| 1. |
- Deiana, Marco, et al.
(författare)
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A Light‐up Logic Platform for Selective Recognition of Parallel G‐Quadruplex Structures via Disaggregation‐Induced Emission
- 2020
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Ingår i: Angewandte Chemie International Edition. - : Wiley-VCH Verlagsgesellschaft. - 1433-7851 .- 1521-3773. ; 59:2, s. 896-902
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Tidskriftsartikel (refereegranskat)abstract
- The design of turn‐on dyes with optical signals sensitive to the formation of supramolecular structures provides fascinating and underexplored opportunities for G‐quadruplex (G4) DNA detection and characterization. Here, we show a new switching mechanism that relies on the recognition‐driven disaggregation (on‐signal) of an ultrabright coumarin‐quinazoline conjugate. The synthesized probe selectively lights‐up parallel G4 DNA structures via the disassembly of its supramolecular state, demonstrating outputs that are easily integrable into a label free molecular logic system. Finally, our molecule preferentially stains the G4‐rich nucleoli of cancer cells.
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| 2. |
- Deiana, Marco, et al.
(författare)
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A Minimalistic Coumarin Turn-On Probe for Selective Recognition of Parallel G-Quadruplex DNA Structures
- 2021
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Ingår i: ACS Chemical Biology. - : American Chemical Society (ACS). - 1554-8929 .- 1554-8937. ; 16:8, s. 1365-1376
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Tidskriftsartikel (refereegranskat)abstract
- G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small “druglike” optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure–activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.
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| 3. |
- Deiana, Marco, et al.
(författare)
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A new G-quadruplex-specific photosensitizer inducing genome instability in cancer cells by triggering oxidative DNA damage and impeding replication fork progression
- 2023
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 51:12, s. 6264-6285
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Tidskriftsartikel (refereegranskat)abstract
- Photodynamic therapy (PDT) ideally relies on the administration, selective accumulation and photoactivation of a photosensitizer (PS) into diseased tissues. In this context, we report a new heavy-atom-free fluorescent G-quadruplex (G4) DNA-binding PS, named DBI. We reveal by fluorescence microscopy that DBI preferentially localizes in intraluminal vesicles (ILVs), precursors of exosomes, which are key components of cancer cell proliferation. Moreover, purified exosomal DNA was recognized by a G4-specific antibody, thus highlighting the presence of such G4-forming sequences in the vesicles. Despite the absence of fluorescence signal from DBI in nuclei, light-irradiated DBI-treated cells generated reactive oxygen species (ROS), triggering a 3-fold increase of nuclear G4 foci, slowing fork progression and elevated levels of both DNA base damage, 8-oxoguanine, and double-stranded DNA breaks. Consequently, DBI was found to exert significant phototoxic effects (at nanomolar scale) toward cancer cell lines and tumor organoids. Furthermore, in vivo testing reveals that photoactivation of DBI induces not only G4 formation and DNA damage but also apoptosis in zebrafish, specifically in the area where DBI had accumulated. Collectively, this approach shows significant promise for image-guided PDT.
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| 4. |
- Deiana, Marco, et al.
(författare)
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A Site-Specific Self-Assembled Light-up Rotor Probe for Selective Recognition and Stabilization of c-MYC G-Quadruplex DNA
- 2020
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Ingår i: Nanoscale. - : Royal Society of Chemistry. - 2040-3364 .- 2040-3372. ; 12:24, s. 12950-12957
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Tidskriftsartikel (refereegranskat)abstract
- Direct and unambiguous evidence of the formation of G-quadruplexes (G4s) in human cells have shown their implication in several key biological events and has emphasized their role as important targets for small-molecule cancer therapeutics. Here, we report on the first example of a self-assembled multitasking molecular-rotor G4-binder able to discriminate between an extensive panel of G4 and non-G4 structures and to selectively light-up (up to 105-fold), bind (nanomolar range), and stabilize the c-MYC promoter G4 DNA. In particular, association with the c-MYC G4 triggers the disassembly of its supramolecular state (disaggregation-induced emission, DIE) and induces geometrical restrictions (motion-induced change in emission, MICE) leading to a significant enhancement of its emission yield. Moreover, this optical reporter is able to selectively stabilize the c-MYC G4 and inhibit DNA synthesis. Finally, by using confocal laser-scanning microscopy (CLSM) we show the ability of this compound to localize primarily in the subnuclear G4-rich compartments of cancer cells. This work provides a benchmark for the future design and development of a new generation of smart sequence-selective supramolecular G4-binders that combine outstanding sensing and stability properties, to be utilized in anti-cancer therapy.
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| 5. |
- Deiana, Marco, et al.
(författare)
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Unravelling the cellular emission fingerprint of the benchmark G-quadruplex-interactive compound Phen-DC3
- 2020
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Ingår i: Chemical Communications. - : Royal Society of Chemistry. - 1359-7345 .- 1364-548X. ; 56:91, s. 14251-14254
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Tidskriftsartikel (refereegranskat)abstract
- Phen-DC3 is among the most commonly used G-quadruplex (G4)-stabilizers in vitro and in cells. Here, we show that the G4-interactive binding interactions enable one to tune the optical properties of Phen-DC3 allowing the detection of G4 structures in cancer cells. This work opens up new directions for the use of Phen-DC3 as a selective G4 fluorescent reporter.
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| 6. |
- Jamroskovic, Jan, et al.
(författare)
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Identification of putative G-quadruplex DNA structures in S. pombe genome by quantitative PCR stop assay
- 2019
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 82
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Tidskriftsartikel (refereegranskat)abstract
- In order to understand in which biological processes the four-stranded G-quadruplex (G4) DNA structures play a role, it is important to determine which predicted regions can actually adopt a G4 structure. Here, to identify DNA regions in Schizosaccharomyces pombe that fold into G4 structures, we first optimized a quantitative PCR (qPCR) assay using the G4 stabilizer, PhenDC3. We call this method the qPCR stop assay, and used it to screen for G4 structures in genomic DNA. The presence of G4 stabilizers inhibited DNA amplification in 14/15 unexplored genomic regions in S. pombe that encompassed predicted G4 structures, suggesting that at these sites the stabilized G4 structure formed an obstacle for the DNA polymerase. Furthermore, the formation of G4 structures was confirmed by complementary in vitro assays. In vivo, the S. pombe G4 unwinder Pif1 helicase, Pfh1, was associated with tested G4 sites, suggesting that the G4 structures also formed in vivo. Thus, we propose that the confirmed G4 structures in S. pombe form an obstacle for replication in vivo, and that the qPCR stop assay is a method that can be used to identify G4 structures. Finally, we suggest that the qPCR stop assay can also be used for identifying G4 structures in other organisms, as well as being adapted to screen for novel G4 stabilizers.
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| 7. |
- Jamroskovic, Jan, et al.
(författare)
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Quinazoline Ligands Induce Cancer Cell Death through Selective STAT3 Inhibition and G-Quadruplex Stabilization
- 2020
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 142:6, s. 2876-2888
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Tidskriftsartikel (refereegranskat)abstract
- The signal transducer and activator of transcription 3 (STAT3) protein is a master regulator of most key hallmarks and enablers of cancer, including cell proliferation and the response to DNA damage. G-Quadruplex (G4) structures are four-stranded noncanonical DNA structures enriched at telomeres and oncogenes' promoters. In cancer cells, stabilization of G4 DNAs leads to replication stress and DNA damage accumulation and is therefore considered a promising target for oncotherapy. Here, we designed and synthesized novel quinazoline-based compounds that simultaneously and selectively affect these two well-recognized cancer targets, G4 DNA structures and the STAT3 protein. Using a combination of in vitro assays, NMR, and molecular dynamics simulations, we show that these small, uncharged compounds not only bind to the STAT3 protein but also stabilize G4 structures. In human cultured cells, the compounds inhibit phosphorylation-dependent activation of STAT3 without affecting the antiapoptotic factor STAT1 and cause increased formation of G4 structures, as revealed by the use of a G4 DNA-specific antibody. As a result, treated cells show slower DNA replication, DNA damage checkpoint activation, and an increased apoptotic rate. Importantly, cancer cells are more sensitive to these molecules compared to noncancerous cell lines. This is the first report of a promising class of compounds that not only targets the DNA damage cancer response machinery but also simultaneously inhibits the STAT3-induced cancer cell proliferation, demonstrating a novel approach in cancer therapy.
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| 8. |
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| 9. |
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| 10. |
- Lekmeechai, Sujinna, et al.
(författare)
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Helicobacter pylori Outer Membrane Vesicles Protect the Pathogen From Reactive Oxygen Species of the Respiratory Burst
- 2018
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Outer membrane vesicles (OMVs) play an important role in the persistence of Helicobacter pylori infection. Helicobacter OMVs carry a plethora of virulence factors, including catalase (KatA), an antioxidant enzyme that counteracts the host respiratory burst. We found KatA to be enriched and surface-associated in OMVs compared to bacterial cells. This conferred OMV-dependent KatA activity resulting in neutralization of H2O2 and NaClO, and rescue of surrounding bacteria from oxidative damage. The antioxidant activity of OMVs was abolished by deletion of KatA. In conclusion, enrichment of antioxidative KatA in OMVs is highly important for efficient immune evasion.
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| 11. |
- Liu, Junfa, et al.
(författare)
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Elevated CpxR~P levels repress the Ysc-Yop type III secretion system of Yersinia pseudotuberculosis
- 2012
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Ingår i: Research in Microbiology. - Elsevier : Elsevier BV. - 0923-2508 .- 1769-7123. ; 163:8, s. 518-530
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Tidskriftsartikel (refereegranskat)abstract
- One way that Gram-negative bacteria respond to extracytoplasmic stress is through the CpxA-CpxR system. An activated CpxA sensor kinase phosphorylates the CpxR response regulator to instigate positive auto-amplification of Cpx pathway activation, as well as synthesis of various bacterial survival factors. In the absence of CpxA, human enteropathogenic Yersinia pseudotuberculosis accumulates high CpxR~P levels aided by the action of low molecular weight phosphodonors such as acetyl~P. Critically, these bacteria are also defective for plasmid encoded Ysc-Yop-dependent type III synthesis and secretion, an essential determinant of virulence. Herein, we investigated whether elevated CpxR~P levels account for lost Ysc-Yop function. Decisively, reducing CpxR~P in Yersinia defective for CpxA phosphatase activity - through incorporating second-site suppressor mutations in ackA-pta or cpxR - dramatically restored Ysc-Yop T3S function. Moreover, the repressive effect of accumulated CpxR~P is a direct consequence of binding to the promoter regions of the T3S genes. Thus, Cpx pathway activation has two consequences in Yersinia; one, to maintain quality control in the bacterial envelope, and the second, to restrict ysc-yop gene expression to those occasions where it will have maximal effect.
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| 12. |
- Liu, Junfa, et al.
(författare)
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Phosphorylated CpxR Restricts Production of the RovA Global Regulator in Yersinia pseudotuberculosis
- 2011
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Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 6:8, s. e23314-
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Tidskriftsartikel (refereegranskat)abstract
- Background: RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression. Methodology/Principal Findings: In this study, we characterized CpxR phosphorylation (CpxR similar to P) in vitro, and determined that phosphorylation was necessary for CpxR to efficiently bind to the PCR-amplified upstream regulatory region of rovA. The precise CpxR similar to P binding site was mapped by a nuclease protection assay and directed mutagenesis confirmed that in vivo binding to the rovA promoter inhibits transcription. Reduced RovA production was most pronounced following CpxR, P accumulation in the Yersinia cytoplasm during chronic Cpx pathway activation and by the indiscriminate phosphodonor action of acetyl phosphate. Conclusions/Significance: Cpx pathway activation restricts levels of the RovA global regulator. The regulatory influence of CpxR similar to P must therefore extend well beyond periplasmic quality control in the Yersinia envelope, to include genes involved in environmental survival and pathogenicity.
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| 13. |
- Obi, Ikenna, et al.
(författare)
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Demarcating SurA activities required for outer membrane targeting of Yersinia pseudotuberculosis adhesins
- 2013
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Ingår i: Infection and Immunity. - Washington : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 81:7, s. 2296-2308
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Tidskriftsartikel (refereegranskat)abstract
- SurA is a periplasmic protein folding factor involved in chaperoning and trafficking of outer membrane proteins across the Gram-negative bacterial periplasm. In addition, SurA also possesses peptidyl-prolyl cis/trans isomerase activity. In enteropathogenic Yersinia pseudotuberculosis, we have previously reported that SurA is needed for bacterial virulence and envelope integrity. In this study, we investigated the role of SurA in the assembly of important Yersinia adhesins. Using genetic mutation, biochemical characterization and an in vitro-based bacterial host cell association assay, we confirmed that surface localization of the invasin adhesin is dependent on SurA. As a surA deletion also has some impact on the levels of individual components of the BAM complex in the Yersinia outer membrane, abolished invasin surface assembly could reflect both a direct loss of SurA-dependent periplasmic targeting as well as a potentially compromised BAM complex assembly platform in the outer membrane. To varying degrees, the assembly of two other adhesins, Ail and the pH 6 antigen fibrillum PsaA also depend on SurA. Consequently, loss of SurA leads to a dramatic reduction in Yersinia attachment to eukaryotic host cells. Genetic complementation of surA deletion mutants indicated a prominent role for SurA chaperone function in outer membrane protein assembly. Significantly, the N-terminus of SurA contributed most of this SurA chaperone function. Despite a dominant chaperoning role, it was also evident that SurA isomerization activity did make a modest contribution to this assembly process.
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| 14. |
- Obi, Ikenna, et al.
(författare)
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Stabilization of G-quadruplex DNA structures in Schizosaccharomyces pombe causes single-strand DNA lesions and impedes DNA replication
- 2020
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 48:19, s. 10998-11015
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Tidskriftsartikel (refereegranskat)abstract
- G-quadruplex (G4) structures are stable noncanonical DNA structures that are implicated in the regulation of many cellular pathways. We show here that the G4-stabilizing compound PhenDC3 causes growth defects in Schizosaccharomyces pombe cells, especially during S-phase in synchronized cultures. By visualizing individual DNA molecules, we observed shorter DNA fragments of newly replicated DNA in the PhenDC3-treated cells, suggesting that PhenDC3 impedes replication fork progression. Furthermore, a novel single DNA molecule damage assay revealed increased single-strand DNA lesions in the PhenDC3-treated cells. Moreover, chromatin immunoprecipitation showed enrichment of the leading-strand DNA polymerase at sites of predicted G4 structures, suggesting that these structures impede DNA replication. We tested a subset of these sites and showed that they form G4 structures, that they stall DNA synthesis in vitro and that they can be resolved by the breast cancerassociated Pif1 family helicases. Our results thus suggest that G4 structures occur in S. pombe and that stabilized/unresolved G4 structures are obstacles for the replication machinery. The increased levels of DNA damage might further highlight the association of the human Pif1 helicase with familial breast cancer and the onset of other human diseases connected to unresolved G4 structures.
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| 15. |
- Obi, Ikenna, et al.
(författare)
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Varying dependency of periplasmic peptidylprolyl cis-trans isomerases in promoting Yersinia pseudotuberculosis stress tolerance and pathogenicity
- 2011
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Ingår i: Biochemical Journal. - United Kingdom : Portland Press Limited. - 0264-6021 .- 1470-8728. ; 439:2, s. 321-332
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Tidskriftsartikel (refereegranskat)abstract
- Periplasmic PPIases (peptidylprolyl cis-trans isomerases) catalyse the cis-trans isomerization of peptidyl-prolyl bonds, which is a rate-limiting step during protein folding. We demonstrate that the surA, ppiA, ppiD, fkpA and fklB alleles each encode a periplasmic PPIase in the bacterial pathogen Yersinia pseudotuberculosis. Of these, four were purified to homogeneity. Purified SurA, FkpA and FklB, but not PpiD, displayed detectable PPIase activity in vitro. Significantly, only Y. pseudotuberculosis lacking surA caused drastic alterations to the outer membrane protein profile and FA (fatty acid) composition. They also exhibited aberrant cellular morphology, leaking LPS (lipopolysaccharide) into the extracellular environment. The SurA PPIase is therefore most critical for maintaining Y. pseudotuberculosis envelope integrity during routine culturing. On the other hand, bacteria lacking either surA or all of the genes ppiA, ppiD, fkpA and fklB were sensitive to hydrogen peroxide and were attenuated in mice infections. Thus Y. pseudotuberculosis exhibits both SurA-dependent and -independent requirements for periplasmic PPIase activity to ensure in vivo survival and a full virulence effect in a mammalian host.
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| 16. |
- Olofsson, Annelie, et al.
(författare)
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Uptake of Helicobacter pylori vesicles is facilitated by clathrin-dependent and clathrin-independent endocytic pathways
- 2014
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Ingår i: mBio. - 2161-2129 .- 2150-7511. ; 5:3, s. e00979-14-
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Tidskriftsartikel (refereegranskat)abstract
- UNLABELLED: Bacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.IMPORTANCE: Bacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.
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| 17. |
- Skoog, Emma C, 1983, et al.
(författare)
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BabA dependent binding of Helicobacter pylori to human gastric mucins cause aggregation that inhibits proliferation and is regulated via ArsS
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Mucins in the gastric mucus layer carry a range of glycan structures, which vary between individuals, can have antimicrobial effect or act as ligands for Helicobacter pylori. Mucins from various individuals and disease states modulate H. pylori proliferation and adhesin gene expression differently. Here we investigate the relationship between adhesin mediated binding, aggregation, proliferation and adhesin gene expression using human gastric mucins and synthetic adhesin ligand conjugates. By combining measurements of optical density, bacterial metabolic activity and live/dead stains, we could distinguish bacterial aggregation from viability changes, enabling elucidation of mechanisms behind the anti-prolific effects that mucins can have. Binding of H. pylori to Leb-glycoconjugates inhibited the proliferation of the bacteria in a BabA dependent manner, similarly to the effect of mucins carrying Leb. Furthermore, deletion of arsS lead to a decrease in binding to Leb-glycoconjugates and Leb-decorated mucins, accompanied by decreased aggregation and absence of anti-prolific effect of mucins and Leb-glycoconjugates. Inhibition of proliferation caused by adhesin dependent binding to mucins, and the subsequent aggregation suggests a new role of mucins in the host defense against H. pylori. This aggregating trait of mucins may be useful to incorporate into the design of adhesin inhibitors and other disease intervention molecules.
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| 18. |
- Thanikkal, Edvin J., et al.
(författare)
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The Yersinia pseudotuberculosis Cpx envelope stress system contributes to transcriptional activation of rovM
- 2019
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Ingår i: Virulence. - : Taylor & Francis Group. - 2150-5594 .- 2150-5608. ; 10:1, s. 37-57
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Tidskriftsartikel (refereegranskat)abstract
- The Gram-negative enteropathogen Yersinia pseudotuberculosis possesses a number of regulatory systems that detect cell envelope damage caused by noxious extracytoplasmic stresses. The CpxA sensor kinase and CpxR response regulator two-component regulatory system is one such pathway. Active Cpx signalling upregulates various factors designed to repair and restore cell envelope integrity. Concomitantly, this pathway also down-regulates key determinants of virulence. In Yersinia, cpxA deletion accumulates high levels of phosphorylated CpxR (CpxR~P). Accumulated CpxR~P directly repressed rovA expression and this limited expression of virulence-associated processes. A second transcriptional regulator, RovM, also negatively regulates rovA expression in response to nutrient stress. Hence, this study aimed to determine if CpxR~P can influence rovA expression through control of RovM levels. We determined that the active CpxR~P isoform bound to the promoter of rovM and directly induced its expression, which naturally associated with a concurrent reduction in rovA expression. Site-directed mutagenesis of the CpxR~P binding sequence in the rovM promoter region desensitised rovM expression to CpxR~P. These data suggest that accumulated CpxR~P inversely manipulates the levels of two global transcriptional regulators, RovA and RovM, and this would be expected to have considerable influence on Yersinia pathophysiology and metabolism.
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| 19. |
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| 20. |
- Yan, Kok-Phen, et al.
(författare)
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The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 49:14, s. 8339-8354
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Tidskriftsartikel (refereegranskat)abstract
- The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 binding proteins from the fission yeast Schizosaccharomyces pombe. Gene ontology analysis of the molecular functions enriched in this pull-down assay included mRNA binding, RNA helicase activity, and translation regulator activity. We focused this study on three of the identified proteins that possessed putative arginine-glycine-glycine (RGG) domains, namely the Stm1 homolog Oga1 and the DEAD box RNA helicases Dbp2 and Ded1. We found that Oga1, Dbp2, and Ded1 bound to both DNA and RNA G4s in vitro. Both Dbp2 and Ded1 bound to G4 structures through the RGG domain located in the C-terminal region of the helicases, and point mutations in this domain weakened the G4 binding properties of the helicases. Dbp2 and Ded1 destabilized less thermostable G4 RNA and DNA structures, and this ability was independent of ATP but dependent on the RGG domain. Our study provides the first evidence that the RGG motifs in DEAD box helicases are necessary for both G4 binding and G4 destabilization.
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