Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems

Abstract

Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS) are a family of defence systems against bacteriophages (hereafter phages) that share ancestry with the cGAS-STING innate immune pathway in animals. CBASS systems are composed of an oligonucleotide cyclase, which generates signalling cyclic oligonucleotides in response to phage infection, and an effector that is activated by the cyclic oligonucleotides and promotes cell death. Cell death occurs before phage replication is completed, therefore preventing the spread of phages to nearby cells. Here, we analysed 38,000 bacterial and archaeal genomes and identified more than 5,000 CBASS systems, which have diverse architectures with multiple signalling molecules, effectors and ancillary genes. We propose a classification system for CBASS that groups systems according to their operon organization, signalling molecules and effector function. Four major CBASS types were identified, sharing at least six effector subtypes that promote cell death by membrane impairment, DNA degradation or other means. We observed evidence of extensive gain and loss of CBASS systems, as well as shuffling of effector genes between systems. We expect that our classification and nomenclature scheme will guide future research in the developing CBASS field.

Extended Data Figure 1. Phylogenetic analysis of oligonucleotide cyclases (CD-NTase) and their CBASS types.

The phylogenetic tree of all cyclases, as depicted and colored in refs (5 and 8) is presented in the center. Each clade is then expanded and presented in the periphery as a circular tree to increase resolution. Outer ring depicts the effector type; middle ring depicts the system type. Numbers next to each clade represent the bootstrap value for that node in the central tree.

Fig. 1. General description of CBASS systems.

a | A general model for the mode of action of CBASS systems. Phage infection is sensed by the cyclase (or by the ancillary genes, together with the cyclase), leading to activation of the cyclase which generates a cyclic oligonucleotide signaling molecule. The signaling molecule is sensed by the effector and activates its cell killing function. CBASS cyclases can generate a variety of cyclic di- and tri-nucleotides, and CBASS effectors can exert cell death through membrane degradation, cleavage of phage and host DNA, formation of membrane-spanning pores, and other means whose mechanisms are yet to be identified. b | Four types of CBASS systems classified according to their ancillary gene content. Box arrows with dashed outlines represent genes that are present in some, but not all, of the systems in the respective CBASS type. Ancillary genes are denotes as Cap genes (CD-NTase-Associated Proteins). c | Representative instances of CBASS operons and their genomic neighborhood. The name of the bacterial species and the accession of the relevant genomic scaffold in the IMG database are indicated on the left.

Fig. 2. Phylogenetic distribution of CBASS types and effectors.

a | Presence of CBASS systems in analyzed genomes, divided by phyla. Data for phyla with >100 genomes in the database are shown. Numbers above each bar represent the number of genomes from the specific phylum that contain CBASS out of the total number of genomes from the specific phylum that are present in the analyzed database. b | Presence of CBASS systems in species, divided by phyla. CBASS was counted as present in a species if at least one genome of that species contained CBASS. Numbers above each bar represent the number of species from the specific phylum that contain CBASS out of the total number of species from the specific phylum that are present in the analyzed database. c | Phyletic distribution of genomes per CBASS type. Data are shown for phyla with >200 genomes in the database. Rightmost bar depicts the phyletic distribution of all 38,167 genomes in the database for comparison purposes. d | Distribution of effector genes in each CBASS type.

Fig. 3. Rapid gain and loss of and gene shuffling in CBASS systems.

a | Presence of different CBASS systems in closely related genomes. Each row represents a different strain of either Escherichia coli (left) or Pseudomonas aeruginosa (right), each column corresponds to a different CBASS type (colored boxes indicate the presence of the CBASS system; grey boxes depict absence of CBASS). Letter within the box represents the effector type (A, phospholipase; B, transmembrane domains; C, endonuclease; E, domain of unknown function DUF4297; F, phosphorylase/nucleosidase). P. aeruginosa strain ST-111 refers to ST-111 38_London_12_VIM_2_08_12. b | Phylogenetic tree of 3 highly similar cyclases (80-85% protein sequence identity) that belong to a type III CBASS. The % identity between each protein and its cognate protein in the neighboring leaf of the tree is depicted.