Exploring the diversity of anti-defense systems across prokaryotes, phages and mobile genetic elements.

The co-evolution of prokaryotes, phages and mobile genetic elements (MGEs) has driven the diversification of defense and anti-defense systems alike. Anti-defense proteins have diverse functional domains, sequences and are typically small, creating a challenge to detect anti-defense homologs across prokaryotic and phage genomes. To date, no tools comprehensively annotate anti-defense proteins within a desired sequence.

Single phage proteins sequester signals from TIR and cGAS-like enzymes.

Prokaryotic anti-phage immune systems use TIR and cGAS-like enzymes to produce 1''-3'-glycocyclic ADP-ribose (1''-3'-gcADPR) and cyclic dinucleotide (CDN) and cyclic trinucleotide (CTN) signalling molecules, respectively, which limit phage replication. However, how phages neutralize these distinct and common systems is largely unclear. Here we show that the Thoeris anti-defence proteins Tad1 and Tad2 both achieve anti-cyclic-oligonucleotide-based anti-phage signalling system (anti-CBASS) activity by simultaneously sequestering CBASS cyclic oligonucleotides.

AcrIF11 is a potent CRISPR-specific ADP-ribosyltransferase encoded by phage and plasmid.

Phage-encoded anti-CRISPR (Acr) proteins inhibit CRISPR-Cas systems to allow phage replication and lysogeny maintenance. Most of the Acrs characterized to date are stable stoichiometric inhibitors, and while enzymatic Acrs have been characterized biochemically, little is known about their potency, specificity, and reversibility. Here, we examine AcrIF11, a widespread phage and plasmid-encoded ADP-ribosyltransferase (ART) that inhibits the Type I-F CRISPR-Cas system.

Diverse viral cas genes antagonize CRISPR immunity.

Prokaryotic CRISPR-Cas immunity is subverted by anti-CRISPRs (Acrs), which inhibit Cas protein activities when expressed during the phage lytic cycle or from resident prophages or plasmids. Acrs often bind to specific cognate Cas proteins, and hence inhibition is typically limited to a single CRISPR-Cas subtype. Furthermore, although acr genes are frequently organized together in phage-associated gene clusters, how such inhibitors initially evolve has remained unclear.

Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides.

Cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is a common immune system that uses cyclic oligonucleotide signals to limit phage replication. In turn, phages encode anti-CBASS (Acb) proteins such as Acb2, which can sequester some cyclic dinucleotides (CDNs) and limit downstream effector activation. Here, we identified that Acb2 sequesters many CDNs produced by CBASS systems and inhibits stimulator of interferon genes (STING) activity in human cells.

Cas9 degradation in human cells using phage anti-CRISPR proteins.

Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune systems, allowing successful invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas systems using a wide variety of mechanisms. AcrIIA1 is encoded by numerous phages and plasmids, binds specifically to the Cas9 HNH domain, and was the first Acr discovered to inhibit SpyCas9.

Identification of an anti-CRISPR protein that inhibits the CRISPR-Cas type I-B system in .

is the widespread anaerobic spore-forming bacterium that is a major cause of potentially lethal nosocomial infections associated with antibiotic therapy worldwide. Due to the increase in severe forms associated with a strong inflammatory response and higher recurrence rates, a current imperative is to develop synergistic and alternative treatments for infections. In particular, phage therapy is regarded as a potential substitute for existing antimicrobial treatments.

F-Type Pyocins Are Diverse Noncontractile Phage Tail-Like Weapons for Killing .

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition.

Core defense hotspots within Pseudomonas aeruginosa are a consistent and rich source of anti-phage defense systems.

Bacteria use a diverse arsenal of anti-phage immune systems, including CRISPR-Cas and restriction enzymes. Recent advances in anti-phage system discovery and annotation tools have unearthed many unique systems, often encoded in horizontally transferred defense islands, which can be horizontally transferred. Here, we developed Hidden Markov Models (HMMs) for defense systems and queried microbial genomes on the NCBI database.

The ϕPA3 phage nucleus is enclosed by a self-assembling 2D crystalline lattice.

To protect themselves from host attack, numerous jumbo bacteriophages establish a phage nucleus-a micron-scale, proteinaceous structure encompassing the replicating phage DNA. Bacteriophage and host proteins associated with replication and transcription are concentrated inside the phage nucleus while other phage and host proteins are excluded, including CRISPR-Cas and restriction endonuclease host defense systems. Here, we show that nucleus fragments isolated from ϕPA3 infected Pseudomonas aeruginosa form a 2-dimensional lattice, having p2 or p4 symmetry.

Bacteriophages inhibit and evade cGAS-like immune function in bacteria.

A fundamental strategy of eukaryotic antiviral immunity involves the cGAS enzyme, which synthesizes 2',3'-cGAMP and activates the effector STING. Diverse bacteria contain cGAS-like enzymes that produce cyclic oligonucleotides and induce anti-phage activity, known as CBASS. However, this activity has only been demonstrated through heterologous expression.

Bacteriophage genome engineering with CRISPR-Cas13a.

Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous 'phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker.

Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor.

Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR-Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR-Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA.

CRISPR-Cas12a targeting of ssDNA plays no detectable role in immunity.

CRISPR-Cas12a (Cpf1) is a bacterial RNA-guided nuclease that cuts double-stranded DNA (dsDNA) at sites specified by a CRISPR RNA (crRNA) guide. Additional activities have been ascribed to this enzyme in vitro: site-specific (cis) single-stranded DNA (ssDNA) cleavage and indiscriminate (trans) degradation of ssDNA, RNA, and dsDNA after activation by a complementary target. The ability of Cas12a to cleave nucleic acids indiscriminately has been harnessed for many applications, including diagnostics, but it remains unknown if it contributes to bacterial immunity.

Lack of Cas13a inhibition by anti-CRISPR proteins from Leptotrichia prophages.

CRISPR systems are prokaryotic adaptive immune systems that use RNA-guided Cas nucleases to recognize and destroy foreign genetic elements. To overcome CRISPR immunity, bacteriophages have evolved diverse families of anti-CRISPR proteins (Acrs). Recently, Lin et al.

A single bacterial enzyme i(NHI)bits phage DNA replication.

In this issue of Cell Host & Microbe, Nayeemul Bari et al. discover an anti-phage immune system in bacteria that uses a single enzyme to accomplish the challenging feat of detecting phage DNA and limiting its replication. Unlike CRISPR-Cas and restriction modification (R-M) systems, which use sequence motifs, nuclease-helicase immunity (Nhi) is proposed to target phage-specific replication intermediates.