Biology subjects

Tamulaitiene, G.

Publications and source records attributed to Tamulaitiene, G..

6 recordsLinked to original sources

bioRxiv2026

Chemical suppression of a bacterial immune system revives repressed phages

Many antiviral immune systems have recently been discovered in bacteria. The mechanisms of several are obscure, as are their individual significance for antiphage defense. To shed light on the mechanism and significance of the two-component type I Thoeris antiphage immune system, we leveraged high-throughput phenotypic screening to identify three small molecule inhibitors. The inhibitors target the ThsA NADase component, inhibiting its 3'-cADPR-activated filamentation. The temporal control afforded by the small-molecule inhibitors allowed us to answer an outstanding question in antiviral immunity--is persistent immunity required to repress phage titers, or do immune systems become unnecessary after eradicating infectious phages? We found that Thoeris immunity must be maintained, as chemical inhibition enabled repressed phages to revive and overtake the bacterial population. Furthermore, due to the cooperative nature of antiviral immunity, we found that Thoeris must be inhibited in only 10% of the bacteria to cause phage-induced lysis of the entire population.

biochemistry↗

bioRxiv2026

Deoxydinucleotides activate the bacterial anti-phage defense system ApeA

Bacteria and archaea encode diverse antiviral defense systems, many of which rely on toxic effector proteins that are activated specifically upon bacteriophage infection. However, the mechanisms by which infection is recognized and coupled to effector activation remain poorly understood for most antiviral systems. Here, we focus on ApeA, a HEPN-domain antiviral protein that confers immunity through cleavage of host tRNAs within their anticodon loops. We show that ApeA proteins form large doughnut-shaped oligomers that are activated upon ligand binding in a conserved protein pocket distinct from the catalytic center. In the Ec2ApeA variant, this pocket specifically recognizes 5'-phosphorylated deoxydinucleotides that likely arise as intermediates of host genome degradation by viral nucleases, thereby enabling Ec2ApeA to achieve a broad protection profile. Together, our results reveal how small-molecule products of virus-induced host cell destruction function as signals that activate bacterial immune defenses.

molecular biology↗

bioRxiv2025

Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation

During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins like Cas4, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a supercomplex with the Cas1-Cas2 integrase and the Csn2 protein. However, the supercomplex structure and the role of the ring-like Csn2 protein remain unknown. Here, we present cryo-electron microscopy structures of the type II-A prespacer selection supercomplex in the DNA-scanning and two different PAM-bound configurations. Our study uncovers the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems, and reveals the role of the accessory protein Csn2, which serves as a platform for the assembly of Cas9 and Cas1-Cas2 integrase on prespacer DNA, reminiscent of the sliding clamp in DNA replication. Repurposing of Cas9 by the CRISPR adaptation machinery for prespacer selection characterized here demonstrates Cas9 plasticity and expands our knowledge of the Cas9 biology.

microbiology↗

bioRxiv2025

Chemical inhibition of a bacterial immune system

The rise of antibiotic resistance motivates a revived interest in phage therapy. However, bacteria possess dozens of anti-bacteriophage immune systems that confer resistance to therapeutic phages. Chemical inhibitors of these anti-phage immune systems could be employed as adjuvants to overcome resistance in phage-based therapies. Here, we report that anti-phage systems can be selectively inhibited by small molecules, thereby sensitizing phage-resistant bacteria to phages. We discovered a class of chemical inhibitors that inhibit the type II Thoeris anti-phage immune system. These inhibitors block the biosynthesis of a histidine-ADPR intracellular alarm signal by ThsB and prevent ThsA from arresting phage replication. These inhibitors promiscuously inhibit type II Thoeris systems from diverse bacteria--including antibiotic-resistant pathogens. Chemical inhibition of the Thoeris defense improved the efficacy of a model phage therapy against a phage-resistant strain of P. aeruginosa in a mouse infection, suggesting a therapeutic potential. Furthermore, these inhibitors may be employed as chemical tools to dissect the importance of the Thoeris system for phage defense in natural microbial communities.

biochemistry↗

bioRxiv2024

Filament formation activates protease and ring nuclease activities of CRISPR SAVED-Lon

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cAn) signaling to activate various auxiliary effectors, including the CRISPR-associated SAVED-Lon protease CalpL, which forms a tripartite effector system together with an anti-{sigma} factor, CalpT, and an ECF-like {sigma} factor, CalpS. Here we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E. coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.

biochemistry↗

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↗