Biology subjects
Publications and source records attributed to Nakamoto, J. A..
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Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless the ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in B. subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in an position analogous to that of S4-domain-containing RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC in facilitating the addition of polyalanine tails to the truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species the YlmH plays a central role in the RQC.
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
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.
Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods lead to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. CRISPR-Cas systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with L. (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 x 10-2 (kDNA) or 5 x 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.
The testis-determining factor (TDF) is an essential transcriptional protein for male differentiation in mammals, expressed along spermatids to early zygotes and, to some extent, in diverse cellular lines. In this study, we developed fluorescent biosensors capable of indicating the presence of TDF. We used in vitro evolution techniques to produce RNA aptamers that bind the recombinantly expressed HMG-box, the DNA binding domain of TDF. Bioinformatic analysis along in vitro evolution setup suggested two predominant aptamer clusters with distinctive motifs. The top ranked aptamer from each cluster, M1 and M2, showed specific binding to TDF. Aptamers were fluorescently modified as molecular beacons. Pre-steady-state kinetics indicated the beacons bind rapidly, within 50 seconds, yet M1 showed better signal to noise ratios than M2. Structural predictions of the aptamer interaction indicated that M1 is composed by three stem loops and likely interact with the HMG-box of TDF through the pocket formed by the three loops. Molecular modelling of M1 beacon shows that binding to TDF entails a conformational change of the sensor resulting in the measured fluorescence changes. To our knowledge, this is the first work describing an RNA beacon for detecting the essential TDF. Potential applications and advantages over alternative methods are provided and discussed.
During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of the stress signalling network termed the stringent response. Screening for (p)ppGpp-binding targets within Staphylococcus aureus identified four ribosome-associated GTPases (RA-GTPases), RsgA, RbgA, Era and HflX, each of which are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) states. Entry into the OFF-state from the ON-state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly. Here, we sought to determine how (p)ppGpp impacts RA-GTPase-ribosome interactions by examining the affinity and kinetics of binding between RA-GTPases and ribosomes in various nucleotide-bound states. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell. Binding to (p)ppGpp reduces stable association of RA-GTPases to ribosomal subunits compared to the GTP-bound state both in vitro and within bacterial cells by inducing the OFF-state conformation. We propose that in this conformation, the G2/switch I loop adopts a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated growth control.
Initiation factor 3 (IF3) is an essential protein that enhances the fidelity and speed of bacterial initiation of mRNA translation. The dynamic interplay between the two independent IF3 domains, their alternative binding sites, and the mechanism that ensures translation initiation fidelity remains elusive. Here, we show that the functional positioning of IF3 domains occurs at velocities ranging over two orders of magnitude, driven by each 30S initiation ligand. IF1 and IF2 rapidly promote the accommodation of IF3 on the 30S platform with the C-terminal domain moving towards the P site. Reversion of this movement is triggered by decoding the mRNA start codon and rate limits translation initiation. Binding of the tRNA results in the concomitant accommodation of the N-terminal domain of IF3, largely dependent on the mRNA and initiator tRNA. 70S initiation complex formation promotes the closing and dissociation of IF3, recycling the factor for a new round of translation initiation. Altogether our results unveil the kinetic spectrum of IF3 conformations and highlight fundamental movements of the factor that ensure accurate translation initiation.
Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable and steadily present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in less than 30 seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a potential biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection.