| 1. |
- Bednarska, Natalia G., et al.
(author)
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Protein aggregation as an antibiotic design strategy
- 2016
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In: Molecular Microbiology. - : WILEY-BLACKWELL. - 0950-382X .- 1365-2958. ; 99:5, s. 849-865
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Journal article (peer-reviewed)abstract
- Taking advantage of the xenobiotic nature of bacterial infections, we tested whether the cytotoxicity of protein aggregation can be targeted to bacterial pathogens without affecting their mammalian hosts. In particular, we examined if peptides encoding aggregation-prone sequence segments of bacterial proteins can display antimicrobial activity by initiating toxic protein aggregation in bacteria, but not in mammalian cells. Unbiased in vitro screening of aggregating peptide sequences from bacterial genomes lead to the identification of several peptides that are strongly bactericidal against methicillin-resistant Staphylococcus aureus. Upon parenteral administration in vivo, the peptides cured mice from bacterial sepsis without apparent toxic side effects as judged from histological and hematological evaluation. We found that the peptides enter and accumulate in the bacterial cytosol where they cause aggregation of bacterial polypeptides. Although the precise chain of events that leads to cell death remains to be elucidated, the ability to tap into aggregation-prone sequences of bacterial proteomes to elicit antimicrobial activity represents a rich and unexplored chemical space to be mined in search of novel therapeutic strategies to fight infectious diseases.
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| 2. |
- Berghoff, Bork A., et al.
(author)
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Two regulatory RNA elements affect TisB-dependent depolarization and persister formation
- 2017
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In: Molecular Microbiology. - : WILEY. - 0950-382X .- 1365-2958. ; 103:6, s. 1020-1033
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Journal article (peer-reviewed)abstract
- Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5 UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity.
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| 3. |
- Brandis, Gerrit, 1985-, et al.
(author)
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Autoregulation of the tufB operon in Salmonella
- 2016
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In: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 100:6, s. 1004-1016
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Journal article (peer-reviewed)abstract
- In Salmonella enterica and related species, translation elongation factor EF-Tu is encoded by two widely separated but near-identical genes, tufA and tufB. Two thirds of EF-Tu is expressed from tufA with the remaining one third coming from tufB. Inactivation of tufA is partly compensated by a doubling in the amount of EF-TuB but the mechanism of this up-regulation is unknown. By experimental evolution selecting for improved growth rate in a strain with an inactive tufA we selected six different noncoding or synonymous point mutations close to the tufB start codon. Based on these results we constructed a total of 161 different point mutations around the tufB start codon, as well as tufB 3′-truncations, and measured tufB expression using tufB-yfp transcriptional and translational fusions. The expression data support the presence of two competing stem-loop structures that can form in the 5′-end of the tufB mRNA. Formation of the ‘closed’ structure leads to Rho-dependent transcriptional termination of the tufB mRNA. We propose a model in which translational speed is used as a sensor for EF-Tu concentration and where the expression of tufB is post-transcriptionally regulated. This model describes for the first time how expression of the most abundant Salmonella protein is autoregulated.
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| 4. |
- Brandis, Gerrit, 1985-, et al.
(author)
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Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella
- 2018
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In: Molecular Microbiology. - : WILEY. - 0950-382X .- 1365-2958. ; 108:6, s. 697-710
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Journal article (peer-reviewed)abstract
- Bacteria can have multiple copies of a gene at separate locations on the same chromosome. Some of these gene families, including tuf (translation elongation factor EF-Tu) and rrl (ribosomal RNA), encode functions critically important for bacterial fitness. Genes within these families are known to evolve in concert using homologous recombination to transfer genetic information from one gene to another. This mechanism can counteract the detrimental effects of nucleotide sequence divergence over time. Whether such mechanisms can also protect against the potentially lethal effects of mobile genetic element insertion is not well understood. To address this we constructed two different length insertion cassettes to mimic mobile genetic elements and inserted these into various positions of the tuf and rrl genes. Wemeasured rates of recombinational repair that removed the inserted cassette and studied the underlying mechanism. Our results indicate that homologous recombination can protect the tuf and rrl genes from inactivation by mobile genetic elements, but forinsertions within shorter gene sequences the efficiency of repair is very low. Intriguingly, we found that physical distance separating genes on the chromosome directly affects the rate of recombinational repair suggesting that relative location will influence the ability of homologous recombination to maintain homogeneity.
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| 5. |
- Brochet, Mathieu, et al.
(author)
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Calcium signalling in malaria parasites
- 2016
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In: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 100:3, s. 397-408
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Journal article (peer-reviewed)abstract
- Ca2+ is a ubiquitous intracellular messenger in malaria parasites with important functions in asexual blood stages responsible for malaria symptoms, the preceding liver‐stage infection and transmission through the mosquito. Intracellular messengers amplify signals by binding to effector molecules that trigger physiological changes. The characterisation of some Ca2+ effector proteins has begun to provide insights into the vast range of biological processes controlled by Ca2+ signalling in malaria parasites, including host cell egress and invasion, protein secretion, motility and cell cycle regulation. Despite the importance of Ca2+ signalling during the life cycle of malaria parasites, little is known about Ca2+ homeostasis. Recent findings highlighted that upstream of stage‐specific Ca2+ effectors is a conserved interplay between second messengers to control critical intracellular Ca2+ signals throughout the life cycle. The identification of the molecular mechanisms integrating stage‐transcending mechanisms of Ca2+ homeostasis in a network of stage‐specific regulator and effector pathways now represents a major challenge for a meaningful understanding of Ca2+ signalling in malaria parasites.
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| 6. |
- Bru, Samuel, et al.
(author)
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Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae
- 2016
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In: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 101:3, s. 367-380
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Journal article (peer-reviewed)abstract
- Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4 Delta) or hydrolyse polyP (ppn1 Delta, ppx1 Delta) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.
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| 7. |
- Derouiche, Abderahmane, 1980, et al.
(author)
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Bacillus subtilisSalA is a phosphorylation-dependent transcription regulator that represses scoC and activates the production of the exoprotease AprE
- 2015
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In: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 97:6, s. 1195-1208
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Journal article (peer-reviewed)abstract
- Bacillus subtilisMrp family protein SalA has been shown to indirectly promote the production of the exoprotease AprE by inhibiting the expression of scoC, which codes for a repressor of aprE. The exact mechanism by which SalA influences scoC expression has not been clarified previously. We demonstrate that SalA possesses a DNA-binding domain (residues 1-60), which binds to the promoter region of scoC. The binding of SalA to its target DNA depends on the presence of ATP and is stimulated by phosphorylation of SalA at tyrosine 327. The B.subtilis protein-tyrosine kinase PtkA interacts specifically with the C-terminal domain of SalAin vivo and in vitro and is responsible for activating its DNA binding via phosphorylation of tyrosine 327. In vivo, a mutant mimicking phosphorylation of SalA (SalA Y327E) exhibited a strong repression of scoC and consequently overproduction of AprE. By contrast, the non-phosphorylatable SalA Y327F and the ΔptkA exhibited the opposite effect, stronger expression of scoC and lower production of the exoprotease. Interestingly, both SalA and PtkA contain the same ATP-binding Walker domain and have thus presumably arisen from the common ancestral protein. Their regulatory interplay seems to be conserved in other bacteria.
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| 8. |
- Engman, Jakob, et al.
(author)
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Regulated protein aggregation, a mechanism to control the activity of the ClpXP adaptor protein YjbH.
- 2015
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In: Molecular Microbiology. - : Wiley. - 1365-2958 .- 0950-382X. ; 95:1, s. 51-63
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Journal article (peer-reviewed)abstract
- Bacteria use stress response pathways to activate diverse target genes to react to a variety of stresses. The Bacillus subtilis Spx protein is a global transcriptional regulator that controls expression of more than 140 genes and operons in response to thiol-specific oxidative stress. Under non-stress conditions the concentration of Spx is kept low by proteolysis catalyzed by the ClpXP complex. Spx protein levels increase in response to disulfide stress, and decrease when the cells cope with the stress. The cytosolic adaptor protein YjbH is required to target Spx for efficient proteolysis by ClpXP. We demonstrate that YjbH aggregates in response to disulfide stress, that is, the YjbH protein is soluble under non-stressed conditions and destabilized during stress leading to aggregation. Stress conditions (heat and ethanol) that cause severe perturbations in protein stability/folding also induced aggregation of YjbH and led to induction of Spx. By heterologous expression of a less aggregation prone YjbH homolog Spx induction was abolished. Thus we show that moderation of YjbH solubility is an important mechanism of signal transduction and represents a new mechanism of controlling the activity of adaptor proteins.
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| 9. |
- Fröjd, Markus J., et al.
(author)
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Apical assemblies of intermediate filament-like protein FilP are highly dynamic and affect polar growth determinant DivIVA in Streptomyces venezuelae
- 2019
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In: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 112:1
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Journal article (peer-reviewed)abstract
- Streptomyces spp. grow as branching hyphae, building the cell wall in restricted zones at hyphal tips. The organization of this mode of polar growth involves three coiled-coil proteins: DivIVA and Scy, which form apical protein complexes referred to as polarisomes; and the intermediate filament-like protein FilP, which influences cell shape and interacts with both Scy and DivIVA. Here, we use live cell imaging of Streptomyces venezuelae to clarify the subcellular localization and dynamics of FilP and its effect on hyphal morphology. By monitoring a FilP-mCherry fusion protein, we show that FilP accumulates in gradient-like zones behind the hyphal tips. The apical gradient pattern of FilP localization is dependent on hyphal tip extension and immediately dissipates upon growth arrest. Fluorescence recovery after photobleaching experiments show that FilP gradients are dynamic and subject to subunit exchange during vegetative growth. Further, the localization of FilP at hyphal tips is not directly dependent on scy, even though the strongly perturbed morphology of most scy mutant hyphae is associated with mislocalization of FilP. Finally, we find that filP has an effect on the size and position of the foci of key polar growth determinant DivIVA. This effect likely contributes to the phenotype of filP mutants.
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| 10. |
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