Biology subjects

Sorek, R.

Publications and source records attributed to Sorek, R..

36 records · Page 2Linked to original sources

bioRxiv2023

Phages overcome bacterial immunity via diverse anti-defense proteins

It was recently shown that bacteria employ, apart from CRISPR-Cas and restriction systems, a considerable diversity of phage resistance systems, but it is largely unknown how phages cope with this multilayered bacterial immunity. Here, we analyzed groups of closely related Bacillus phages that showed differential sensitivity to bacterial defense systems, and identified multiple families of anti-defense proteins that inhibit the Gabija, Thoeris, and Hachiman systems. We show that these proteins efficiently cancel the defensive activity when co-expressed with the respective defense system or introduced into phage genomes. Homologs of these anti-defense proteins are found in hundreds of phages that infect taxonomically diverse bacterial species. We show that an anti-Gabija protein, denoted Gad1, blocks the ability of the Gabija defense complex to cleave phage-derived DNA. Our data further reveal an anti-Thoeris protein, denoted Tad2, which is a "sponge" that sequesters the immune signaling molecules produced by Thoeris TIR-domain proteins in response to phage. Our results demonstrate that phages encode an arsenal of anti-defense proteins that can disable a variety of bacterial defense mechanisms.

genetics↗

bioRxiv2023

Structural basis of Gabija anti-phage defense and viral immune evasion

Bacteria encode hundreds of diverse defense systems that protect from viral infection and inhibit phage propagation1-5. Gabija is one of the most prevalent anti-phage defense systems, occurring in >15% of all sequenced bacterial and archaeal genomes1,6,7, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-EM to define how Gabija proteins assemble into an [~]500 kDa supramolecular complex that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defense complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajAB assembly that is essential for phage resistance in vivo. We show that a phage-encoded protein Gabija anti-defense 1 (Gad1) directly binds the Gabija GajAB complex and inactivates defense. A cryo-EM structure of the virally inhibited state reveals that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defense complex and define a unique mechanism of viral immune evasion.

microbiology↗

bioRxiv2023

A conserved family of immune effectors cleaves cellular ATP upon viral infection

During viral infection, cells can deploy immune strategies that deprive viruses of molecules essential for their replication. In this study, we report a family of immune effectors in bacteria which, in response to phage infection, degrade cellular ATP and dATP by cleaving the N-glycosidic bond between the adenine and sugar moieties. These ATP nucleosidase effectors are widely distributed within multiple bacterial defense systems including CBASS, prokaryotic argonautes and NLR-like proteins, and we show that degradation of (d)ATP during infection halts phage propagation and aborts infection. By analyzing homologs of the immune ATP nucleosidase domain, we discover and characterize Detocs, a new family of bacterial defense systems with a two-component phosphotransfer signaling architecture. The immune ATP nucleosidase domain is also encoded within a diverse set of eukaryotic proteins that have immune-like architectures, and we show biochemically that these eukaryotic homologs preserve the ATP nucleosidase activity. Our findings suggest that ATP and dATP degradation is a cell-autonomous innate immune strategy conserved across the tree of life.

microbiology↗

bioRxiv2022

Discovery of phage determinants that confer sensitivity to bacterial immune systems

Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. While the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 192 phage mutants that escape 19 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.

microbiology↗

bioRxiv2022

Cryo-EM structure of the RADAR supramolecular anti-phage defense complex

RADAR is a two-protein bacterial defense system which was reported to defend against phage by editing messenger RNA. Here we determine cryo-EM structures of the RADAR defense complex, revealing RdrA as a heptameric, two-layered AAA+ ATPase and RdrB as a dodecameric, hollow complex with twelve surface-exposed deaminase active sites. RdrA and RdrB join to form a giant assembly up to 10 MDa, with RdrA docked as a funnel over the RdrB active site. Surprisingly, our structures reveal a RdrB active site that targets mononucleotides, not RNA. We show that RdrB catalyzes ATP-to-ITP conversion in vitro and induces the accumulation of inosine mononucleotides during phage infection in vivo, limiting phage replication. Our results define ATP mononucleotide deamination as a determinant of RADAR immunity and reveal supramolecular assembly of a nucleotide-modifying machine as a novel mechanism of anti-phage defense.

biochemistry↗

bioRxiv2022

The defence island repertoire of the Escherichia coli pan-genome

In recent years it has become clear that anti-phage defence systems cluster non-randomly within bacterial genomes in so-called "defence islands". Despite serving as a valuable tool for the discovery of novel defence systems, the nature and distribution of defence islands themselves remain poorly understood. In this study, we comprehensively mapped the repertoire of defence islands within >1,300 strains of Escherichia coli, the most widely studied organism in terms of phage-bacteria interactions. We found that defence islands preferentially integrate at several dozens of dedicated integration hotspots in the E. coli genome. Defence islands are usually carried on mobile genetic elements including prophages, integrative conjugative elements and transposons, as well as on other genetic elements whose nature of mobilisation is unclear. Each type of mobile genetic element has a preferred integration position but can carry a diverse variety of defensive cargo. On average, an E. coli genome has 4.5 genomic hotspots occupied by a defence system-containing mobile element, with some strains possessing up to eight defensively occupied hotspots. Our data show that the overwhelming majority of the E. coli pan-immune system is carried on mobile genetic elements that integrate at a discrete set of genomic hotspots, and explains why the immune repertoire substantially varies between different strains of the same species.

microbiology↗

bioRxiv2022

An expanding arsenal of immune systems that protect bacteria from phages

Bacterial anti-phage defense systems are frequently clustered in microbial genomes, forming defense islands. This genomic property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms in bacteria is still unknown. In this study we report the discovery of 21 new defense systems that protect bacteria from phages, based on computational genomic analyses and phage infection experiments. We find multiple systems with protein domains known to be involved in eukaryotic anti-viral immunity, including ISG15-like proteins, dynamin-like proteins, and SEFIR domains, and show that these domains participate in bacterial defense against phages. Additional systems include protein domains predicted to manipulate DNA and RNA molecules, as well as multiple toxin-antitoxin systems shown here to function in anti-phage defense. The systems we discovered are widely distributed in bacterial and archaeal genomes, and in some bacteria form a considerable fraction of the immune arsenal. Our data substantially expand the known inventory of defense systems utilized by bacteria to counteract phage infection.

microbiology↗

bioRxiv2022

Viruses inhibit TIR gcADPR signaling to overcome bacterial defense

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants, and animals. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signaling molecule whose molecular structure remained elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function. We identified a large family of phage-encoded proteins, denoted here Thoeris anti-defense 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are "sponges" that bind and sequester the immune signaling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. A high-resolution crystal structure of Tad1 bound to the signaling molecule revealed that its chemical structure is 1'-2' glycocyclic ADPR (gcADPR), a unique molecule not previously described in other biological systems. Our results define the chemical structure of a central immune signaling molecule, and reveal a new mode of action by which pathogens can suppress host immunity.

microbiology↗

bioRxiv2021

Class I DISARM provides anti-phage and anti-conjugation activity by unmethylated DNA recognition

Bacteriophages impose a strong evolutionary pressure on microbes for the development of mechanisms of survival. Multiple new mechanisms of innate defense have been described recently, with the molecular mechanism of most of them remaining uncharacterized. Here, we show that a Class 1 DISARM (defense island system associated with restriction-modification) system from Serratia sp. provides broad protection from double-stranded DNA phages, and drives a population of single-stranded phages to extinction. We identify that protection is not abolished by deletion of individual DISARM genes and that the absence of methylase genes drmMI and drmMII does not result in autoimmunity. In addition to antiphage activity we also observe that DISARM limits conjugation, and this activity is linked to the number of methylase cognate sites in the plasmid. Overall, we show that Class 1 DISARM provides robust anti-phage and anti-plasmid protection mediated primarily by drmA and drmB, which provide resistance to invading nucleic acids using a mechanism enhanced by the recognition of unmethylated cognate sites of the two methylases drmMI and drmMII.

microbiology↗

bioRxiv2021

Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion

Defense-associated sirtuins (DSR) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbor an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2-resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defense systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

microbiology↗

bioRxiv2021

Sir2-domain associated short prokaryotic Argonautes provide defence against invading mobile genetic elements through NAD+ depletion

Argonaute (Ago) proteins are found in all three domains of life. The so-called long Agos are composed of four major domains (N, PAZ, MID, and PIWI) and contribute to RNA silencing in eukaryotes (eAgos) or defence against invading mobile genetic elements in prokaryotes (pAgos). The majority (~60%) of pAgos identified bioinformatically are shorter (comprised of only MID and PIWI domains) and are typically associated with Sir2, Mrr or TIR domain-containing proteins. The cellular function and mechanism of short pAgos remain enigmatic. Here, we show that Geobacter sulfurreducens short pAgo and the NAD+-bound Sir2-protein form a stable heterodimeric complex. The GsSir2/Ago complex presumably recognizes invading plasmid or phage DNA and activates the Sir2 subunit, which triggers endogenous NAD+ depletion and cell death, and prevents the propagation of invading DNA. We reconstituted NAD+ depletion activity in vitro and showed that activated GsSir2/Ago complex functions as a NADase that hydrolyses NAD+ to ADPR. Thus, short Sir2-associated pAgos provide defence against phages and plasmids and underscores the diversity of mechanisms of prokaryotic Agos.

molecular biology↗

bioRxiv2021

The DarTG toxin-antitoxin system provides phage defense by ADP-ribosylating viral DNA

Toxin-antitoxin (TA) systems are broadly distributed, yet poorly conserved, genetic elements whose biological functions are unclear and controversial. Some TA systems may provide bacteria with immunity to infection by their ubiquitous viral predators, the bacteriophage. To identify TA systems that protect E. coli MG1655 against phage, we searched for those frequently encoded near known phage defense genes in bacterial genomes. Two of the systems tested provide strong protection against phage infection and are homologs of DarTG, a recently discovered family of TA systems whose biological functions and natural activating conditions were unclear. We demonstrate that phage infection triggers the release of DarT toxin, a DNA ADP-ribosyltransferase, to modify viral DNA and prevent replication, thereby blocking the production of mature virions. Phages can evolve to overcome DarTG defense either through mutations to their DNA polymerase or to an anti-DarT factor, gp61.2, encoded by many T-even phages. Collectively, our results indicate that phage defense may be a common function for TA systems and reveal the mechanism by which DarTG systems inhibit phage infection.

microbiology↗

bioRxiv2021

Bacterial gasdermins reveal an ancient mechanism of cell death

Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. Here we discover gasdermin homologs encoded in bacteria that execute prokaryotic cell death. Structures of bacterial gasdermins reveal a conserved pore-forming domain that is stabilized in the inactive state with a buried lipid modification. We demonstrate that bacterial gasdermins are activated by dedicated caspase-like proteases that catalyze site-specific cleavage and removal of an inhibitory C-terminal peptide. Release of autoinhibition induces the assembly of >200 [A] pores that form a mesh-like structure and disrupt membrane integrity. These results demonstrate that caspase-mediated activation of gasdermins is an ancient form of regulated cell death shared between bacteria and animals.

microbiology↗

bioRxiv2021

Antiviral defense via nucleotide depletion in bacteria

DNA viruses and retroviruses need to consume large quantities of deoxynucleotides (dNTPs) when replicating within infected cells. The human antiviral factor SAMHD1 takes advantage of this vulnerability in the viral life cycle, and inhibits viral replication by degrading dNTPs into their constituent deoxynucleosides and inorganic phosphate. In this study we report that bacteria employ a similar strategy to defend against phage infection. We found a family of defensive dCTP deaminase proteins that, in response to phage infection, convert dCTP into deoxy-uracil nucleotides. A second family of phage resistance genes encode dGTPase enzymes, which degrade dGTP into phosphate-free deoxy-guanosine (dG) and are distant homologs of the human SAMHD1. Our results show that the defensive proteins completely eliminate the specific deoxynucleotide (either dCTP or dGTP) from the nucleotide pool during phage infection, thus starving the phage of an essential DNA building block and halting its replication. Both defensive genes are found in a diverse set of bacterial species and are specifically enriched in Vibrio genomes. Our study demonstrates that manipulation of the deoxynucleotide pool is a potent antiviral strategy shared by both prokaryotes and eukaryotes.

microbiology↗

bioRxiv2021

Antiviral activity of bacterial TIR domains via signaling molecules that trigger cell death

The Toll/interleukin-1 receptor (TIR) domain is a canonical component of animal and plant immune systems. In plants, intracellular pathogen sensing by immune receptors triggers their TIR domains to generate a molecule which is a variant of cyclic ADP-ribose (v-cADPR). This molecule is hypothesized to activate plant cell death via a yet unresolved pathway. TIR domains were recently also shown to be involved in a bacterial anti-phage defense system called Thoeris, but the mechanism of Thoeris defense remained unknown. In this study we report that phage infection triggers Thoeris TIR-domain proteins to produce an isomer of cyclic ADP-ribose. This molecular signal activates a second protein, ThsA, which then depletes the cell of the essential molecule nicotinamide adenine dinucleotide (NAD) and leads to abortive infection and cell death. We further show that similar to eukaryotic innate immune systems, bacterial TIR-domain proteins determine the immunological specificity to the invading pathogen. Our results describe a new antiviral signaling pathway in bacteria, and suggest that generation of intracellular signaling molecules is an ancient immunological function of TIR domains conserved in both plant and bacterial immunity.

microbiology↗

bioRxiv2020

Bacterial retrons function in anti-phage defense

Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA. The RT uses the non-coding RNA as a template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. In this study we report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the non-coding RNA, and an effector protein. Retron-containing systems are abundant in genomic "defense islands", suggesting a role for most retrons in phage resistance. By cloning multiple retron systems into a retron-less Escherichia coli strain, we show that these systems confer defense against a broad range of phages, with different retrons defending against different phages. Focusing on a single retron, Ec48, we show evidence that it is a "guardian" of RecBCD, a complex with central anti-phage functions in the bacterial cell. Inhibition of RecBCD by dedicated phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed. Our results expose a new family of anti-phage defense systems abundant in bacteria.

microbiology↗

bioRxiv2019

The somatic genetic and epigenetic mutation rate in a wild long-lived perennial Populus trichocarpa

BackgroundPlants can transmit somatic mutations and epimutations to offspring, which in turn can affect fitness. Knowledge of the rate at which these variations arise is necessary to understand how plant development contributes to local adaption in an eco-evolutionary context, particularly in long-lived perennials. ResultsHere, we generated a new high-quality reference genome from the oldest branch of a wild Populus trichocarpa tree with two dominant stems which have been evolving independently for 330 years. By sampling multiple, age-estimated branches of this tree, we used a multi-omics approach to quantify age-related somatic changes at the genetic, epigenetic and transcriptional level. We show that the per-year somatic mutation and epimutation rates are lower than in annuals and that transcriptional variation is mainly independent of age divergence and cytosine methylation. Furthermore, a detailed analysis of the somatic epimutation spectrum indicates that transgenerationally heritable epimutations originate mainly from DNA methylation maintenance errors during mitotic rather than during meiotic cell divisions. ConclusionTaken together, our study provides unprecedented insights into the origin of nucleotide and functional variation in a long-lived perennial plant.

genomics↗

bioRxiv2019

Peptide-based quorum sensing systems in Paenibacillus polymyxa

Paenibacillus polymyxa is an agriculturally important plant growth-promoting rhizobacterium. Many Paenibacillus species are known to be engaged in complex bacteria-bacteria and bacteria-host interactions, which in other species were shown to necessitate quorum sensing communication. However, to date no quorum sensing systems have been described in Paenibacillus. Here we show that the type strain P. polymyxa ATCC 842 encodes at least 16 peptide-based communication systems. Each of these systems is comprised of a pro-peptide that is secreted to the growth medium and processed to generate a mature short peptide. Each peptide has a cognate intracellular receptor of the RRNPP family, and we show that external addition of P. polymyxa communication peptides leads to reprogramming of the transcriptional response. We found that these quorum sensing systems are conserved across hundreds of species belonging to the Paenibacillaceae family, with some species encoding more than 25 different peptide-receptor pairs, representing a record number of quorum sensing systems encoded in a single genome.

microbiology↗