Biology subjects

Kurata, T.

Publications and source records attributed to Kurata, T..

11 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

Nanobody-mediated modulation of long RSH enzymes Rel and RelA catalysis by restriction of their conformational landscape

Long RSH enzymes, Rel and RelA, are the master regulators of (p)ppGpp alarmone levels in bacteria. Their catalytic activity is governed by transitions between a compact, hydrolysis-competent (HDON) state and an elongated, synthesis-competent (SYNTHON) state. The equilibrium between these states is modulated by factors such as "starved" ribosomes and regulatory proteins DarB, EIIANTR, ACP and YtfK. Here, we identify and characterize camelid nanobodies that act as selective allosteric modulators by trapping Rel/RelA enzymes in distinct conformational states. Nanobodies that lock the TGS domain of RelA and prevent its activation by deacylated tRNA on starved ribosomes, strongly inhibit (p)ppGpp synthesis and suppress the virulence of E. coli in an animal model. Nb898 stabilizes Rel in the open SYNTHON state, enhancing synthesis activity while suppressing hydrolysis. Conversely, Nb585 traps Rel in a HDON conformation, strongly inhibiting alarmone synthesis while promoting (p)ppGpp hydrolysis. Structural and biochemical analyses reveal that nanobodies, like natural allosteric regulators, act by restricting the RSH enzymes conformational landscape. These findings establish nanobodies as powerful tools for dissecting RSH function and provide potential leads for developing protein-based RSH modulators.

microbiology↗

bioRxiv2024

Toxic Small Alarmone Synthetase FaRel2 inhibits translation by pyrophosphorylating tRNAGly and tRNAThr

Translation-targeting toxic Small Alarmone Synthetases (toxSAS) are effectors of bacterial Toxin-Antitoxin systems that pyrophosphorylate the 3-CCA end of tRNA to prevent aminoacylation. toxSAS are implicated in antiphage immunity: phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNAGly and tRNAThr. The 1st, 2nd, 4th and 5th base pairs the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRelSJ46 differ in tRNA specificity from FaRel2, and rationalise this through structural modelling: while the universal 3-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defences, we hypothesise that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

microbiology↗

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↗

bioRxiv2022

Establishment of Measles Virus Receptor-expressing Vero Cells Lacking Functional Poliovirus Receptors

Global efforts are underway to eliminate measles and rubella and active viral surveillance is key to achieving this goal. In addition to this, the World Health Organization announced guidelines for handling materials potentially infectious for poliovirus (PV) to minimize the risk of PV reintroduction and achieve PV eradication. To support the global efforts, we have established new PV-non-susceptible cell lines useful for the isolation of measles virus (MeV) and rubella virus (RuV) (Vero {Delta}PVR1/2 hSLAM+). In the cell lines, MeV and RuV replicated efficiently with no concern about PV replication.

microbiology↗

bioRxiv2022

Direct activation of an innate immune system in bacteria by a viral capsid protein

Bacteria have evolved sophisticated and diverse immunity mechanisms to protect themselves against a nearly constant onslaught of bacteriophages1-3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns (PAMPs)4, many bacterial immune systems that respond to bacteriophage infection require a phage-specific trigger to be activated. However, the identities of such triggers and the mechanistic basis of sensing remain almost completely unknown. Here, we discover and investigate the anti-phage function of a fused toxin-antitoxin (TA) system called CapRelSJ46 that protects E. coli against diverse phages. Through genetic, biochemical, and structural analysis, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both an antitoxin element and a phage-infection sensor. Following infection by certain phages, the newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to then pyrophosphorylate tRNAs, which blocks translation to restrict viral infection. Collectively, our results reveal the molecular mechanism by which a bacterial immune system directly senses a conserved, essential component of phages, suggesting a PAMP-like sensing model for TA-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a Red Queen conflict5, revealing a new front in the intense coevolutionary battle being waged by phage and bacteria. With capsid proteins of some eukaryotic viruses known to stimulate innate immune signaling in mammalian hosts6-10, our results now reveal an ancient, deeply conserved facet of immunity.

microbiology↗

bioRxiv2021

Panacea: a hyperpromiscuous antitoxin protein domain for the neutralisation of diverse toxin domains

Toxin-Antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids, as well as bacteriophages. They act as regulatory switches, with the toxin limiting the growth of bacteria and archaea by compromising diverse essential cellular targets, and the antitoxin counteracting the toxic effect. To uncover previously uncharted TA diversity across microbes and bacteriophages, we analysed the conservation of genomic neighbourhoods using our computational tool FlaGs (for Flanking Genes), which allows high-throughput detection of TA-like operons. Focussing on the widespread but poorly experimentally characterised antitoxin domain DUF4065, our in silico analyses indicated that DUF4065-containing proteins serve as broadly distributed antitoxin components in putative TA-like operons with dozens of different toxic domains with multiple different folds. Given the versatility of DUF4065, we have renamed the domain to Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralised TA pairs. While the majority of validated PanA-neutralised toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises replication and translation, as well as inducing RelA-dependent accumulation of the nucleotide alarmone (p)ppGpp. We find that Panacea-containing antitoxins form a complex with their diverse cognate toxins, characteristic of the direct neutralisation mechanisms employed by Type II TA systems. Finally, through directed evolution we have selected PanA variants that can neutralise non-cognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain. SignificanceToxin-antitoxin systems are enigmatic and diverse elements of bacterial and bacteriophage genomes. We have uncovered remarkable versatility of an antitoxin protein domain, that has evolved to neutralise dozens of different toxin domains. We find that antitoxins carrying this domain - Panacea - form complexes with their cognate toxins, indicating a direct neutralisation mechanism, and that Panacea can be evolved to neutralise a non-cognate and non-homologous toxin with just two amino acid substitutions. This raises the possibility that this domain could be an adaptable universal, or semi-universal protein neutraliser with significant biotechnological and medical potential.

microbiology↗

bioRxiv2021

RelA-SpoT Homologue toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of Small Alarmone Synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analogue of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest employed by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2 and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some Small Alarmone Hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as a novel class of RNA-modifying enzymes.

microbiology↗

bioRxiv2020

Subtilase activity in the intrusive cells mediates haustorium maturation in parasitic plants

Parasitic plants that infect crops are devastating to agriculture throughout the world. They develop a unique inducible organ called the haustorium, which connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically towards the host vasculature. Then, some of the intrusive cells re-differentiate to form a xylem bridge that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells are unknown. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-Sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and used promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of an SBT inhibitor in the intrusive cells inhibited their development, inhibited the development of the xylem bridge, and reduced auxin response levels near the site where the xylem bridge normally develops. Therefore, we propose that subtilase activity plays an important role in haustorium development in this parasitic plant. One sentence summaryTissue-specific analysis showed that the subtilases specifically expressed in intrusive cells regulate auxin-mediated host-parasite connections in the parasitic plant Phtheirospermum japonicum.

plant biology↗