phages can use to overcome TA-mediated defense are largely unknown.

In this work, I characterize the role of an E. coli toxin-antitoxin system called toxIN, containing the endoribonuclease toxin ToxN and the short, repetitive RNA antitoxin toxI, in the arms race between phages and bacteria. Using RNA sequencing to track ToxN activity during T4 infection, I show that, following phage infection, ToxN is liberated from toxI and cleaves T4 mRNAs, thereby disrupting T4 translation and the assembly of new viral particles. I then demonstrate that host transcription shutoff is the activating trigger for toxIN. Because T4 robustly inhibits host transcription, cells cannot replenish the antitoxin toxI, which is rapidly degraded. Next, by isolating toxIN-resistant clones of T4, I demonstrate that T4 can overcome ToxN by increasing the expression of a small phage protein encoded by the gene tifA. TifA is a bona fide antitoxin for ToxN that is conserved in the genomes of many T4-like phages, including the coliphages T2, T6, and RB69. Interestingly, TifA is an RNA-binding protein that forms ribonucleoprotein complexes with ToxN, suggesting that TifA may sequester RNA-bound ToxN to prevent cleavage of phage transcripts during infection. Taken together, these results reveal the native targets and activation mechanism of an inducible, phage-defensive TA system and uncover a unique mechanism by which phages can overcome TA-mediated defense.