Biology subjects

Shyrokova, L.

Publications and source records attributed to Shyrokova, L..

6 recordsLinked to original sources

bioRxiv2025

Characterization of five environmental phages infecting Escherichia coli K-12 isolated during a phage biology training course

Phage collections are essential tools for discovering and dissecting bacterial anti-phage defense systems. Here, we report the isolation and characterization of five environmental Escherichia coli-infecting phages, obtained during the 2023 Fundamentals of Basic and Applied Phage Biology course at Lund University. The phages were isolated using a motile E. coli K-12 BW25113 strain, whose motility is conferred by an IS5 insertion upstream of the flhDC operon, the master regulator of flagellar synthesis. The isolated Escherichia phages include Lubas (LuPh1) and Lucat (LuPh2) of the genus Tequatrovirus; Lupin (LuPh3) and Lucris (LuPh4) of the genus Tequintavirus; and Kompetensportalen (LuPh5) of the genus Chivirus. Transmission electron microscopy confirmed myovirus and siphovirus morphologies consistent with these genera. As expected for phages in the flagellotropic Chivirus genus, LuPh5 failed to infect a poorly motile BW25113 strain lacking the IS5 element upstream of flhDC. By testing a panel of eight previously described anti-phage defense systems, we found that LuPh1 and LuPh2 are inhibited by the toxin-antitoxin-chaperone CmdTAC system; LuPh5 is inhibited by both the restriction-modification system EcoRI and the abortive infection reverse transcriptase AbiK; and all five phages are sensitive to the hybrid artificial CmdTA-HigC system. Collectively, our findings expand the toolkit for probing phage-host interactions and underscore the pedagogical value of incorporating phage isolation into practical training for emerging researchers.

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

The Viral AlphaFold Database of monomers and homodimers reveals conserved protein folds in viruses of bacteria, archaea, and eukaryotes

Viruses are among the most abundant and genetically diverse entities on Earth, yet the functions and evolutionary origins of most viral proteins remain poorly understood. Their rapid evolution often obscures evolutionary relationships, making it difficult to assign functions using sequence-based methods alone. Although conservation of protein fold can reveal deep homologies undetectable by sequence comparison, viral proteins remain vastly underrepresented in structural databases, limiting our ability to explore them at the structural level. Here, we address this gap by clustering all unique viral sequences from the NCBI RefSeq database and predicting the structures of [~]27,000 representative proteins using AlphaFold2, creating a large-scale viral structural resource, the Viral AlphaFold Database (VAD). We uncover [~]10,000 proteins belonging to clusters that share folds across viruses infecting bacteria, archaea, and eukaryotes, revealing shared protein folds across diverse host-infecting viruses. We also predict oligomeric states using AlphaFold2-based homodimer modelling, alongside structural comparisons to the Protein Data Bank, providing valuable new data on the potential for viral proteins to oligomerise. We further reveal that large regions of the viral protein universe remain functionally dark and report the discovery and experimental validation of a previously uncharacterised antiviral toxin-antitoxin (TA) system. VAD is a resource that provides a foundation for exploring viral structure-function relationships, including ancient folds that shape viral interactions across all life. Predicted structures used in this study are available at data-sharing.atkinson-lab.com/vad/.

molecular biology↗

bioRxiv2024

Mechanism of phage sensing and abortion by toxin-antitoxin-chaperone systems

Toxin-antitoxins (TAs) are prokaryotic two-gene systems comprised of a toxin neutralised by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilises the antitoxin by recognising its chaperone addiction (ChAD) element. TACs have been shown to mediate antiphage defence, but the mechanisms of viral sensing and restriction are unexplored. We identify and characterise two Escherichia coli antiphage TAC systems containing HigBA and CmdTA TA units, HigBAC and CmdTAC. The HigBAC is triggered through recognition of the gpV major tail protein of phage {lambda}. Both the ChAD and gpV are recognised by the HigC chaperone through analogous aromatic molecular patterns, explaining the mechanism of activation. We show that the CmdT ADP-ribosyltransferase toxin modifies mRNA to shut down protein synthesis. We establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with the HigC chaperone phage sensor. HighlightsE. coli HigBAC and CmdTAC are translation-targeting phage immunity TAC systems HigC chaperone recognises phage {lambda} major tail protein to trigger HigBAC toxicity CmdT ADP-ribosyltransferase toxin abrogates translation through modification of mRNA HigC combined with CmdTA yields hybrid broad-spectrum antiphage defence system

microbiology↗

bioRxiv2024

Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

biochemistry↗

bioRxiv2023

The structural basis of hyperpromiscuity in a core combinatorial network of Type II toxin-antitoxin and related phage defence systems

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralising antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins) we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralisation by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralising mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralisation, while in multidomain antitoxins the neutralisation is mediated by other domains, such as PAD1, Phd-C and ZFD. We hypothesise that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 new NetFlax TA systems. We used functional domain annotations and with metabolic labelling assays to predict their potential mechanisms of toxicity (such as disruption of membrane integrity, inhibition of cell division and abrogation of protein synthesis) as well as biological functions (such as antiphage defence). The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/. Significance statementToxin-antitoxin systems are enigmatic components of microbial genomes, with their biological functions being a conundrum of debate for decades. Increasingly, TAs are being found to have a role in defence against bacteriophages. By mapping and experimentally validating a core combinatorial network of TA systems and high-throughput prediction of structural interfaces, we uncover the evolutionary scale of TA partner swapping and discover new toxic effectors. We validate the predicted toxin:antitoxin complex interfaces of four TA systems, uncovering the evolutionary malleable mechanism of toxin neutralisation by Panacea-containing PanA antitoxins. We find TAs are evolutionarily related to several other phage defence systems, cementing their role as important molecular components of the arsenal of microbial warfare.

microbiology↗