Systematic discovery of antiphage defense systems in the microbial pangenome

Maps of defense arsenals in microbial genomes To survive the attack of foreign invaders such as viruses and plasmids, bacteria and archaea fight back with immune systems that are usually clustered in “defense islands” in their genomes. Doron et al. took advantage of this property to map microbial defense systems systematically (see the Perspective by Kim). Candidate immune systems were then experimentally validated for their activities. Like well-known defense arsenals such as restriction-modification and CRISPR systems, these additional immune systems now require mechanistic investigation and could potentially be engineered into useful molecular tools in the future. Science, this issue p. eaar4120; see also p. 993 Bioinformatics and experimental validation identify nine antiphage and one antiplasmid immune defense systems in microbes. INTRODUCTION Bacteria and archaea are frequently attacked by viruses (phages) and as a result have developed multiple, sophisticated lines of active defense that can collectively be referred to as the prokaryotic “immune system. ” Although bacterial defense against phages has been studied for decades, it was suggested that many currently unknown defense systems reside in the genomes of nonmodel bacteria and archaea and await discovery. RATIONALE Antiphage defense systems are known to be frequently physically clustered in microbial genomes such that, for example, genes encoding restriction enzymes commonly reside in the vicinity of genes encoding other phage resistance systems. The observation that defense systems are clustered in genomic “defense islands” has led to the hypothesis that genes of unknown function residing within such defense islands may also participate in antiphage defense. In this study, we aimed to comprehensively identify and experimentally verify new defense systems based on their enrichment within defense islands in an attempt to systematically map the arsenal of defense tools that are at the disposal of microbes in their fight against phages. RESULTS We searched for gene cassettes of unknown function that are enriched near known defense systems in more than 45,000 available bacterial and archaeal genome sequences. Such gene cassettes were defined as candidate defense systems and were systematically engineered into model bacteria, which were then infected by an array of phages to test for antiphage activities. This yielded the discovery of nine new families of antiphage defense systems and one additional family of antiplasmid systems that are widespread in microbes and shown to strongly protect against foreign DNA invasion. The systems discovered include ones that seem to have adopted components of the bacterial flagella and chromosome maintenance complexes and use these components for defensive capacities. Our data also show that genes with Toll-interleukin receptor (TIR) domains are involved in bacterial defense against phages, providing evidence for a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria. CONCLUSIONS Our study expands the known arsenal of defense systems used by prokaryotes for protection against phages, exposing tens of thousands of instances of defense systems that were so far unknown. Some of these systems appear to employ completely new mechanisms of defense against phages. In the past, the discovery and mechanistic understanding of antiphage defense systems led to the development of important biotechnological tools, as exemplified by the use of restriction enzymes and CRISPR-Cas for biotechnological and biomedical applications. One may envision that some of the systems discovered in the current study, once their mechanism is deciphered, will also be adapted into useful molecular tools in the future. A pipeline for systematic discovery of defense systems. Microbial genomes (more than 45,000 in the current study) are mined for genetic systems that are physically enriched next to known defense systems such as restriction-modification and CRISPR-Cas. Candidate predicted systems are cloned into model bacteria, and these bacteria are then infected by an array of phages from various families to determine whether they provide defense. The arms race between bacteria and phages led to the development of sophisticated antiphage defense systems, including CRISPR-Cas and restriction-modification systems. Evidence suggests that known and unknown defense systems are located in “defense islands” in microbial genomes. Here, we comprehensively characterized the bacterial defensive arsenal by examining gene families that are clustered next to known defense genes in prokaryotic genomes. Candidate defense systems were systematically engineered and validated in model bacteria for their antiphage activities. We report nine previously unknown antiphage systems and one antiplasmid system that are widespread in microbes and strongly protect against foreign invaders. These include systems that adopted components of the bacterial flagella and condensin complexes. Our data also suggest a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria.