| 1. |
- Zhang, Tong, et al.
(författare)
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Direct activation of a bacterial innate immune system by a viral capsid protein
- 2022
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 612:7938, s. 132-140
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Tidskriftsartikel (refereegranskat)abstract
- Bacteria have evolved diverse immunity mechanisms to protect themselves against the constant onslaught of bacteriophages1-3. Similar to how eukaryotic innate immune systems sense foreign invaders through pathogen-associated molecular patterns4 (PAMPs), many bacterial immune systems that respond to bacteriophage infection require phage-specific triggers to be activated. However, the identities of such triggers and the sensing mechanisms remain largely unknown. Here we identify and investigate the anti-phage function of CapRelSJ46, a fused toxin-antitoxin system that protects Escherichia coli against diverse phages. Using genetic, biochemical and structural analyses, we demonstrate that the C-terminal domain of CapRelSJ46 regulates the toxic N-terminal region, serving as both antitoxin and phage infection sensor. Following infection by certain phages, newly synthesized major capsid protein binds directly to the C-terminal domain of CapRelSJ46 to relieve autoinhibition, enabling the toxin domain to 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 toxin-antitoxin-mediated innate immunity in bacteria. We provide evidence that CapRels and their phage-encoded triggers are engaged in a 'Red Queen conflict'5, revealing a new front in the intense coevolutionary battle between phages and bacteria. Given that capsid proteins of some eukaryotic viruses are known to stimulate innate immune signalling in mammalian hosts6-10, our results reveal a deeply conserved facet of immunity.
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| 2. |
- Jimmy, Steffi, 1988-
(författare)
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Novel RelA-SpoT Homolog toxin-antitoxin systems that inhibit bacterial growth through production of toxic alarmone ppApp
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The stress alarmone nucleotides guanosine pentaphosphate (pppGpp) and tetraphosphate (ppGpp), collectively known as (p)ppGpp, are the molecular mediators of the bacterial stringent response — a bacterial stress response implicated in virulence, antibiotic tolerance and biofilm formation. At high concentrations, (p)ppGpp halts bacterial growth, inhibits protein synthesis and rewires transcription and metabolism. In Escherichia coli (E. coli ) the (p)ppGpp levels are controlled by two large multi-domain proteins RelA and SpoT, the namesakes of Rel-SpoT Homolog (RSH) protein family. During amino acid starvation, RelA directly senses the acylation status of the A-site tRNA on the ribosome. In the presence of uncharged tRNA, RelA is activated to synthesize (p)ppGpp using ATP and either GDP or GTP as substrates; SpoT opposes the activity of RelA by hydrolyzing the alarmone. The RSH family also includes single domain, monofunctional enzymes: Small Alarmone Synthetases (SASs), which can synthesize (p)ppGpp, and Small Alarmone Hydrolases (SAHs), which can hydrolyze (p)ppGpp. Acting together with ‘long’ RSHs such as RelA and SpoT, these enzymes control the intracellular alarmone levels. Using conservation of genomic neighborhoods analysis of RSH sequences, we have identified several families of SAS factors encoded in conserved bicistronic architectures that are similar to the so-called toxin-antitoxin operons. We experimentally validated five of these SASs as being the toxins (toxSASs) which are neutralized by the products of the six neighboring antitoxin genes. The SAS enzyme from Cellulomonas marina (C. marina ) FaRel inhibits the growth of E. coli cells by synthesizing alarmones ppGpp and ppApp, which in turn leads to the depletion of cellular ATP and GTP. These toxic effects can be countered by the C. marina SAH antitoxin through degradation of ppGpp and ppApp alarmones.Since (p)ppGpp plays such a crucial role in bacterial virulence and antibiotic tolerance, the (p)ppGpp-mediated signaling has emerged as a target for developing new antibacterials. ppGpp-mimetics are a promising strategy for direct inhibition of RSH enzymes. We tested a targeted chemical library of ppGpp analogs in enzymatic assays with purified E. coli RelA activated by the ribosome. Although the screen has yielded several potent inhibitors, none of them were effective in live bacterial cells. Despite their limited utility as antibacterials, these compounds are useful tools for future structural and biochemical work. We took an alternative approach and developed a High Throughput Screening (HTS) assay which utilized amino acid auxotroph Bacillus subtilis lacking (p)ppGpp. We have performed an HTS screen with a diverse compound library and identified a set of compounds sharing a common 4-(6-(phenoxyl) alkyl)-3,5-dimethyl-1H-pyrazole core as possible stringent response inhibitors. Our follow-up characterization of these compounds as well as reported potential inhibitors — the ppGpp analog Relacin and cationic peptide 1018 — revealed that neither compound is sufficiently specific to warrant further development.
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| 3. |
- Leslie, David J., et al.
(författare)
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Nutritional Control of DNA Replication Initiation through the Proteolysis and Regulated Translation of DnaA
- 2015
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Ingår i: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 11:7
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Tidskriftsartikel (refereegranskat)abstract
- Bacteria can arrest their own growth and proliferation upon nutrient depletion and under various stressful conditions to ensure their survival. However, the molecular mechanisms responsible for suppressing growth and arresting the cell cycle under such conditions remain incompletely understood. Here, we identify post-transcriptional mechanisms that help enforce a cell-cycle arrest in Caulobacter crescentus following nutrient limitation and during entry into stationary phase by limiting the accumulation of DnaA, the conserved replication initiator protein. DnaA is rapidly degraded by the Lon protease following nutrient limitation. However, the rate of DnaA degradation is not significantly altered by changes in nutrient availability. Instead, we demonstrate that decreased nutrient availability downregulates dnaA translation by a mechanism involving the 5' untranslated leader region of the dnaA transcript; Lon-dependent proteolysis of DnaA then outpaces synthesis, leading to the elimination of DnaA and the arrest of DNA replication. Our results demonstrate how regulated translation and constitutive degradation provide cells a means of precisely and rapidly modulating the concentration of key regulatory proteins in response to environmental inputs.
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| 4. |
- 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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