Anti-phage Defense Research Articles

Chemical inhibition of cell surface modification sensitizes bacteria to phage infection.

Many bacteriophages that infect Gram-positive bacteria rely on the bacterial cell surface polymer wall teichoic acid (WTA) as a receptor. However, some bacteria modulate their cell wall with d-alanine residues, which can disrupt phage adsorption. The prevalence and significance of WTA alanylation as an anti-phage defense is unknown. A chemical inhibitor of WTA d-alanylation could be employed to efficiently screen phage-host combinations for those that exhibit alanylation-dependent infections. Since the incorporation of d-alanine residues into the cell wall requires the activity of d-alanine:alanyl carrier protein ligase (DltA), a DltA inhibitor was employed as this tool. Herein, we found that a chemical probe inhibiting DltA activity impeded bacterial cell wall alanylation and enhanced infectivity of many phages against Bacillus subtilis, including phages Phi29, SPP1, SPO1, SP50, and Goe2. This finding reveals the breadth of immunity conferred by WTA alanylation in B. subtilis, which was previously known to impact only phages Phi29 and SPP1, but not SPO1, SP50, or Goe2. DltA inhibition selectively promoted infection by several phages that bind WTA, having no impact on the flagellotropic phage PBS1. Unexpectedly, DltA inhibition also had no effect on phage SP10, which binds to WTA. This selective chemical tool has the potential to unravel bacteriophage interactions with bacteria, leading to improved phage therapies in the future.

Structure and mechanism of Zorya anti-phage defense system

Structure and mechanism of Zorya anti-phage defense system

Assembly-mediated activation of the SIR2-HerA supramolecular complex for anti-phage defense

SIR2-HerA, a bacterial two-protein anti-phage defense system, induces bacterial death by depleting NAD+ upon phage infection. Biochemical reconstitution of SIR2, HerA, and the SIR2-HerA complex reveals a dynamic assembly process. Unlike other ATPases, HerA can form various oligomers, ranging from dimers to nonamers. When assembled with SIR2, HerA forms a hexamer and converts SIR2 from a nuclease to an NAD+ hydrolase, representing an unexpected regulatory mechanism mediated by protein assembly. Furthermore, high concentrations of ATP can inhibit NAD+ hydrolysis by the SIR2-HerA complex. Cryo-EM structures of the SIR2-HerA complex reveal a giant supramolecular assembly up to 1 MDa, with SIR2 as a dodecamer and HerA as a hexamer, crucial for anti-phage defense. Unexpectedly, the HerA hexamer resembles a spiral staircase and exhibits helicase activities toward dual-forked DNA. Together, we reveal the supramolecular assembly of SIR2-HerA as a unique mechanism for switching enzymatic activities and bolstering anti-phage defense strategies.

Multiple enzymatic activities of a Sir2-HerA system cooperate for anti-phage defense

In response to the persistent exposure to phage infection, bacteria have evolved diverse antiviral defense mechanisms. In this study, we report a bacterial two-component defense system consisting of a Sir2 NADase and a HerA helicase. Cryo-electron microscopy reveals that Sir2 and HerA assemble into a ∼1 MDa supramolecular octadecamer. Unexpectedly, this complex exhibits various enzymatic activities, including ATPase, NADase, helicase, and nuclease, which work together in a sophisticated manner to fulfill the antiphage function. Therefore, we name this defense system "Nezha" after a divine warrior in Chinese mythology who employs multiple weapons to defeat enemies. Our findings demonstrate that Nezha could sense phage infections, self-activate to arrest cell growth, eliminate phage genomes, and subsequently deactivate to allow for cell recovery. Collectively, Nezha represents a paradigm of sophisticated and multifaceted strategies bacteria use to defend against viral infections.

Structural and functional investigation of GajB protein in Gabija anti-phage defense.

Bacteriophages (phages) are viruses that infect bacteria and archaea. To fend off invading phages, the hosts have evolved a variety of anti-phage defense mechanisms. Gabija is one of the most abundant prokaryotic antiviral systems and consists of two proteins, GajA and GajB. GajA has been characterized experimentally as a sequence-specific DNA endonuclease. Although GajB was previously predicted to be a UvrD-like helicase, its function is unclear. Here, we report the results of structural and functional analyses of GajB. The crystal structure of GajB revealed a UvrD-like domain architecture, including two RecA-like core and two accessory subdomains. However, local structural elements that are important for the helicase function of UvrD are not conserved in GajB. In functional assays, GajB did not unwind or bind various types of DNA substrates. We demonstrated that GajB interacts with GajA to form a heterooctameric Gabija complex, but GajB did not exhibit helicase activity when bound to GajA. These results advance our understanding of the molecular mechanism underlying Gabija anti-phage defense and highlight the role of GajB as a component of a multi-subunit antiviral complex in bacteria.

Abortive infection antiphage defense systems: separating mechanism and phenotype.

Bacteria have evolved a wide array of mechanisms that allow them to eliminate phage infection. 'Abortive infection' (abi) systems are an expanding category of such mechanisms, defined as those which induce programmed cell death (or dormancy) upon infection, and thus halt phage propagation within a bacterial population. This definition entails two requirements - a phenotypic observation (cell death upon infection), and a mechanistic determination of its sources (system-induced death). The phenotypic and mechanistic aspects of abi are often implicitly assumed to be tightly linked, and studies regularly tend to establish one and deduce the other. However, recent evidence points to a complicated relationship between the mechanism of defense and the phenotype observed upon infection. We argue that rather than viewing the abi phenotype as an inherent quality of a set of defense systems, it should be more appropriately thought of as an attribute of interactions between specific phages and bacteria under given conditions. Consequently, we also point to potential pitfalls in the prevailing methods for ascertaining the abi phenotype. Overall, we propose an alternative framework for parsing interactions between attacking phages and defending bacteria.

OLD family nuclease function across diverse anti-phage defense systems.

Bacteriophages constitute a ubiquitous threat to bacteria, and bacteria have evolved numerous anti-phage defense systems to protect themselves. These systems include well-studied phenomena such as restriction endonucleases and CRISPR, while emerging studies have identified many new anti-phage defense systems whose mechanisms are unknown or poorly understood. Some of these systems involve overcoming lysogenization defect (OLD) nucleases, a family of proteins comprising an ABC ATPase domain linked to a Toprim nuclease domain. Despite being discovered over 50 years ago, OLD nuclease function remained mysterious until recent biochemical, structural, and bioinformatic studies revealed that OLD nucleases protect bacteria by functioning in diverse anti-phage defense systems including the Gabija system and retrons. In this review we will highlight recent discoveries in OLD protein function and their involvement in multiple discrete anti-phage defense systems.

Characterization of novel recombinant mycobacteriophages derived from homologous recombination between two temperate phages.

Comparative analyses of mycobacteriophage genomes reveals extensive genetic diversity in genome organization and gene content, contributing to widespread mosaicism. We previously reported that the prophage of mycobacteriophage Butters (cluster N) provides defense against infection by Island3 (subcluster I1). To explore the anti-Island3 defense mechanism, we attempted to isolate Island3 defense escape mutants on a Butters lysogen, but only uncovered phages with recombinant genomes comprised of regions of Butters and Island3 arranged from left arm to right arm as Butters-Island3-Butters (BIBs). Recombination occurs within two distinct homologous regions that encompass lysin A, lysin B, and holin genes in one segment, and RecE and RecT genes in the other. Structural genes of mosaic BIB genomes are contributed by Butters while the immunity cassette is derived from Island3. Consequently, BIBs are morphologically identical to Butters (as shown by transmission electron microscopy) but are homoimmune with Island3. Recombinant phages overcome antiphage defense and silencing of the lytic cycle. We leverage this observation to propose a stratagem to generate novel phages for potential therapeutic use.

Biochemical characterization of type I-E anti-CRISPR proteins, AcrIE2 and AcrIE4

In bacteria and archaea, CRISPRs and Cas proteins constitute an adaptive immune system against invading foreign genetic materials, such as bacteriophages and plasmids. To counteract CRISPR-mediated immunity, bacteriophages encode anti-CRISPR (Acr) proteins that neutralize the host CRISPR–Cas systems. Several Acr proteins that act against type I-E CRISPR–Cas systems have been identified. Here, we describe the biochemical characterization of two type I-E Acr proteins, AcrIE2 and AcrIE4. We determined the crystal structure of AcrIE2 using single-wavelength anomalous diffraction and performed a structural comparison with the previously reported AcrIE2 structures solved by different techniques. Binding assays with type I-E Cas proteins were carried out for the target identification of AcrIE2. We also analyzed the interaction between AcrIE4 and its target Cas component using biochemical methods. Our findings corroborate and expand the knowledge on type I-E Acr proteins, illuminating diverse molecular mechanisms of inhibiting CRISPR-mediated prokaryotic anti-phage defense.

The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin–antitoxin and related phage defense systems

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.

Atypical bacterial Argonautes regulate antiphage defense.

Atypical bacterial Argonautes regulate antiphage defense.

Immunology: NACHT domain proteins get a prokaryotic origin story

The NACHT domain is found in eukaryotic pattern recognition receptors that promote anti-microbial defense, mediating their oligomerization into immune signaling complexes. Kibby etal. uncover a superfamily of prokaryotic NACHT-containing proteins and demonstrate that some members of this family mediate anti-phage defense.

Phages are unrecognized players in the ecology of the oral pathogen Porphyromonas gingivalis

BackgroundPorphyromonas gingivalis (hereafter “Pg”) is an oral pathogen that has been hypothesized to act as a keystone driver of inflammation and periodontal disease. Although Pg is most readily recovered from individuals with actively progressing periodontal disease, healthy individuals and those with stable non-progressing disease are also colonized by Pg. Insights into the factors shaping the striking strain-level variation in Pg, and its variable associations with disease, are needed to achieve a more mechanistic understanding of periodontal disease and its progression. One of the key forces often shaping strain-level diversity in microbial communities is infection of bacteria by their viral (phage) predators and symbionts. Surprisingly, although Pg has been the subject of study for over 40 years, essentially nothing is known of its phages, and the prevailing paradigm is that phages are not important in the ecology of Pg.ResultsHere we systematically addressed the question of whether Pg are infected by phages—and we found that they are. We found that prophages are common in Pg, they are genomically diverse, and they encode genes that have the potential to alter Pg physiology and interactions. We found that phages represent unrecognized targets of the prevalent CRISPR-Cas defense systems in Pg, and that Pg strains encode numerous additional mechanistically diverse candidate anti-phage defense systems. We also found that phages and candidate anti-phage defense system elements together are major contributors to strain-level diversity and the species pangenome of this oral pathogen. Finally, we demonstrate that prophages harbored by a model Pg strain are active in culture, producing extracellular viral particles in broth cultures.ConclusionThis work definitively establishes that phages are a major unrecognized force shaping the ecology and intra-species strain-level diversity of the well-studied oral pathogen Pg. The foundational phage sequence datasets and model systems that we establish here add to the rich context of all that is already known about Pg, and point to numerous avenues of future inquiry that promise to shed new light on fundamental features of phage impacts on human health and disease broadly.B5C3jjutXjtawSKFEwKi2zVideo

Toxic antiphage defense proteins inhibited by intragenic antitoxin proteins

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here, we report that these proteins are toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic RpnL. The crystal structure of RpnAS revealed a dimerization interface encompassing α helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document that plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose that many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms.

Molecular basis of assembly, operation and regulation of a novel anti-phage defense system-RADAR

Molecular basis of assembly, operation and regulation of a novel anti-phage defense system-RADAR

CBASS to cGAS-STING: The Origins and Mechanisms of Nucleotide Second Messenger Immune Signaling.

Host defense against viral pathogens is an essential function for all living organisms. In cell-intrinsic innate immunity, dedicated sensor proteins recognize molecular signatures of infection and communicate to downstream adaptor or effector proteins to activate immune defense. Remarkably, recent evidence demonstrates that much of the core machinery of innate immunity is shared across eukaryotic and prokaryotic domains of life. Here, we review a pioneering example of evolutionary conservation in innate immunity: the animal cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) signaling pathway and its ancestor in bacteria, CBASS (cyclic nucleotide-based antiphage signaling system) antiphage defense. We discuss the unique mechanism by which animal cGLRs (cGAS-like receptors) and bacterial CD-NTases (cGAS/dinucleotide-cyclase in Vibrio (DncV)-like nucleotidyltransferases) in these pathways link pathogen detection with immune activation using nucleotide second messenger signals. Comparing the biochemical, structural, and mechanistic details of cGAS-STING, cGLR signaling, and CBASS, we highlight emerging questions in the field and examine evolutionary pressures that may have shaped the origins of nucleotide second messenger signaling in antiviral defense.

Prophage identification and molecular analysis in the genomes of Pseudomonas aeruginosa strains isolated from critical care patients.

Prophages are bacteriophages integrated into the bacterial host's chromosome. This research aims to analyze and characterize the existing prophages within a collection of 53 Pseudomonas aeruginosa strains from intensive care units (ICUs) in Portugal and Spain. A total of 113 prophages were localized in the collection, with 18 of them being present in more than one strain simultaneously. After annotation, five of them were discarded as incomplete, and the 13 remaining prophages were characterized. Of 13, 10 belonged to the siphovirus tail morphology group, 2 to the podovirus tail morphology group, and 1 to the myovirus tail morphology group. All prophages had a length ranging from 20,199 to 63,401 bp and a GC% between 56.2% and 63.6%. The number of open reading frames (ORFs) oscillated between 32 and 88, and in 3/13 prophages, more than 50% of the ORFs had an unknown function. With our findings, we show that prophages are present in the majority of the P. aeruginosa strains isolated from Portuguese and Spanish critically ill patients, many of them found in more than one circulating strain at the same time and following a similar clonal distribution pattern. Although a great sum of ORFs had an unknown function, number of proteins in relation to viral defense (anti-CRISPR proteins, toxin/antitoxin modules, proteins against restriction-modification systems) as well as to prophage interference into their host's quorum sensing system and regulatory cascades were found. This supports the idea that prophages have an influence in bacterial pathogenesis and anti-phage defense. IMPORTANCE Despite being known for decades, prophages remain understudied when compared to the lytic phages employed in phage therapy. This research aims to shed some light into the nature, composition, and role of prophages found within a set of circulating strains of Pseudomas aeruginosa, with special attention to high-risk clones. Given the fact that prophages can effectively influence bacterial pathogenesis, prophage basic research constitutes a topic of growing interest. Furthermore, the abundance of viral defense and regulatory proteins within prophage genomes detected in this study evidences the importance of characterizing the most frequent prophages in circulating clinical strains and in high-risk clones if phage therapy is to be used.

Viral sponges sequester nucleotide signals to inactivate immunity.

Bacteria synthesize specialized nucleotide signals to control anti-phage defense. Two papers - by Huiting et al. and Jenson et al. - now reveal that bacteriophages encode protein 'sponges' that sequester cyclic oligonucleotide immune signals and inactivate host antiviral immunity.

Everything OLD is new again: How structural, functional, and bioinformatic advances have redefined a neglected nuclease family.

Overcoming lysogenization defect (OLD) proteins are a conserved family of ATP-powered nucleases that function in anti-phage defense. Recent bioinformatic, genetic, and crystallographic studies have yielded new insights into the structure, function, and evolution of these enzymes. Here we review these developments and propose a new classification scheme to categorize OLD homologs that relies on gene neighborhoods, biochemical properties, domain organization, and catalytic machinery. This taxonomy reveals important similarities and differences between family members and provides a blueprint to contextualize future in vivo and in vitro findings. We also detail how OLD nucleases are related to PARIS and Septu anti-phage defense systems and discuss important mechanistic questions that remain unanswered.

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. Out of the 30 species with >200 completely sequenced genomes, our analysis found Pseudomonas aeruginosa exhibits the greatest diversity of anti-phage systems, as measured by Shannon entropy. Using network analysis to identify the common neighbors of anti-phage systems, we identified two core defense hotspot loci (cDHS1 and cDHS2). cDHS1 is up to 224 kb (median: 26 kb) with varied arrangements of more than 30 distinct immune systems across isolates, while cDHS2 has 24 distinct systems (median: 6 kb). Both cDHS regions are occupied in a majority of P. aeruginosa isolates. Most cDHS genes are of unknown function potentially representing new anti-phage systems, which we validated by identifying a novel anti-phage system (Shango) commonly encoded in cDHS1. Identifying core genes flanking immune islands could simplify immune system discovery and may represent popular landing spots for diverse MGEs carrying anti-phage systems.