| 1. |
- Dominguez-Molina, Lucia, et al.
(författare)
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Biochemical and X-ray analyses of the players involved in the faRel2/aTfaRel2 toxin-antitoxin operon
- 2023
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Ingår i: Acta crystallographica. Section F, Structural biology communications. - 2053-230X. ; 79:10
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Tidskriftsartikel (refereegranskat)abstract
- The aTfaRel2/faRel2 operon from Coprobacillus sp. D7 encodes a bicistronic type II toxin-antitoxin (TA) module. The FaRel2 toxin is a toxic small alarmone synthetase (toxSAS) that inhibits translation through the pyrophosphorylation of uncharged tRNAs at the 3'-CCA end. The toxin is neutralized by the antitoxin ATfaRel2 through the formation of an inactive TA complex. Here, the production, biophysical analysis and crystallization of ATfaRel2 and FaRel2 as well as of the ATfaRel2-FaRel2 complex are reported. ATfaRel2 is monomeric in solution. The antitoxin crystallized in space group P21212 with unit-cell parameters a = 53.3, b = 34.2, c = 37.6 Å, and the best crystal diffracted to a resolution of 1.24 Å. Crystals of FaRel2 in complex with APCPP, a nonhydrolysable ATP analogue, belonged to space group P21, with unit-cell parameters a = 31.5, b = 60.6, c = 177.2 Å, β = 90.6°, and diffracted to 2.6 Å resolution. The ATfaRel2-FaRel2Y128F complex forms a heterotetramer in solution composed of two toxins and two antitoxins. This complex crystallized in two space groups: F4132, with unit-cell parameters a = b = c = 227.1 Å, and P212121, with unit-cell parameters a = 51.7, b = 106.2, c = 135.1 Å. The crystals diffracted to 1.98 and 2.1 Å resolution, respectively.
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| 2. |
- Dominguez-Molina, Lucia, et al.
(författare)
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Mechanisms of neutralization of toxSAS from toxin-antitoxin modules
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Ingår i: Nature Chemical Biology. - 1552-4469.
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Tidskriftsartikel (refereegranskat)abstract
- Toxic small alarmone synthetase (toxSAS) enzymes constitute a family of bacterial effectors present in toxin-antitoxin and secretion systems. toxSASs act through either translation inhibition mediated by pyrophosphorylation of transfer RNA (tRNA) CCA ends or synthesis of the toxic alarmone adenosine pentaphosphate ((pp)pApp) and adenosine triphosphate (ATP) depletion, exemplified by FaRel2 and FaRel, respectively. However, structural bases of toxSAS neutralization are missing. Here we show that the pseudo-Zn 2+ finger domain (pZFD) of the ATfaRel2 antitoxin precludes access of ATP to the pyrophosphate donor site of the FaRel2 toxin, without affecting recruitment of the tRNA pyrophosphate acceptor. By contrast, (pp)pApp-producing toxSASs are inhibited by Tis1 antitoxin domains though occlusion of the pyrophosphate acceptor-binding site. Consequently, the auxiliary pZFD of AT2faRel is dispensable for FaRel neutralization. Collectively, our study establishes the general principles of toxSAS inhibition by structured antitoxin domains, with the control strategy directly coupled to toxSAS substrate specificity.
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| 3. |
- 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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| 4. |
- 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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| 5. |
- Koller, Timm O, et al.
(författare)
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Structural basis for HflXr-mediated antibiotic resistance in Listeria monocytogenes
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 1362-4962 .- 0305-1048. ; 50:19, s. 11285-11300
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Tidskriftsartikel (refereegranskat)abstract
- HflX is a ubiquitous bacterial GTPase that splits and recycles stressed ribosomes. In addition to HflX, Listeria monocytogenes contains a second HflX homolog, HflXr. Unlike HflX, HflXr confers resistance to macrolide and lincosamide antibiotics by an experimentally unexplored mechanism. Here, we have determined cryo-EM structures of L. monocytogenes HflXr-50S and HflX-50S complexes as well as L. monocytogenes 70S ribosomes in the presence and absence of the lincosamide lincomycin. While the overall geometry of HflXr on the 50S subunit is similar to that of HflX, a loop within the N-terminal domain of HflXr, which is two amino acids longer than in HflX, reaches deeper into the peptidyltransferase center. Moreover, unlike HflX, the binding of HflXr induces conformational changes within adjacent rRNA nucleotides that would be incompatible with drug binding. These findings suggest that HflXr confers resistance using an allosteric ribosome protection mechanism, rather than by simply splitting and recycling antibiotic-stalled ribosomes.
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| 6. |
- Kumar, Pravin, et al.
(författare)
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Clinically observed deletions in SARS-CoV-2 Nsp1 affect its stability and ability to inhibit translation
- 2022
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Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 596:9, s. 1203-1213
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Tidskriftsartikel (refereegranskat)abstract
- Nonstructural protein 1 (Nsp1) of SARS-CoV-2 inhibits host cell translation through an interaction between its C-terminal domain and the 40S ribosome. The N-terminal domain (NTD) of Nsp1 is a target of recurring deletions, some of which are associated with altered COVID-19 disease progression. Here, we characterize the efficiency of translational inhibition by clinically observed Nsp1 deletion variants. We show that a frequent deletion of residues 79–89 severely reduces the ability of Nsp1 to inhibit translation while not abrogating Nsp1 binding to the 40S. Notably, while the SARS-CoV-2 5′ untranslated region enhances translation of mRNA, it does not protect from Nsp1-mediated inhibition. Finally, thermal stability measurements and structure predictions reveal a correlation between stability of the NTD and the efficiency of translation inhibition.
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| 7. |
- 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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| 8. |
- Kurata, Tatsuaki, et al.
(författare)
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RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis
- 2021
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Ingår i: Molecular Cell. - : Cell Press. - 1097-2765 .- 1097-4164. ; 81:15, s. 3160-3170.e9
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Tidskriftsartikel (refereegranskat)abstract
- 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 analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used 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 RNA-modifying enzymes.
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| 9. |
- Mets, Toomas, et al.
(författare)
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Mechanism of phage sensing and restriction by toxin-antitoxin-chaperone systems
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Ingår i: Cell Host and Microbe. - 1934-6069.
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Tidskriftsartikel (refereegranskat)abstract
- Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense.
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| 10. |
- Takada, Hiraku, et al.
(författare)
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Expression of Bacillus subtilis ABCF antibiotic resistance factor VmlR is regulated by RNA polymerase pausing, transcription attenuation, translation attenuation and (p)ppGpp
- 2022
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 50:11, s. 6174-6189
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Tidskriftsartikel (refereegranskat)abstract
- Since antibiotic resistance is often associated with a fitness cost, bacteria employ multi-layered regulatory mechanisms to ensure that expression of resistance factors is restricted to times of antibiotic challenge. In Bacillus subtilis, the chromosomally-encoded ABCF ATPase VmlR confers resistance to pleuromutilin, lincosamide and type A streptogramin translation inhibitors. Here we show that vmlR expression is regulated by translation attenuation and transcription attenuation mechanisms. Antibiotic-induced ribosome stalling during translation of an upstream open reading frame in the vmlR leader region prevents formation of an anti-antiterminator structure, leading to the formation of an antiterminator structure that prevents intrinsic termination. Thus, transcription in the presence of antibiotic induces vmlR expression. We also show that NusG-dependent RNA polymerase pausing in the vmlR leader prevents leaky expression in the absence of antibiotic. Furthermore, we demonstrate that induction of VmlR expression by compromised protein synthesis does not require the ability of VmlR to rescue the translational defect, as exemplified by constitutive induction of VmlR by ribosome assembly defects. Rather, the specificity of induction is determined by the antibiotic's ability to stall the ribosome on the regulatory open reading frame located within the vmlR leader. Finally, we demonstrate the involvement of (p)ppGpp-mediated signalling in antibiotic-induced VmlR expression.
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