Biology subjects

Marraffini, L. A.

Publications and source records attributed to Marraffini, L. A..

15 recordsLinked to original sources

bioRxiv2026

A lipid fenestration-gated membrane depolarization mechanism expands the repertoire of CRISPR-mediated anti-phage defense strategies

Prokaryotic Type III CRISPR-Cas systems synthesize cyclic oligoadenylate (cOA) second messengers that activate CRISPR-associated Rossmann fold (CARF) immune effectors during viral infection. Here we characterize Chp1, a membrane-embedded CARF effector that assembles as a tetrameric pore harboring autoinhibitory lipid fenestrations that occlude the pore in the inactive state. cOA binding triggers a structural rearrangement that (i) closes the fenestrations, (ii) eliminates the lipid obstruction of the pore to open it, and (iii) remodels the pore entrance from hydrophobic to polar to allow ion permeation. This conformational switch triggers depolarization of the membrane of the infected cell, which enters growth arrest and becomes inhospitable for viral replication. Our results uncover a lipid-gated mechanism that expands the repertoire of CRISPR-mediated defense strategies.

microbiology↗

bioRxiv2026

Mre11-Rad50 enhances spacer acquisition in a haloarchaeal Type I-B CRISPR-Cas system

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (cas) genes provide adaptive immunity to bacteria and archaea. CRISPR-Cas systems acquire short DNA fragments from the genomes of infecting plasmids and viruses, which are inserted into the CRISPR locus as a "spacer" sequence in between repeats. Spacers constitute a memory of infection that is used to recognize and attack invading genetic elements in future infections. Despite the evolutionarily divergent genetic backgrounds of bacteria and archaea, the same CRISPR-Cas systems are functional in both of these prokaryotic domains. In bacteria, efficient spacer acquisition requires the DNA repair nucleases RecBCD/AddAB. These nucleases, however, are not present in archaea. Here we investigated the importance of the DNA repair systems in the Haloferax volcanii Type I-B CRISPR-Cas response. We found that elimination of the DNA repair nuclease Mre11-Rad50, but not Fen1, substantially reduces spacer acquisition. CRISPR immunity against H. volcanii pleomorphic virus 1 (HFPV-1), on the other hand, was not affected by these deletions. Our results describe how CRISPR-Cas systems have adapted to provide anti-viral defense to hosts from different domains of life.

microbiology↗

bioRxiv2026

A 5-hydroxymethylcytosine DNA glycosylase provides defense against T-even bacteriophages

The most abundant prokaryotic mechanisms of defense against phage predation involve the recognition and destruction of the infecting DNA. One method of counter-defense is the incorporation of modified nucleobases into the phage genome to avoid interaction with enzymes that target the viral DNA. T-even coliphages replace cytosine with 5-hydroxymethylcytosine (5hmC) that in some cases are further decorated with glucosyl groups. To explore the diversity of immunity genes that recognize 5hmC, we infected a library of metagenomic DNA inserts from uncultured, non-sequenced soil bacteria with a mutant T4 phage that harbored only non-glucosylated 5hmC on its genome. Bacteria that resisted infection carried a DNA glycosylase, Brig3, that specifically excises 5hmC nucleobases to generate abasic sites in the phage genome and prevent viral proliferation. The crystal structure of Brig3 bound to its substrate revealed a catalytic mechanism in which the 5hmC nucleobase is flipped out of the DNA into the active site and replaced by an asparagine residue that inserts into the double helix to contact the complementary guanosine. Brig3 is encoded within an operon that also encodes BapA, a hydrolase that removes glucosyl groups from glucosyl-5hmC present in the genome of otherwise Brig3-resistant T-even phages carrying this hypermodified base. Our results uncover a defense strategy in which the combined action of BapA and Brig3 widens the immune response to restrict the infection of T-even phages with genomes that are either partially or completely glucosylated.

microbiology↗

bioRxiv2025

Cap1 forms a cyclic tetra-adenylate-induced membrane pore during the type III-A CRISPR-Cas immune response

Type III CRISPR-Cas systems synthesize cyclic oligoadenylates (cOAs) signaling molecules upon RNA-guided recognition of viral (phage) transcripts. cOAs bind to and activate CARF effectors, many of which contain transmembrane domains that cause membrane depolarization to prevent phage propagation. How ligand binding induces the activity of these effectors is poorly understood at the molecular level. Here we report the structure and function of the Cap1 effector, composed of a pair of transmembrane helices (TM1/2), a CARF-like (CARFL) domain and a domain of unknown function (DUF4579). In vivo, Cap1 activation results in membrane depolarization, a growth arrest of the bacterial host and the abrogation of the viral lytic cycle. Cryo-EM studies on apo- and cOA-bound states of Cap1 in glyco-diosgenin detergent revealed the formation of tetrameric complexes in both states, with one cOA molecule bound in a pocket composed of four CARFL domains. Ligand binding triggers conformational changes that widen an otherwise narrow transmembrane pore formed by the four TM1/2 domains. Our findings reveal at the atomic level the mechanism of pore opening and membrane depolarization mediated by cyclic nucleotide signaling during the type III CRISPR-Cas response.

microbiology↗

bioRxiv2025

A CARF-HAD phosphatase effector provides immunity during the type III-A CRISPR-Cas response

CRISPR-Cas systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, Type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain, which we named Chp. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.

microbiology↗

bioRxiv2024

Deep mutational scanning identifies variants of Cas1 and Cas2 that increase spacer acquisition in type II-A CRISPR-Cas systems

A remarkable feature of CRISPR-Cas systems is their ability to acquire short sequences from invading viruses to create a molecular record of infection. These sequences, known as spacers, are inserted into the CRISPR locus by the Cas1-Cas2 integrase complex and mediate sequence- specific immunity in prokaryotes. Spacer acquisition has been used to develop unique biotechnological applications such as the immunization of industrially relevant bacteria against bacteriophage infection and the recording of biological signals into stable genetic information. These technologies, however, are constrained by the low efficiency of the spacer acquisition process. To overcome this limitation, we developed a genetic system that combined deep mutational scanning (DMS) of cas genes from the Streptococcus pyogenes type II-A CRISPR- Cas system with a method that selects bacteria that acquire new spacers. This procedure enabled the identification of cas mutations that support up to a sevenfold increase in the levels of spacer acquisition and a significant enhancement of immunity against phage infection. In addition, our analysis revealed key interactions at the Cas1-Cas2 interface critical for spacer integration. Our findings provide insights into the molecular determinants of spacer acquisition and offer a platform to improve CRISPR-Cas-based applications.

microbiology↗

bioRxiv2024

MtvS Regulates the Francisella Type V-A CRISPR-Cas System

Bacteria and archaea often harbor multiple CRISPR-Cas loci to defend against mobile genetic elements. Little is known, however, about whether and how different CRISPR-Cas systems are differentially regulated, in many instances due to the impossibility of studying CRISPR immunity in native hosts. Here we investigated the regulation of the endogenous type II-B and type V-A CRISPR-Cas systems present in the opportunistic human pathogen Francisella novicida U112. We found that while the type II-B system is constitutively expressed, the type V-A system is differentially expressed at stationary phase and high cell density. We identified MtvS1 and MtvS2 as factors required for this regulation, as well as for the modulation of many additional genes in stationary phase, some of which are required for Francisella virulence. Both Francisella MtvS proteins bind to RNA polymerase. MtvS1 is predicted to interact with the {beta} subunit of the RNA polymerase, and MtvS2 with multiple RNA polymerase subunits as well as MtvS1. We propose that MtvS1 and MtvS2 constitute noncanonical alternative sigma factors involved in the regulation of the expression of the type V-A CRISPR locus and other genes in Francisella. Last, we show that the MtvS1 homolog YgfB is required for expression of the type I-E CRISPR-Cas system in E. coli, a result that suggests a broader role in gene regulation for these alternative sigma factors.

microbiology↗

bioRxiv2024

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 {Phi}80 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.

microbiology↗

bioRxiv2024

The Cas10 nuclease activity relieves host dormancy to facilitate spacer acquisition and retention during type III-A CRISPR immunity

A hallmark of CRISPR immunity is the acquisition of short viral DNA sequences, known as spacers, that are transcribed into guide RNAs to recognize complementary sequences. The staphylococcal type III-A CRISPR-Cas system uses guide RNAs to locate viral transcripts and start a response that displays two mechanisms of immunity. When immunity is triggered by an early-expressed phage RNA, degradation of viral ssDNA can cure the host from infection. In contrast, when the RNA guide targets a late-expressed transcript, defense requires the activity of Csm6, a non-specific RNase. Here we show that Csm6 triggers a growth arrest of the host that provides immunity at the population level which hinders viral propagation to allow the replication of non-infected cells. We demonstrate that this mechanism leads to defense against not only the target phage but also other viruses present in the population that fail to replicate in the arrested cells. On the other hand, dormancy limits the acquisition and retention of spacers that trigger it. We found that the ssDNase activity of type III-A systems is required for the re-growth of a subset of the arrested cells, presumably through the degradation of the phage DNA, ending target transcription and inactivating the immune response. Altogether, our work reveals a built-in mechanism within type III-A CRISPR-Cas systems that allows the exit from dormancy needed for the subsistence of spacers that provide broad-spectrum immunity.

microbiology↗

bioRxiv2023

Bacterial cGAS senses a viral RNA to initiate immunity

CBASS immunity protects prokaryotes from viral (phage) attack through the production of cyclic dinucleotides which activate effector proteins that trigger the death of the infected host. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a highly structured 400-nt RNA, termed CBASS-activating bacteriophage RNA (cabRNA), that binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP. Phages that escape CBASS immunity harbor mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. Since the mammalian cyclase OAS1 also binds viral dsRNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defense pathways.

microbiology↗

bioRxiv2020

Integration of prophages into CRISPR loci remodels viral immunity in Streptococcus pyogenes

CRISPR loci are composed of short DNA repeats separated by sequences that match the genomes of phages and plasmids, known as spacers. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defense, phages can produce small proteins that inhibit these nucleases. Here we demonstrate that the {Phi}AP1.1 temperate phage utilizes an alternate approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection this phage expresses a canonical anti-CRISPR, AcrIIA23 that prevents Cas9 function, allowing {Phi}AP1.1 to integrate into the direct repeats of the CRISPR locus and neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transfer of spacers, enabling a complex modulation of the type II-A CRISPR immune response.

microbiology↗

bioRxiv2020

Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6

Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers to activate downstream effectors including Csm6-family ribonucleases via their CARF domains. Here we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator and report the crystal structure of EiCsm6 bound to a cA6 mimic. The structure, combined with biochemical and in vivo functional assays, reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.

biochemistry↗

bioRxiv2019

Expansion of CRISPR loci with multiple memories of infection enables the survival of structured bacterial communities

Type II CRISPR-Cas systems provide immunity against phages and plasmids that infect bacteria. Following infection, a short sequence of the phage genome known as the \"spacer\" is inserted into the CRISPR locus to capture a memory of the infection and immunize the host. Spacers are transcribed and processed into guide RNAs that direct the Cas9 nuclease to its target on the invader. Thousands of spacers are acquired to target the viral genome at multiple locations and neutralize phage mutants that evade the immunity specified by a single guide RNA. In liquid cultures, where phages and their hosts are constantly mixed, spacer diversity is generated at the population level, and a single immunization per cell is sufficient to confer robust immunity. Although rare, bacteria that acquire multiple spacers can also be found, demonstrating that type II CRISPR-Cas systems also have the capability of generating spacer diversity at the cellular level. However, conditions in which this feature is important for survival are poorly understood. Here we found that when phage infections occur on solid media, a high proportion of the surviving colonies display sectored morphologies that contain individual cells with multiple spacers. We show that this is the result of the bacteria-host co-evolution, in which the immunity provided by the initial acquired spacer is easily overcome by escaper phages that decimate all the progeny of the founder cell that do not acquire additional spacers. Our results reveal the versatility of type II CRISPR-Cas immunity, which can respond with both single or multiple spacer acquisition schemes to solve challenges presented by different environments.

microbiology↗

bioRxiv2018

Dynamics of Cas10 Govern Discrimination between Self and Nonself in Type III CRISPR-Cas Immunity

Adaptive immune systems are required to accurately distinguish between self and nonself in order to defend against invading pathogens while avoiding autoimmunity. Type III CRISPR-Cas systems employ guide RNAs that recognize complementary RNA molecules to trigger the degradation of both the target transcript and its template DNA. These systems can broadly eliminate foreign targets with multiple mutations, but still effectively curb immunity against the host. The molecular basis for these unique features remains unknown. Here we use single-molecule fluorescence microscopy to study the interaction between a type III-A ribonucleoprotein complex and various RNA substrates. We find that Cas10--the DNase effector of the complex--displays rapid conformational fluctuations on foreign RNA targets, but is locked in a static configuration on self RNA. Single-stranded DNA promotes Cas10s occupancy at a selected set of conformational states, which is also sensitively modulated by target mutations and predictive of CRISPR interference activity. These findings highlight the central role of the internal dynamics of CRISPR-Cas complexes in self/nonself discrimination and target specificity.

biophysics↗

bioRxiv2018

Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks.

The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double strand break (DSB), Cas9 remains stably bound to it. Here we show persistent Cas9 binding blocks access to DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches one direction towards the Cas9-DSB complex. By exploiting these RNA polymerase-Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of PAM sequences and a simple method of improving selection of highly active sgRNA for genome editing.

molecular biology↗