Biology subjects

Kranzusch, P. J.

Publications and source records attributed to Kranzusch, P. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Bacterial diadenylate cyclase domains synthesize diverse nucleotide signals in anti-phage defense

Bacterial diadenylate cyclase (DAC) enzymes synthesize the nucleotide signal 3'3' cyclic di-AMP (3'3'-c-di-AMP) to control osmoregulation, cell-wall homeostasis, and DNA-damage responses. Here we discover specialized roles for DAC enzymes in bacterial immunity and define a Panoptes-like system we name Panoptoo as a DAC-containing anti-phage defense that guards against viral immune evasion. The Panoptoo protein PanS is a minimal DAC that constitutively synthesizes 3'3' cyclic UMP-AMP (3'3'-cUA) or 3'3'-c-di-AMP to negatively regulate a partnering PanE S2TM{beta} membrane-targeting effector. We show that Panoptoo decoy signaling acts as a counter-defense to detect phage immune evasion proteins that inhibit nucleotide immune signals. A 1.5 [A] crystal structure of PanS in complex with 3'3'-cUA explains how a symmetry break in the canonical DAC active site enables synthesis of asymmetric signaling molecules. Together, our results uncover a role for DAC domains in bacterial anti-phage defense and expand our understanding of nucleotide signaling in antiviral immunity.

microbiology↗

bioRxiv2026

Human interferon stimulated genes target ancient features of animal and bacterial viral replication

Animal and bacterial cells defend against viral infection by rapidly activating antiviral restriction factors. In human cells, antiviral immunity is initiated by interferon signaling that results in expression of hundreds of interferon-stimulated genes (ISGs)1,2,3,4,5,6. Complex regulatory networks and co-evolution of viral evasion strategies complicate analysis of immune proteins under native conditions and the function of most individual ISGs remains unknown7,8,9,10,11,12. Here we discover that heterologous expression of ISGs in bacteria is sufficient to protect against infection by diverse bacteriophages demonstrating properties of antiviral restriction preserved across billions of years of viral evolution. A screen of 306 human ISGs against 11 E. coli phages reveals that ISGs can restrict phage replication with potency equal to endogenous bacterial defense systems. We select for phage mutants that escape ISG restriction and identify the phage DNA primase-helicase complex as a target of human SPSB1. A 2.0 [A] crystal structure of the SPSB1-primase complex uncovers a recognition mechanism of foreign DxNxN protein motifs found in many animal and bacterial viral replication proteins. We show that SPSB1 in human cells recognizes and induces degradation of protein targets containing this motif from norovirus and poxvirus pathogens. Our results establish a cross-kingdom approach to studying immune function and reveal that human ISGs target features of viral replication common across kingdoms of life.

immunology↗

bioRxiv2026

Phage protease enzymes activate CBASS antiphage immunity

Cyclic oligonucleotide-based antiphage signaling systems (CBASS) are immunity pathways in bacteria that use a cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme to sense phage infection and initiate antiviral defense. Bacteria encode thousands of diverse CBASS operons, demonstrating a critical role for CD-NTase activation in controlling the prokaryotic response to viral infection. Here we discover proteolytic cleavage by phage prohead proteases as a mechanism of CD-NTase activation and demonstrate that diverse CBASS operons function as molecular sensors of protease activity. We reconstitute CBASS recognition of phage T4 infection in vitro and identify proteolytic cleavage of a surface exposed CD-NTase activation loop as a trigger of enzyme catalysis and nucleotide immune signal synthesis. Phage prohead proteases are sufficient to activate CBASS in vivo and explain how immune signaling is initiated during late stages of viral infection. Combining biochemical and structure-based phylogenetic analyses, we map activation loops in Clade A, D, and G CD-NTase enzymes and define critical residues that control CBASS recognition of distinct phage families. Our results define CBASS recognition of phage protease activity as a widespread mechanism of antiviral defense.

microbiology↗

bioRxiv2025

Giant DNA viruses encode a hallmark translation initiation complex of eukaryotic life

In contrast to living organisms, viruses were long thought to lack protein synthesis machinery and instead depend on host factors to translate viral transcripts. Here, we discover that giant DNA viruses encode a distinct and functional IF4F translation initiation complex to drive protein synthesis, thereby blurring the line between cellular and acellular biology. During infection, eukaryotic IF4F on host ribosomes is replaced by an essential viral IF4F that regulates viral translation, virion formation, and replication plasticity during altered host states. Structural dissection of viral IF4F reveals that the mRNA cap-binding subunit mediates exclusive interactions with viral mRNAs, constituting a molecular switch from translating host to viral proteins. Thus, our study establishes that viruses express a eukaryotic translation initiation complex for protein synthesis, illuminating a series of evolutionary innovations to a core process of life.

microbiology↗

bioRxiv2025

A choanoflagellate cGLR-STING pathway reveals evolutionary links between bacterial and animal immunity

Animal innate immunity evolved from ancient pathways in bacterial anti-phage defense. How bacterial immune components were first acquired and adapted within eukaryotic cells remains poorly understood. Here we identify a complete cGLR-STING signaling axis in choanoflagellates, the closest living relatives of animals, that exhibits a mosaic of features from both bacterial and animal immunity. Comparative genomics reveals choanoflagellate cGLR and STING genes organized in operon-like arrangements reminiscent of bacterial defense loci. Reconstitution of choanoflagellate cGLR-STING signaling in vitro demonstrates that activation occurs through the conserved nucleotide immune signal 2'3'-cGAMP. Structural analysis of a choanoflagellate STING-2'3'-cGAMP complex explains how retention of bacterial-like features in early eukaryotic proteins shapes ligand specificity and receptor activation. We analyze cGLR and STING evolution in unicellular eukaryotes and identify further STING homologs in choanoflagellates and fungi that support additional independent acquisition events. Our results reveal molecular fossils that bridge bacterial and animal immunity and illuminate early eukaryotic immune system evolution.

immunology↗

bioRxiv2025

Structural basis of QueC-family protein function in qatABCD anti-phage defense

QueC proteins are nucleoside biosynthesis enzymes required for production of the 7-deazaguanine derivative queuosine. Recently, QueC-family proteins were also shown to catalyze a deazaguanylation protein-nucleobase conjugation reaction in type IV CBASS bacterial anti-phage defense. Here we determine the structural basis of QueC-family protein function in a distinct bacterial immunity system named qatABCD. We demonstrate that the QueC-family protein QatC forms a specific complex with the immunity protein QatB and that this complex is minimally required for qatABCD defense. Crystal structures of the QatBC complex enable direct comparison of qatABCD and type IV CBASS defense and support a shared role for QueC-family proteins in targeting protein substrates for N-terminal modification. We show that the QatB unstructured N-terminus and N-terminal glycine motif are essential for qatABCD defense in vivo, suggesting a modification occurs analogous to CBASS deazaguanylation. These findings highlight broad roles of QueC proteins beyond nucleoside biosynthesis and suggest that adaptation of QueC-like proteins for specialized biochemical functions is a common strategy in bacterial anti-phage immunity.

biochemistry↗

bioRxiv2025

Functional diversity of phage sponge proteins that sequester host immune signals

Multiple bacterial immune systems, including CBASS, Thoeris, and Pycsar, employ signaling molecules that activate the immune response following phage infection. Phages counteract bacterial immune signaling using sponge proteins that bind and sequester the immune signals, but the breadth of immune signals targeted by phage sponges is unclear. Here we study the functional versatility of Acb2, Tad1 and Tad2, three families of sponge proteins known to inhibit CBASS and Thoeris signaling. Eighty-four proteins representing the phylogenetic diversity of these sponge families were tested for their ability to inhibit immunity by sequestering 3'3'-cGAMP and 3'3'-cUA (CBASS), cCMP and cUMP (Pycsar), and 3'cADPR, His-ADPR and N7-cADPR (types I, II and IV Thoeris, respectively). While Acb2 proteins were so far reported to inhibit only CBASS systems, we found Acb2 homologs that bind 3'cADPR and inhibit Thoeris defense. In addition, we discovered sponge proteins that inhibit Pycsar and type IV Thoeris by binding cUMP and N7-cADPR, respectively. Using crystal structures, structural modeling and biochemical analyses, we explain the molecular basis for signal-binding specificities in members of these sponge families. Our study reports the first sponges inhibiting Pycsar and type IV Thoeris, and demonstrates how phage sponges evolve to obtain diverse specificities.

microbiology↗

bioRxiv2025

Nuclease-NTPase systems use shared molecular features to controlbacterial anti-phage defense

Bacteria encode an enormous diversity of defense systems including restriction-modification and CRISPR-Cas that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate many recently identified anti-phage defense operons are comprised of a predicted nuclease and an accessory NTPase protein, suggesting additional classes of nucleic acid targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyze 16 nuclease-NTPase systems and define shared features that control anti-phage defense. Purification, biochemical characterization, and in vitro reconstitution of nucleic acid targeting for each system demonstrate protein-protein complex formation is a universal feature of nuclease-NTPase systems and explain patterns of phage targeting and susceptibility. We show that some nuclease-NTPase systems use highly degenerate recognition site preferences to enable exceptionally broad nucleic acid degradation. Our results uncover shared principles of anti-phage defense system function and provide a foundation to explain the widespread role of nuclease-NTPase systems in bacterial immunity.

microbiology↗

bioRxiv2025

Activating and inhibiting nucleotide signals coordinate bacterial anti-phage defense

The cellular nucleotide pool is a major focal point of the host immune response to viral infection. Immune effector proteins that disrupt the nucleotide pool allow animal and bacterial cells to broadly restrict diverse viruses, but reduced nucleotide availability induces cellular toxicity and can limit host fitness(Ahmad et al., 1998; Goldstone et al., 2011; Hsueh et al., 2022; Itsko & Schaaper, 2014; Tal et al., 2022). Here we discover a bacterial anti-phage defense system named Clover that overcomes this tradeoff by encoding a deoxynucleoside triphosphohydrolase enzyme (CloA) that dynamically responds to both an activating phage cue and an inhibitory nucleotide immune signal produced by a partnering regulatory enzyme (CloB). Analysis of Clover phage restriction in cells and reconstitution of enzymatic function in vitro demonstrate that CloA is a dGTPase that responds to viral enzymes that increase cellular levels of dTTP. To restrain CloA activation in the absence of infection, we show that CloB synthesizes a dTTP-related inhibitory nucleotide signal p3diT (5'-triphosphothymidyl-3'5'-thymidine) that binds to CloA and suppresses activation. Cryo-EM structures of CloA in activated and suppressed states reveal how dTTP and p3diT control distinct allosteric sites and regulate effector function. Our results define how nucleotide signals coordinate both activation and inhibition of antiviral immunity and explain how cells balance defense and immune-mediated toxicity.

microbiology↗

bioRxiv2025

Deazaguanylation is a nucleobase-protein conjugation required for type IV CBASS immunity

7-deazapurines are nucleobase analogs essential for nucleic acid modifications in nearly all cellular life. Here, we discover a role for 7-deazapurines in protein modification within type IV CBASS anti-phage defense and define functions for CBASS ancillary proteins Cap9 and Cap10 in nucleobase-protein conjugation. A structure of Cap10 reveals a tRNA transglycosylase-family enzyme remodeled to bind the modified N-terminus of a partner cGAS/DncV-like nucleotidyltransferase linked to a 7-amido-7-deazaguanine (NDG) nucleobase. The structure of Cap9 explains how this QueC-like enzyme co-opts a 7-deazapurine biosynthetic reaction mechanism for NDG conjugation. We demonstrate that Cap9, Cap10, and NDG conjugation are essential for host defense against phage infection. Our results define a previously unknown 7-deazapurine protein modification and explain how nucleobase biosynthetic machinery has been repurposed for antiviral immunity.

microbiology↗

bioRxiv2024

A widespread family of viral sponge proteins reveals specific inhibition of nucleotide signals in anti-phage defense

Cyclic oligonucleotide-based antiviral signaling systems (CBASS) are bacterial anti-phage defense operons that use nucleotide signals to control immune activation. Here we biochemically screen 57 diverse E. coli and Bacillus phages for the ability to disrupt CBASS immunity and discover anti-CBASS 4 (Acb4) from the Bacillus phage SPO1 as the founding member of a large family of >1,300 immune evasion proteins. A 2.1 [A] crystal structure of Acb4 in complex with 3'3'-cGAMP reveals a tetrameric assembly that functions as a sponge to sequester CBASS signals and inhibit immune activation. We demonstrate Acb4 alone is sufficient to disrupt CBASS activation in vitro and enable immune evasion in vivo. Analyzing phages that infect diverse bacteria, we explain how Acb4 selectively targets nucleotide signals in host defense and avoids disruption of cellular homeostasis. Together, our results reveal principles of immune evasion protein evolution and explain a major mechanism phages use to inhibit host immunity.

microbiology↗

bioRxiv2024

Cell-free reconstitution of HIV-1 innate immune sensing reveals the capsid is a molecular cloak that protects reverse transcripts from cGAS

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect viral genomes from cGAS even after completing reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, the capsid inhibitor lenacapavir also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

microbiology↗

bioRxiv2024

TIR domains produce histidine-ADPR conjugates as immune signaling molecules in bacteria

TIR domains are central components of pattern recognition immune proteins across all domains of life. In both bacteria and plants, TIR-domain proteins were shown to recognize pathogen invasion and then produce immune signaling molecules exclusively comprising nucleotide moieties. Here we show that the TIR domain protein of the type II Thoeris defense system in bacteria produces a unique signaling molecule comprising the amino acid histidine conjugated to ADP-ribose (His-ADPR). His-ADPR is generated in response to phage infection and activates the cognate Thoeris effector by binding a Macro domain located at the C-terminus of the effector protein. By determining the crystal structure of a ligand-bound Macro domain, we describe the structural basis for His-ADPR recognition. Our analyses furthermore reveal a family of phage proteins that bind and sequester His-ADPR signaling molecules, allowing phages to evade TIR- mediated immunity. These data demonstrate diversity in bacterial TIR signaling and reveal a new class of TIR-derived immune signaling molecules combining nucleotide and amino acid moieties.

microbiology↗

bioRxiv2023

Bacterial SEAL domains undergo autoproteolysis and function in regulated intramembrane proteolysis

Gram-positive bacteria use SigI/RsgI-family sigma factor/anti-sigma factor pairs to sense and respond to cell wall defects and plant polysaccharides. In Bacillus subtilis this signal transduction pathway involves regulated intramembrane proteolysis (RIP) of the membrane-anchored anti-sigma factor RsgI. However, unlike most RIP signaling pathways, site-1 cleavage of RsgI on the extracytoplasmic side of the membrane is constitutive and the cleavage products remain stably associated, preventing intramembrane proteolysis. The regulated step in this pathway is their dissociation, which is hypothesized to involve mechanical force. Release of the ectodomain enables intramembrane cleavage by the RasP site-2 protease and activation of SigI. The constitutive site-1 protease has not been identified for any RsgI homolog. Here, we report that RsgIs extracytoplasmic domain has structural and functional similarities to eukaryotic SEA domains that undergo autoproteolysis and have been implicated in mechanotransduction. We show that site-1 proteolysis in B. subtilis and Clostridial RsgI family members is mediated by enzyme-independent autoproteolysis of these SEA-like (SEAL) domains. Importantly, the site of proteolysis enables retention of the ectodomain through an undisrupted {beta}-sheet that spans the two cleavage products. Autoproteolysis can be abrogated by relief of conformational strain in the scissile loop, in a mechanism analogous to eukaryotic SEA domains. Collectively, our data support the model that RsgI-SigI signaling is mediated by mechanotransduction in a manner that has striking parallels with eukaryotic mechanotransducive signaling pathways. SIGNIFICANCESEA domains are broadly conserved among eukaryotes but absent in bacteria. They are present on diverse membrane-anchored proteins some of which have been implicated in mechanotransducive signaling pathways. Many of these domains have been found to undergo autoproteolysis and remain noncovalently associated following cleavage. Their dissociation requires mechanical force. Here, we identify a family of bacterial SEA-like (SEAL) domains that arose independently from their eukaryotic counterparts but have structural and functional similarities. We show these SEAL domains autocleave and the cleavage products remain stably associated. Importantly, these domains are present on membrane-anchored anti-sigma factors that have been implicated in mechanotransduction pathways analogous to those in eukaryotes. Our findings suggest that bacterial and eukaryotic signaling systems have evolved a similar mechanism to transduce mechanical stimuli across the lipid bilayer.

microbiology↗

bioRxiv2023

CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

Caspase recruitment domains (CARDs) and pyrin domains are important facilitators of inflammasome activity and pyroptosis. Upon pathogen recognition by NLR proteins, CARDs recruit and activate caspases, which, in turn, activate gasdermin pore forming proteins to and induce pyroptotic cell death. Here we show that CARD-like domains are present in defense systems that protect bacteria against phage. The bacterial CARD is essential for protease-mediated activation of certain bacterial gasdermins, which promote cell death once phage infection is recognized. We further show that multiple anti-phage defense systems utilize CARD-like domains to activate a variety of cell death effectors. We find that these systems are triggered by a conserved immune evasion protein that phages use to overcome the bacterial defense system RexAB, demonstrating that phage proteins inhibiting one defense system can activate another. We also detect a phage protein with a predicted CARD-like structure that can inhibit the CARD-containing bacterial gasdermin system. Our results suggest that CARD domains represent an ancient component of innate immune systems conserved from bacteria to humans, and that CARD-dependent activation of gasdermins is conserved in organisms across the tree of life.

microbiology↗

bioRxiv2023

Convergent mutations in phage virion assembly proteins enable evasion of Type I CBASS immunity

CBASS is an anti-phage defense system that protects bacteria from phage infection and is evolutionarily related to human cGAS-STING immunity. cGAS-STING signaling is initiated by viral DNA but the stage of phage replication which activates bacterial CBASS remains unclear. Here we define the specificity of Type I CBASS immunity using a comprehensive analysis of 975 operon-phage pairings and show that Type I CBASS operons composed of distinct CD-NTases, and Cap effectors exhibit striking patterns of defense against dsDNA phages across five diverse viral families. We demonstrate that escaper phages evade CBASS immunity by acquiring mutations in structural genes encoding the prohead protease, capsid, and tail fiber proteins. Acquired CBASS resistance is highly operon-specific and typically does not affect overall fitness. However, we observe that some resistance mutations drastically alter phage infection kinetics. Our results define late-stage virus assembly as a critical determinant of CBASS immune activation and evasion by phages.

microbiology↗

bioRxiv2023

A novel virus-induced cyclic dinucleotide, 2'3'-c-di-GMP, mediates STING-dependent antiviral immunity in Drosophila

In mammals, the enzyme cGAS senses the presence of cytosolic DNA and synthesizes the cyclic dinucleotide (CDN) 2'3'-cGAMP. This CDN binds to and activates the protein STING to trigger immunity. We recently discovered in the model organism Drosophila melanogaster two cGAS-like receptors (cGLRs) that activate STING-dependent antiviral immunity and can produce 3'2'-cGAMP, in addition to 2'3'-cGAMP. Here we explore CDN-mediated immunity in 14 different Drosophila species covering 50 million years of evolution and report that 2'3'-cGAMP and 3'2'-cGAMP fail to control infection by Drosophila C virus in D. serrata, D. sechellia and D. mojavensis. Using an accurate and sensitive mass spectrometry method, we discover an unexpected diversity of CDNs produced in a cGLR-dependent manner in response to viral infection in D. melanogaster, including a novel CDN, 2'3'-c-di-GMP. We show that 2'3'-c-di-GMP is the most potent STING agonist identified so far in D. melanogaster and that this molecule also activates a strong antiviral transcriptional response in D. serrata. Our results shed light on the evolution of cGLRs in flies and provide a basis for the understanding of the function and regulation of this emerging family of PRRs in animal innate immunity.

immunology↗

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↗