| 1. |
- Ernits, Karin, et al.
(författare)
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The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin-antitoxin and related phage defense systems
- 2023
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - 1091-6490 .- 0027-8424. ; 120:33, s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- 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 neutralizing 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 neutralization 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 neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.
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| 2. |
- Jimmy, Steffi, et al.
(författare)
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A widespread toxin-antitoxin system exploiting growth control via alarmone signaling
- 2020
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:19, s. 10500-10510
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Tidskriftsartikel (refereegranskat)abstract
- Under stressful conditions, bacterial RelA-SpoT Homolog (RSH) enzymes synthesize the alarmone (p)ppGpp, a nucleotide second messenger. (p)ppGpp rewires bacterial transcription and metabolism to cope with stress, and, at high concentrations, inhibits the process of protein synthesis and bacterial growth to save and redirect resources until conditions improve. Single-domain small alarmone synthetases (SASs) are RSH family members that contain the (p)ppGpp synthesis (SYNTH) domain, but lack the hydrolysis (HD) domain and regulatory C-terminal domains of the long RSHs such as Rel, RelA, and SpoT. We asked whether analysis of the genomic context of SASs can indicate possible functional roles. Indeed, multiple SAS subfamilies are encoded in widespread conserved bicistronic operon architectures that are reminiscent of those typically seen in toxin-antitoxin (TA) operons. We have validated five of these SASs as being toxic (toxSASs), with neutralization by the protein products of six neighboring antitoxin genes. The toxicity of Cellulomonas marina toxSAS FaRel is mediated by the accumulation of alarmones ppGpp and ppApp, and an associated depletion of cellular guanosine triphosphate and adenosine triphosphate pools, and is counteracted by its HD domain-containing antitoxin. Thus, the ToxSAS-antiToxSAS system with its multiple different antitoxins exemplifies how ancient nucleotide-based signaling mechanisms can be repurposed as TA modules during evolution, potentially multiple times independently.
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| 3. |
- Kurata, Tatsuaki, et al.
(författare)
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A hyperpromiscuous antitoxin protein domain for the neutralization of diverse toxin domains
- 2022
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 119:6
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Tidskriftsartikel (refereegranskat)abstract
- Toxin–antitoxin (TA) gene pairs are ubiquitous in microbial chromosomal genomes and plasmids as well as temperate 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 analyzed the conservation of genomic neighborhoods using our computational tool FlaGs (for flanking genes), which allows high-throughput detection of TA-like operons. Focusing on the widespread but poorly experimentally characterized 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 named the domain Panacea (and proteins containing the domain, PanA) after the Greek goddess of universal remedy. We have experimentally validated nine PanA-neutralized TA pairs. While the majority of validated PanA-neutralized toxins act as translation inhibitors or membrane disruptors, a putative nucleotide cyclase toxin from a Burkholderia prophage compromises transcription 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 neutralization mechanisms employed by Type II TA systems. Finally, through directed evolution, we have selected PanA variants that can neutralize noncognate TA toxins, thus experimentally demonstrating the evolutionary plasticity of this hyperpromiscuous antitoxin domain.
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| 4. |
- Mangano, Kyle, et al.
(författare)
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Context-based sensing of orthosomycin antibiotics by the translating ribosome
- 2022
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Ingår i: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 18:11, s. 1277-1286
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Tidskriftsartikel (refereegranskat)abstract
- Orthosomycin antibiotics inhibit protein synthesis by binding to the large ribosomal subunit in the tRNA accommodation corridor, which is traversed by incoming aminoacyl-tRNAs. Structural and biochemical studies suggested that orthosomycins block accommodation of any aminoacyl-tRNAs in the ribosomal A-site. However, the mode of action of orthosomycins in vivo remained unknown. Here, by carrying out genome-wide analysis of antibiotic action in bacterial cells, we discovered that orthosomycins primarily inhibit the ribosomes engaged in translation of specific amino acid sequences. Our results reveal that the predominant sites of orthosomycin-induced translation arrest are defined by the nature of the incoming aminoacyl-tRNA and likely by the identity of the two C-terminal amino acid residues of the nascent protein. We show that nature exploits this antibiotic-sensing mechanism for directing programmed ribosome stalling within the regulatory open reading frame, which may control expression of an orthosomycin-resistance gene in a variety of bacterial species.
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| 5. |
- Mohamad, Merianne, et al.
(författare)
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Sal-type ABC-F proteins : intrinsic and common mediators of pleuromutilin resistance by target protection in staphylococci
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:4, s. 2128-2142
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Tidskriftsartikel (refereegranskat)abstract
- The first member of the pleuromutilin (PLM) class suitable for systemic antibacterial chemotherapy in humans recently entered clinical use, underscoring the need to better understand mechanisms of PLM resistance in disease-causing bacterial genera. Of the proteins reported to mediate PLM resistance in staphylococci, the least-well studied to date is Sal(A), a putative ABC-F NTPase that—by analogy to other proteins of this type—may act to protect the ribosome from PLMs. Here, we establish the importance of Sal proteins as a common source of PLM resistance across multiple species of staphylococci. Sal(A) is revealed as but one member of a larger group of Sal-type ABC-F proteins that vary considerably in their ability to mediate resistance to PLMs and other antibiotics. We find that specific sal genes are intrinsic to particular staphylococcal species, and show that this gene family is likely ancestral to the genus Staphylococcus. Finally, we solve the cryo-EM structure of a representative Sal-type protein (Sal(B)) in complex with the staphylococcal 70S ribosome, revealing that Sal-type proteins bind into the E site to mediate target protection, likely by displacing PLMs and other antibiotics via an allosteric mechanism.
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| 6. |
- Murina, Victoriia, et al.
(författare)
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ABCF ATPases Involved in Protein Synthesis, Ribosome Assembly and Antibiotic Resistance : Structural and Functional Diversification across the Tree of Life
- 2019
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Ingår i: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 431:18, s. 3568-3590
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Tidskriftsartikel (refereegranskat)abstract
- Within the larger ABC superfamily of ATPases, ABCF family members eEF3 in Saccharomyces cerevisiae and EttA in Escherichia coli have been found to function as ribosomal translation factors. Several other ABCFs including biochemically characterized VgaA, LsaA and MsrE confer resistance to antibiotics that target the peptidyl transferase center and exit tunnel of the ribosome. However, the diversity of ABCF subfamilies, the relationships among subfamilies and the evolution of antibiotic resistance (ARE) factors from other ABCFs have not been explored. To address this, we analyzed the presence of ABCFs and their domain architectures in 4505 genomes across the tree of life. We find 45 distinct subfamilies of ABCFs that are widespread across bacterial and eukaryotic phyla, suggesting that they were present in the last common ancestor of both. Surprisingly, currently known ARE ABCFs are not confined to a distinct lineage of the ABCF family tree, suggesting that ARE can readily evolve from other ABCF functions. Our data suggest that there are a number of previously unidentified ARE ABCFs in antibiotic producers and important human pathogens. We also find that ATPase-deficient mutants of all four E. coli ABCFs (EttA, YbiT, YheS and Uup) inhibit protein synthesis, indicative of their ribosomal function, and demonstrate a genetic interaction of ABCFs Uup and YheS with translational GTPase BipA involved in assembly of the 50S ribosome subunit. Finally, we show that the ribosome-binding resistance factor VmlR from Bacillus subtilis is localized to the cytoplasm, ruling out a role in antibiotic efflux.
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| 7. |
- Palmer, Tracy, et al.
(författare)
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A holin/peptidoglycan hydrolase-dependent protein secretion system
- 2021
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Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 115:3, s. 345-355
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Tidskriftsartikel (refereegranskat)abstract
- Gram-negative bacteria have evolved numerous pathways to secrete proteins across their complex cell envelopes. Here, we describe a protein secretion system which uses a holin membrane protein in tandem with a cell wall editing enzyme to mediate the secretion of substrate proteins from the periplasm to the cell exterior. The identity of the cell wall editing enzymes employed was found to vary across biological systems. For instance, the chitinase secretion pathway of Serratia marcescens uses an endopeptidase to facilitate secretion, whereas the secretion of Typhoid toxin in Salmonella enterica serovar Typhi relies on a muramidase. Various families of holins are also predicted to be involved. Genomic analysis indicates that this pathway is conserved and implicated in the secretion of hydrolytic enzymes and toxins for a range of bacteria. The pairing of holins from different families with various types of peptidoglycan hydrolases suggests that this secretion pathway evolved multiple times. We suggest that the complementary bodies of evidence presented is sufficient to propose that the pathway be named the Type 10 Secretion System (TXSS). Potential mechanisms for secretion across the outer membrane are discussed.
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| 8. |
- Saha, Chayan Kumar, 1988-
(författare)
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Bioinformatics, evolution and revolution in our understanding of toxin-antitoxin systems
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacteria experience a wide range of natural challenges during their life cycles, to which they must respond and adapt to live. Under stressed conditions such as amino acid starvation, bacteria slow down their growth mechanism by producing small alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively referred to as (p)ppGpp. Accumulation of (p)ppGpp results in a comprehensive alteration in cellular metabolism. The alarmone (p)ppGpp is produced and degraded by enzymes belonging to the RelA/SpoT Homologue (RSH) protein family, named for their sequence similarity to the RelA and SpoT enzymes of Escherichia coli. The members of the RSH protein family can be classified into long multi-domain RSHs and short single-domain RSHs. Long RSH enzymes such as RelA and SpoT, carry both the (p)ppGpp hydrolysis (HD) domain and (p)ppGpp synthesis (SYNTH) domain in the N-terminal domain enzymatic region (NTD), in combination with additional domains in the C-terminal domain regulatory region (CTD). Short single-domain RSHs can be divided into small alarmone hydrolases (SAHs) carrying the HD domain, and small alarmone synthetases (SASs) that only have the SYNTH domain. At the beginning of my PhD, I studied the diversity of RSH proteins across the tree of life. I identified 35,615 RSH proteins from analyses of 24,072 genomes. I used large-scale phylogenetic analyses to classify the RSH proteins into 13 long RSHs, 11 SAHs and 30 SASs subfamilies. To address why bacteria often carry multiple SASs in the same genome and predict new functions, I developed a computational tool called FlaGs – standing for Flanking Genes – to analyse the conservation of genomic neighbourhoods in large datasets. I also developed a web-based version of FlaGs, called webFlaGs, that is publicly accessible and is used by biologists all over the world. The application of FlaGs to SAS RSHs led to the discovery that multiple SAS subfamilies are encoded in conserved and frequently overlapping two or three-gene architecture, reminiscent of toxin−antitoxin (TA) systems. Five SAS representatives from the FaRel, FaRel2, PhRel, PhRel2 and CapRel subfamilies were experimentally validated as toxins (toxSASs) that are neutralised by the products of six neighbouring antitoxin genes. The toxSAS enzyme FaRel from Cellulomonas marina is encoded as the central gene of a conserved three-gene architecture and acts through the production of nucleotide alarmone ppGpp and its unusual toxic analogue ppApp, causing a significant depletion of ATP and GTP. FaRel toxicity can be countered by both downstream and upstream cognate antitoxins. The latter contains a SAH domain that neutralises toxicity through degradation of ppGpp as well as ppApp. Combining phylogenetic and FlaGs analyses we have discovered that the DUF4065 domain of unknown function is a widely distributed antitoxin domain in putative TA-like operons with dozens of distinct toxic domains. Nine DUF4065-containing antitoxins and their cognate toxins were experimentally validated as TA pairs using toxicity neutralisation assays. These antitoxins form complexes with their diverse cognate toxins. Given the versatility of DUF4065, we have renamed this domain Panacea. We hypothesise that there are multiple hyperpromiscuous antitoxins like Panacea that can be associated with many non-homologous toxin domains, which may also be hyperpromiscuous. Thus, TA systems across all bacteria can be represented as a network of toxin and antitoxin domain combinations, with hyperpromiscuous domains being hubs in the network. To test this and compute a network, I developed a new, iterative version of FlaGs, called NetFlax (standing for Network-FlaGs for toxins and antitoxins), that can identify TA-like gene architectures in an unsupervised manner and generate a toxin-antitoxin domain interaction network. The results from NetFlax verify our strategy since we have rediscovered multiple previously characterised TAs as well as brand new ones. We find that Panacea is unusual but not unique in its hyperpromiscuity and our network indicates the presence of novel hyperpromiscuous domains still to be explored. Our findings also demonstrate how a core network of TAs is evolutionarily linked to various accessory genome systems, including conjugative transfer and phage defence mechanisms. The existing network can potentially be a framework on which future discoveries of the biological role of TAs can be mapped.
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| 9. |
- Saha, Chayan Kumar, et al.
(författare)
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FlaGs and webFlaGs : discovering novel biology through the analysis of gene neighbourhood conservation
- 2020
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Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1367-4811 .- 1460-2059. ; 37:9, s. 1312-1314
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of conservation of gene neighbourhoods over different evolutionary levels is important for understanding operon and gene cluster evolution, and predicting functional associations. Our tool FlaGs (standing for Flanking Genes) takes a list of NCBI protein accessions as input, clusters neighbourhood-encoded proteins into homologous groups using sensitive sequence searching, and outputs a graphical visualization of the gene neighbourhood and its conservation, along with a phylogenetic tree annotated with flanking gene conservation. FlaGs has demonstrated utility for molecular evolutionary analysis, having uncovered a new toxin-antitoxin system in prokaryotes and bacteriophages. The web tool version of FlaGs (webFlaGs) can optionally include a BLASTP search against a reduced RefSeq database to generate an input accession list and analyse neighbourhood conservation within the same run.
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| 10. |
- Saha, Chayan Kumar, et al.
(författare)
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NetFlax: identification of a core combinatorial network of proteinaceous toxin-antitoxin systems in microbes
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Toxin-antitoxin (TA) systems are a large group of evolutionary unrelated small genetic modules, typically consisting of two genes that encode a toxin whose overexpression leads to growth arrest and an antitoxin that neutralises the toxin. They are present in bacteria, archaea as well as in genomes of temperate phage, and involved in important biological functions including genomic element stabilization and defence against phages. Previously, using the prediction by our developed bioinformatics tool FlaGs – that can perform conservation of gene neighbourhood analyses at long evolutionary distance – we discovered a novel class of proteinaceous TA systems related to RelA-SpoT Homolog (RSH) protein family of housekeeping stress-response enzymes. Next, by combining phylogenetic analyses of a diverse set of genomes across the tree of life and predictions from FlaGs, we have discovered a hyperpromiscuous antitoxin domain Panacea, that is widely distributed and capable of neutralising dozens of distinct toxic domains. We asked if Panacea is unique in its hyperpromiscuity. To answer this question, we performed a bioinformatic analysis across the tree of prokaryotic life using our developed tool NetFlax standing for Network-FlaGs for toxins and antitoxins. NetFlax can predict TA-like arrangements and group them into homologous clusters of toxins and antitoxins in an unsupervised manner. In the present study, we have identified 43 clusters that are predicted to be toxins and 34 clusters of predicted antitoxins using the NetFlax pipeline. 21 of these 77 predicted clusters are homologous to previously discovered toxins and antitoxins, which validates the NetFlax strategy. Together, the interconnected clusters constitute a core network of TA-like pairs across all microbial life, where the hyperpromiscuous clusters serve as hubs. The NetFlax network indicates the presence of novel hyperpromiscuous toxin and antitoxin domains still to be explored. The interactive version of the network can be accessed at http://netflax.webflags.se/.
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