Anti-phage Defense Research Articles

Evasion of antiviral bacterial immunity by phage tRNAs.

Retrons are bacterial genetic elements that encode a reverse transcriptase and, in combination with toxic effector proteins, can serve as antiphage defense systems. However, the mechanisms of action of most retron effectors, and how phages evade retrons, are not well understood. Here, we show that some phages can evade retrons and other defense systems by producing specific tRNAs. We find that expression of retron-Eco7 effector proteins (PtuA and PtuB) leads to degradation of tRNATyr and abortive infection. The genomes of T5 phages that evade retron-Eco7 include a tRNA-rich region, including a highly expressed tRNATyr gene, which confers protection against retron-Eco7. Furthermore, we show that other phages (T1, T7) can use a similar strategy, expressing a tRNALys, to counteract a tRNA anticodon defense system (PrrC170).

Comparative Genomic Analysis Reveals Distinct Virulence and Resistance Mechanisms in 21 Bacterial Fish Pathogens

The rising bacterial infections threaten world aquaculture and wild fish populations, making it imperative to increase the understanding of the mechanisms of pathogenic virulence and resistance. This study applies comparative genomic analysis to 21 bacterial fish pathogens, using whole-genome sequences from public genomic resources and sophisticated bioinformatics tools for screening of virulence factors, mobile genetic elements, antibiotic resistance genes, anti-phage defense mechanisms and secretory systems. We have seen that the different pathogens depict a wide range of variability regarding virulence and resistance potential, which may be attributed to species-specific adaptation. Notably, Streptococcus agalactiae and Mycobacterium salmoniphilum were found to possess high offensive and defensive virulence potential, but at different regulative controls. We also found diverse secretion systems and intricate mechanisms for antibiotic resistance, which have provided very important insights into how pathogens adapt to their environments. By categorizing functional genes and finding anti-phage systems, our analysis has revealed new insights into the complex interactions among bacterial virulence, resistance, and host defense mechanisms. These findings not only shed new light on the bacterial pathogenesis process in aquaculture but also provide the bases for focused, therapeutically-based strategies and genomic surveillance programs able to improve disease management and sustainability in aquaculture environments.

Mechanistic basis for the allosteric activation of NADase activity in the Sir2-HerA antiphage defense system

Sir2-HerA is a widely distributed antiphage system composed of a RecA-like ATPase (HerA) and an effector with potential NADase activity (Sir2). Sir2-HerA is believed to provide defense against phage infection in Sir2-dependent NAD+ depletion to arrest the growth of infected cells. However, the detailed mechanism underlying its antiphage activity remains largely unknown. Here, we report functional investigations of Sir2-HerA from Staphylococcus aureus (SaSir2-HerA), unveiling that the NADase function of SaSir2 can be allosterically activated by the binding of SaHerA, which then assembles into a supramolecular complex with NADase activity. By combining the cryo-EM structure of SaSir2-HerA in complex with the NAD+ cleavage product, it is surprisingly observed that Sir2 protomers that interact with HerA are in the activated state, which is due to the opening of the α15-helix covering the active site, allowing NAD+ to access the catalytic pocket for hydrolysis. In brief, our study provides a comprehensive view of an allosteric activation mechanism for Sir2 NADase activity in the Sir2-HerA immune system.

Topological rearrangements activate the HerA-DUF anti-phage defense system.

Leveraging the rich structural information provided by AlphaFold, we used integrated experimental approaches to characterize the HerA-DUF4297 (DUF) anti-phage defense system, in which DUF is of unknown function. To infer the function of DUF, we performed structure-guided genomic analysis and found that DUF homologs are universally present in bacterial immune defense systems. One notable homolog of DUF is Cap4, a universal effector with nuclease activity in CBASS, the most prevalent anti-phage system in bacteria. To test the inferred nuclease function of DUF, we performed biochemical experiments and discovered that the DUF only exhibits activity against DNA substrates when it is bound by HerA. To understand how HerA activates DUF, we determined the structures of DUF and the HerA-DUF complex. DUF forms large oligomeric assemblies with or without HerA, suggesting that oligomerization per se is not sufficient for DUF activation. Instead, DUF activation requires dramatic topological rearrangements that propagate from HerA to the entire HerA-DUF complex, leading to reorganization of DUF for effective DNA cleavage. We further validated these structural insights by structure- guided mutagenesis. Together, these findings reveal dramatic topological rearrangements throughout the HerA-DUF complex, challenge the long-standing dogma that protein oligomerization alone activates immune signaling, and may inform the activation mechanism of CBASS.

Systematic, high-throughput characterization of bacteriophage gene essentiality on diverse hosts.

Understanding core and conditional gene essentiality is crucial for decoding genotype-phenotype relationships in organisms. We present PhageMaP, a high-throughput method to create genome-scale phage knockout libraries for systematically assessing gene essentiality in bacteriophages. Using PhageMaP, we generate gene essentiality maps across hundreds of genes in the model phage T7 and the non-model phage Bas63, on diverse hosts. These maps provide fundamental insights into genome organization, gene function, and host-specific conditional essentiality. By applying PhageMaP to a collection of anti-phage defense systems, we uncover phage genes that either inhibit or activate eight defenses and offer novel mechanistic hypotheses. Furthermore, we engineer synthetic phages with enhanced infectivity by modular transfer of a PhageMaP-discovered defense inhibitor from Bas63 to T7. PhageMaP is generalizable, as it leverages homologous recombination, a universal cellular process, for locus-specific barcoding. This versatile tool advances bacteriophage functional genomics and accelerates rational phage design for therapy.

Mechanisms of Antiphage Defense in Prokaryotes

The globalization of the problem of the formation of bacterial strains poly- and pan-resistant to known antimicrobial drugs creates high risks in the healthcare sector. The threat of a return to the «pre-antibiotic» era dictates the need to search for alternative forms of antibacterial therapy. Phage therapy, based on the use of a natural, widely distributed in the environment, accessible antimicrobial agent, is again becoming relevant. The article highlights the mechanisms of antiviral protection of prokaryotes at various stages of interaction between the virus and the target cell. Revealing the secrets of the confrontation between bacteria and viruses, their co-evolution is necessary to increase the effectiveness of phage therapy and develop modern means of overcoming bacterial resistance to antimicrobial agents.

Genome integrity sensing by the broad-spectrum Hachiman antiphage defense complex.

Hachiman is a broad-spectrum antiphage defense system of unknown function. We show here that Hachiman is a heterodimeric nuclease-helicase complex, HamAB. HamA, previously a protein of unknown function, is the effector nuclease. HamB is the sensor helicase. HamB constrains HamA activity during surveillance of intact double-stranded DNA (dsDNA). When the HamAB complex detects DNA damage, HamB helicase activity activates HamA, unleashing nuclease activity. Hachiman activation degrades all DNA in the cell, creating "phantom" cells devoid of both phage and host DNA. We demonstrate Hachiman activation in the absence of phage by treatment with DNA-damaging agents, suggesting that Hachiman responds to aberrant DNA states. Phylogenetic similarities between the Hachiman helicase and enzymes from eukaryotes and archaea suggest deep functional symmetries with other important helicases across domains of life.

Genome sequences of Mycobacterium sp. Elmwood and accompanying phage, isolated from a public swimming pool in Nebraska.

Genome sequencing of a non-tuberculosis Mycobacterium species, isolated from a public pool, shows that the genome contains several genes for antibiotic resistance and anti-phage defense, which are absent from other related Mycobacteria. Metagenomic binning also provided the genome of the accompanying phage, which is distinct from other mycobacterial phages.

Control of lysogeny and antiphage defense by a prophage-encoded kinase-phosphatase module

The filamentous ‘Pf’ bacteriophages of Pseudomonas aeruginosa play roles in biofilm formation and virulence, but mechanisms governing Pf prophage activation in biofilms are unclear. Here, we identify a prophage regulatory module, KKP (kinase-kinase-phosphatase), that controls virion production of co-resident Pf prophages and mediates host defense against diverse lytic phages. KKP consists of Ser/Thr kinases PfkA and PfkB, and phosphatase PfpC. The kinases have multiple host targets, one of which is MvaU, a host nucleoid-binding protein and known prophage-silencing factor. Characterization of KKP deletion and overexpression strains with transcriptional, protein-level and prophage-based approaches indicates that shifts in the balance between kinase and phosphatase activities regulate phage production by controlling MvaU phosphorylation. In addition, KKP acts as a tripartite toxin-antitoxin system that provides defense against some lytic phages. A conserved lytic phage replication protein inhibits the KKP phosphatase PfpC, stimulating toxic kinase activity and blocking lytic phage production. Thus, KKP represents a phosphorylation-based mechanism for prophage regulation and antiphage defense. The conservation of KKP gene clusters in >1000 diverse temperate prophages suggests that integrated control of temperate and lytic phage infection by KKP-like regulatory modules may play a widespread role in shaping host cell physiology.

Resistance against aminoglycoside antibiotics via drug or target modification enables community-wide antiphage defense.

The ongoing arms race between bacteria and phages has forced bacteria to evolve a sophisticated set of antiphage defense mechanisms that constitute the bacterial immune system. In our previous study, we highlighted the antiphage properties of aminoglycoside antibiotics, which are naturally secreted by Streptomyces. Successful inhibition of phage infection was achieved by addition of pure compounds and supernatants from a natural producer strain emphasizing the potential for community-wide antiphage defense. However, given the dual functionality of these compounds, neighboring bacterial cells require resistance to the antibacterial activity of aminoglycosides to benefit from the protection they confer against phages. In this study, we tested a variety of different aminoglycoside resistance mechanisms acting via drug or target (16S rRNA) modification and demonstrated that they do not interfere with the antiphage properties of the molecules. Furthermore, we confirmed the antiphage impact of aminoglycosides in a community context by coculturing phage-susceptible, apramycin-resistant Streptomyces venezuelae with the apramycin-producing strain Streptoalloteichus tenebrarius. Given the prevalence of aminoglycoside resistance among natural bacterial isolates, this study highlights the ecological relevance of chemical defense via aminoglycosides at the community level.

An enterococcal phage protein inhibits type IV restriction enzymes involved in antiphage defense

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.

Structural insights into activation mechanisms on NADase of the bacterial DSR2 anti-phage defense system.

As a sirtuin (SIR2) family protein, defense-associated sirtuin2 (DSR2) has been demonstrated to participate in bacterial anti-phage resistance via depleting nicotinamide adenine dinucleotide (NAD+) of infected cells, which can be activated by tail tube protein (TTP) and inhibited by DSR anti-defense 1 (DSAD1) of diverse phages. However, the regulating mechanism remains elusive. Here, we determined the cryo-electron microscopy structure of apo DSR2, as well as the respective complex structures with TTP and DSAD1. Structural analyses and biochemical studies reveal that DSR2 forms a tetramer with a SIR2 central core and two distinct conformations. Monomeric TTP preferentially binds to the closed conformation of DSR2, inducing conformational distortions on SIR2 tetramer assembly to activate its NADase activity. DSAD1 combines with the open conformation of DSR2, directly or allosterically inhibiting TTP activation on DSR2 NAD+ hydrolysis. Our findings decipher the detailed molecule mechanisms for DSR2 NADase activity regulation and lay a foundation for in-depth understanding of the DSR2 anti-phage defense system.

Animal and bacterial viruses share conserved mechanisms of immune evasion

Animal and bacterial cells sense and defend against viral infections using evolutionarily conserved antiviral signaling pathways. Here, we show that viruses overcome host signaling using mechanisms of immune evasion that are directly shared across the eukaryotic and prokaryotic kingdoms of life. Structures of animal poxvirus proteins that inhibit host cGAS-STING signaling demonstrate architectural and catalytic active-site homology shared with bacteriophage Acb1 proteins, which inactivate CBASS anti-phage defense. In bacteria, phage Acb1 proteins are viral enzymes that degrade host cyclic nucleotide immune signals. Structural comparisons of poxvirus protein-2'3'-cGAMP and phage Acb1-3'3'-cGAMP complexes reveal a universal mechanism of host nucleotide immune signal degradation and explain kingdom-specific additions that enable viral adaptation. Chimeric bacteriophages confirm that animal poxvirus proteins are sufficient to evade immune signaling in bacteria. Our findings identify a mechanism of immune evasion conserved between animal and bacterial viruses and define shared rules that explain host-virus interactions across multiple kingdoms of life.

Nucleotide Immune Signaling in CBASS, Pycsar, Thoeris, and CRISPR Antiphage Defense.

Bacteria encode an arsenal of diverse systems that defend against phage infection. A common theme uniting many prevalent antiphage defense systems is the use of specialized nucleotide signals that function as second messengers to activate downstream effector proteins and inhibit viral propagation. In this article, we review the molecular mechanisms controlling nucleotide immune signaling in four major families of antiphage defense systems: CBASS, Pycsar, Thoeris, and type III CRISPR immunity. Analyses of the individual steps connecting phage detection, nucleotide signal synthesis, and downstream effector function reveal shared core principles of signaling and uncover system-specific strategies used to augment immune defense. We compare recently discovered mechanisms used by phages to evade nucleotide immune signaling and highlight convergent strategies that shape host-virus interactions. Finally, we explain how the evolutionary connection between bacterial antiphage defense and eukaryotic antiviral immunity defines fundamental rules that govern nucleotide-based immunity across all kingdoms of life.

A bacterial TIR-based immune system senses viral capsids to initiate defense.

Toll/interleukin-1 receptor (TIR) domains are present in immune systems that protect prokaryotes from viral (phage) attack. In response to infection, TIRs can produce a cyclic adenosine diphosphate-ribose (ADPR) signaling molecule, which activates an effector that depletes the host of the essential metabolite NAD+ to limit phage propagation. How bacterial TIRs recognize phage infection is not known. Here we describe the sensing mechanism for the staphylococcal Thoeris defense system, which consists of two TIR domain sensors, ThsB1 and ThsB2, and the effector ThsA. We show that the major capsid protein of phage Φ80α forms a complex with ThsB1 and ThsB2, which is sufficient for the synthesis of 1"-3' glycocyclic ADPR (gcADPR) and subsequent activation of NAD+ cleavage by ThsA. Consistent with this, phages that escape Thoeris immunity harbor mutations in the capsid that prevent complex formation. We show that capsid proteins from staphylococcal Siphoviridae belonging to the capsid serogroup B, but not A, are recognized by ThsB1/B2, a result that suggests that capsid recognition by Sau-Thoeris and other anti-phage defense systems may be an important evolutionary force behind the structural diversity of prokaryotic viruses. More broadly, since mammalian toll-like receptors harboring TIR domains can also recognize viral structural components to produce an inflammatory response against infection, our findings reveal a conserved mechanism for the activation of innate antiviral defense pathways.

The structural basis of the activation and inhibition of DSR2 NADase by phage proteins

DSR2, a Sir2 domain-containing protein, protects bacteria from phage infection by hydrolyzing NAD+. The enzymatic activity of DSR2 is triggered by the SPR phage tail tube protein (TTP), while suppressed by the SPbeta phage-encoded DSAD1 protein, enabling phages to evade the host defense. However, the molecular mechanisms of activation and inhibition of DSR2 remain elusive. Here, we report the cryo-EM structures of apo DSR2, DSR2-TTP-NAD+ and DSR2-DSAD1 complexes. DSR2 assembles into a head-to-head tetramer mediated by its Sir2 domain. The C-terminal helical regions of DSR2 constitute four partner-binding cavities with opened and closed conformation. Two TTP molecules bind to two of the four C-terminal cavities, inducing conformational change of Sir2 domain to activate DSR2. Furthermore, DSAD1 competes with the activator for binding to the C-terminal cavity of DSR2, effectively suppressing its enzymatic activity. Our results provide the mechanistic insights into the DSR2-mediated anti-phage defense system and DSAD1-dependent phage immune evasion.

An enterococcal phage protein broadly inhibits type IV restriction enzymes involved in antiphage defense.

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.

Diverse Antiphage Defenses Are Widespread Among Prophages and Mobile Genetic Elements.

Bacterial viruses known as phages rely on their hosts for replication and thus have developed an intimate partnership over evolutionary time. The survival of temperate phages, which can establish a chronic infection in which their genomes are maintained in a quiescent state known as a prophage, is tightly coupled with the survival of their bacterial hosts. As a result, prophages encode a diverse antiphage defense arsenal to protect themselves and the bacterial host in which they reside from further phage infection. Similarly, the survival and success of prophage-related elements such as phage-inducible chromosomal islands are directly tied to the survival and success of their bacterial host, and they also have been shown to encode numerous antiphage defenses. Here, we describe the current knowledge of antiphage defenses encoded by prophages and prophage-related mobile genetic elements.

Structural characterization of macro domain-containing Thoeris antiphage defense systems.

Thoeris defense systems protect bacteria from infection by phages via abortive infection. In these systems, ThsB proteins serve as sensors of infection and generate signaling nucleotides that activate ThsA effectors. Silent information regulator and SMF/DprA-LOG (SIR2-SLOG) containing ThsA effectors are activated by cyclic ADP-ribose (ADPR) isomers 2'cADPR and 3'cADPR, triggering abortive infection via nicotinamide adenine dinucleotide (NAD+) depletion. Here, we characterize Thoeris systems with transmembrane and macro domain (TM-macro)-containing ThsA effectors. We demonstrate that ThsA macro domains bind ADPR and imidazole adenine dinucleotide (IAD), but not 2'cADPR or 3'cADPR. Combining crystallography, in silico predictions, and site-directed mutagenesis, we show that ThsA macro domains form nucleotide-induced higher-order oligomers, enabling TM domain clustering. We demonstrate that ThsB can produce both ADPR and IAD, and we identify a ThsA TM-macro-specific ThsB subfamily with an active site resembling deoxy-nucleotide and deoxy-nucleoside processing enzymes. Collectively, our study demonstrates that Thoeris systems with SIR2-SLOG and TM-macro ThsA effectors trigger abortive infection via distinct mechanisms.

Regulation of anti-phage defense mechanisms by using cinnamaldehyde as a quorum sensing inhibitor.

Multidrug-resistant bacteria and the shortage of new antibiotics constitute a serious health problem. This problem has led to increased interest in the use of bacteriophages, which have great potential as antimicrobial agents but also carry the risk of inducing resistance. The objective of the present study was to minimize the development of phage resistance in Klebsiella pneumoniae strains by inhibiting quorum sensing (QS) and thus demonstrate the role of QS in regulating defense mechanisms. Cinnamaldehyde (CAD) was added to K. pneumoniae cultures to inhibit QS and thus demonstrate the role of the signaling system in regulating the anti-phage defense mechanism. The QS inhibitory activity of CAD in K. pneumoniae was confirmed by a reduction in the quantitative expression of the lsrB gene (AI-2 pathway) and by proteomic analysis. The infection assays showed that the phage was able to infect a previously resistant K. pneumoniae strain in the cultures to which CAD was added. The results were confirmed using proteomic analysis. Thus, anti-phage defense-related proteins from different systems, such as cyclic oligonucleotide-based bacterial anti-phage signaling systems (CBASS), restriction-modification (R-M) systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, and bacteriophage control infection (BCI), were present in the cultures with phage but not in the cultures with phage and CAD. When the QS and anti-phage defense systems were inhibited by the combined treatment, proteins related to phage infection and proliferation, such as the tail fiber protein, the cell division protein DamX, and the outer membrane channel protein TolC, were detected. Inhibition of QS reduces phage resistance in K. pneumoniae, resulting in the infection of a previously resistant strain by phage, with a significant increase in phage proliferation and a significant reduction in bacterial growth. QS inhibitors could be considered for therapeutic application by including them in phage cocktails or in phage-antibiotic combinations to enhance synergistic effects and reduce the emergence of antimicrobial resistance.