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Publications (10 of 28) Show all publications
Sharma, A., Omer Aden, R., Puhar, A. & Cisneros, D. A. (2023). CRISPR-cas-guided mutagenesis of chromosome and virulence plasmid in Shigella flexneri by cytosine base editing. mSystems, 8(1), Article ID e01045-22.
Open this publication in new window or tab >>CRISPR-cas-guided mutagenesis of chromosome and virulence plasmid in Shigella flexneri by cytosine base editing
2023 (English)In: mSystems, E-ISSN 2379-5077, Vol. 8, no 1, article id e01045-22Article in journal (Refereed) Published
Abstract [en]

Shigella is a Gram-negative bacterium that invades the human gut epithelium. The resulting infection, shigellosis, is the deadliest bacterial diarrheal disease. Much of the information about the genes dictating the pathophysiology of Shigella, both on the chromosome and the virulence plasmid, was obtained by classical reverse genetics. However, technical limitations of the prevalent mutagenesis techniques restrict the generation of mutants in a single reaction to a small number, preventing large-scale targeted mutagenesis of Shigella and the subsequent assessment of phenotype. We adopted a CRISPR-Cas-dependent approach, where a nickase Cas9 and cytidine deaminase fusion is guided by single guide RNA (sgRNA) to introduce targeted C→T transitions, resulting in internal stop codons and premature termination of translation. In proof-of-principle experiments using an mCherry fluorescent reporter, we were able to generate loss-of-function mutants in both Escherichia coli and Shigella flexneri with up to 100% efficacy. Using a modified fluctuation assay, we determined that under optimized conditions, the frequency of untargeted mutations introduced by the Cas9-deaminase fusion was in the same range as spontaneous mutations, making our method a safe choice for bacterial mutagenesis. Furthermore, we programmed the method to mutate well-characterized chromosomal and plasmid-borne Shigella flexneri genes and found the mutant phenotype to be similar to those of the reported gene deletion mutants, with no apparent polar effects at the phenotype level. This method can be used in a 96-well-plate format to increase the throughput and generate an array of targeted loss-of-function mutants in a few days.

Place, publisher, year, edition, pages
American Society for Microbiology, 2023
Keywords
CRISPR, AID, mutagenesis, base editing, Shigella flexneri, Escherichia coli
National Category
Bioinformatics and Computational Biology Biochemistry Molecular Biology Microbiology
Research subject
Genetics; Microbiology
Identifiers
urn:nbn:se:umu:diva-205339 (URN)10.1128/msystems.01045-22 (DOI)000901485800003 ()36541764 (PubMedID)2-s2.0-85149152715 (Scopus ID)
Funder
Carl Tryggers foundation , CTS 18-65The Kempe Foundations, JCK-2031.3The Kempe Foundations, SMK 1860The Kempe Foundations, SMK-1532.2Knut and Alice Wallenberg Foundation, KAW 2015.0225Novo Nordisk Foundation, NNF17OC0026486Swedish Society for Medical Research (SSMF), PD20-0022Swedish Research Council, 2016-06598
Available from: 2023-03-02 Created: 2023-03-02 Last updated: 2025-02-20Bibliographically approved
Tadala, L., Langenbach, D., Dannborg, M., Cervantes-Rivera, R., Sharma, A., Vieth, K., . . . Puhar, A. (2022). Infection-induced membrane ruffling initiates danger and immune signaling via the mechanosensor PIEZO1. Cell Reports, 40(6), Article ID 111173.
Open this publication in new window or tab >>Infection-induced membrane ruffling initiates danger and immune signaling via the mechanosensor PIEZO1
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2022 (English)In: Cell Reports, E-ISSN 2211-1247, Vol. 40, no 6, article id 111173Article in journal (Refereed) Published
Abstract [en]

Microorganisms are generally sensed by receptors recognizing microbial molecules, which evoke changes in cellular activities and gene expression. Bacterial pathogens induce secretion of the danger signal ATP as an early alert response of intestinal epithelial cells, initiating overt inflammation. However, what triggers ATP secretion during infection is unclear. Here we show that the inherently mechanosensitive plasma membrane channel PIEZO1 acts as a sensor for bacterial entry. PIEZO1 is mechanically activated by invasion-induced membrane ruffles upstream of Ca2+ influx and ATP secretion. Mimicking mechanical stimuli of pathogen uptake with sterile beads equally elicits ATP secretion. Chemical or genetic PIEZO1 inactivation inhibits mechanically induced ATP secretion. Moreover, chemical or mechanical PIEZO1 activation evokes gene expression in immune and barrier pathways. Thus, mechanosensation of invasion-induced plasma membrane distortion initiates immune signaling upon infection, independently of detection of microbial molecules. Hence, PIEZO1-dependent detection of infection is driven by physical signals instead of chemical ligands.

Place, publisher, year, edition, pages
Elsevier, 2022
Keywords
mechanosensing, immune detection, danger signals, invasive pathogens, Shigella, Listeria, plasma membrane ruffles, PIEZO1, extracellular ATP, intestinal epithelial cells
National Category
Cell and Molecular Biology Immunology in the medical area Microbiology in the medical area
Research subject
Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-198648 (URN)10.1016/j.celrep.2022.111173 (DOI)000881382400003 ()2-s2.0-85135700972 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation, KAW 2015.0225The Kempe Foundations, JCK-1528The Kempe Foundations, SMK-1859The Kempe Foundations, JCK-2031.3Swedish Research Council, 2016-06598Carl Tryggers foundation , CTS 18-65The Kempe Foundations, SMK-1860The Kempe Foundations, SMK-1532.2Swedish Society for Medical Research (SSMF), PD20-0022
Available from: 2022-08-15 Created: 2022-08-15 Last updated: 2024-01-17Bibliographically approved
Graffeuil, A., Guerrero-Castro, J., Assefa, A., Uhlin, B. E. & Cisneros, D. A. (2022). Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a. Microbial Cell Factories, 21(1), Article ID 139.
Open this publication in new window or tab >>Polar mutagenesis of polycistronic bacterial transcriptional units using Cas12a
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2022 (English)In: Microbial Cell Factories, E-ISSN 1475-2859, Vol. 21, no 1, article id 139Article in journal (Refereed) Published
Abstract [en]

Background: Functionally related genes in bacteria are often organized and transcribed as polycistronic transcrip‑ tional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci.

Results: Cas12a‑mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator’s sequence, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (ΔatpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non‑ polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase.

Conclusions: We conclude that Cas12a‑mediated mutagenesis is an efficient simple system to generate polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intra‑ cellular ATP in the markerless ΔatpIBEFHAGDC mutant.

Place, publisher, year, edition, pages
BioMed Central, 2022
Keywords
Cas12a, CRISPR mutagenesis, Polycistronic operons, Intracellular ATP, Markerless genome editing
National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-198173 (URN)10.1186/s12934-022-01844-y (DOI)000824657800001 ()35831865 (PubMedID)2-s2.0-85133996452 (Scopus ID)
Funder
Carl Tryggers foundation , CTS 15-96Carl Tryggers foundation , CTS 18-65The Kempe Foundations, JCK-1724The Kempe Foundations, SMK 1860Swedish Research Council, 2015-03007Swedish Research Council, 2019-01720Swedish Research Council, 2007-8673Swedish Research Council, 2016-06598Novo Nordisk, NNF17OC0026486
Available from: 2022-07-16 Created: 2022-07-16 Last updated: 2024-07-04Bibliographically approved
Morales, C., Ruiz-Torres, M., Rodríguez-Acebes, S., Lafarga, V., Rodríguez-Corsino, M., Megías, D., . . . Losada, A. (2020). PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection. Journal of Biological Chemistry, 295(1), 146-157
Open this publication in new window or tab >>PDS5 proteins are required for proper cohesin dynamics and participate in replication fork protection
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2020 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 295, no 1, p. 146-157Article in journal (Refereed) Published
Abstract [en]

Cohesin is a chromatin-bound complex that mediates sister chromatid cohesion and facilitates long-range interactions through DNA looping. How the transcription and replication machineries deal with the presence of cohesin on chromatin remains unclear. The dynamic association of cohesin with chromatin depends on WAPL cohesin release factor (WAPL) and on PDS5 cohesin-associated factor (PDS5), which exists in two versions in vertebrate cells, PDS5A and PDS5B. Using genetic deletion in mouse embryo fibroblasts and a combination of CRISPR-mediated gene editing and RNAi-mediated gene silencing in human cells, here we analyzed the consequences of PDS5 depletion for DNA replication. We found that either PDS5A or PDS5B is sufficient for proper cohesin dynamics and that their simultaneous removal increases cohesin's residence time on chromatin and slows down DNA replication. A similar phenotype was observed in WAPL-depleted cells. Cohesin down-regulation restored normal replication fork rates in PDS5-deficient cells, suggesting that chromatin-bound cohesin hinders the advance of the replisome. We further show that PDS5 proteins are required to recruit WRN helicase-interacting protein 1 (WRNIP1), RAD51 recombinase (RAD51), and BRCA2 DNA repair associated (BRCA2) to stalled forks and that in their absence, nascent DNA strands at unprotected forks are degraded by MRE11 homolog double-strand break repair nuclease (MRE11). These findings indicate that PDS5 proteins participate in replication fork protection and also provide insights into how cohesin and its regulators contribute to the response to replication stress, a common feature of cancer cells.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology, 2020
Keywords
DNA replication, cell cycle, genomic instability, BRCA2, microscopy, cohesin, fork protection, fork reversal, fork stalling, replication stress, replisome
National Category
Cell Biology Biochemistry Molecular Biology
Identifiers
urn:nbn:se:umu:diva-174302 (URN)10.1074/jbc.RA119.011099 (DOI)000505982700012 ()31757807 (PubMedID)2-s2.0-85077476314 (Scopus ID)
Available from: 2020-08-20 Created: 2020-08-20 Last updated: 2025-02-20Bibliographically approved
Hagberg, A., Rzhepishevska, O. I., Semenets, A., Cisneros, D. A. & Ramstedt, M. (2020). Surface analysis of bacterial systems using cryo-X-ray photoelectron spectroscopy. Surface and Interface Analysis, 52, 792-801
Open this publication in new window or tab >>Surface analysis of bacterial systems using cryo-X-ray photoelectron spectroscopy
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2020 (English)In: Surface and Interface Analysis, ISSN 0142-2421, E-ISSN 1096-9918, Vol. 52, p. 792-801Article in journal (Refereed) Published
Abstract [en]

Surface analysis of biological systems using XPS often requires dehydration of the sample for it to be compatible with the ultrahigh vacuum of the spectrometer. However, if samples are frozen to liquid-nitrogen temperature prior to and during analysis, water can be retained in the sample and the organization of the sample surface should be preserved to a higher degree than in desiccated samples. This article presents recent developments of cryo-X-ray photoelectron spectroscopy (cryo-XPS) for analyses of hydrated biological samples at liquid nitrogen temperature. We describe experiments on bacterial cells, bacterial biofilms, and bacterial outer membrane vesicles using a variety of bacterial species. Differences and similarities in surface chemistry are monitored depending on growth in liquid culture, on culture plates, as well as in biofilms, and are discussed. Two data treatment methods providing decomposition of the C 1s spectra into lipid, polysaccharide, and peptide/peptidoglycan content are used and compared.

Place, publisher, year, edition, pages
John Wiley & Sons, 2020
National Category
Analytical Chemistry Microbiology
Identifiers
urn:nbn:se:umu:diva-173400 (URN)10.1002/sia.6854 (DOI)000544872100001 ()2-s2.0-85087293673 (Scopus ID)
Funder
Swedish Research Council Formas, 2017-00403The Kempe Foundations, JCK-1720
Note

Special Issue: SI

Available from: 2020-07-06 Created: 2020-07-06 Last updated: 2024-04-09Bibliographically approved
Muhar, M., Ebert, A., Neumann, T., Umkehrer, C., Jude, J., Wieshofer, C., . . . Zuber, J. (2018). SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis. Science, 360(6390), 800-805
Open this publication in new window or tab >>SLAM-seq defines direct gene-regulatory functions of the BRD4-MYC axis
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2018 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 360, no 6390, p. 800-805Article in journal (Refereed) Published
Abstract [en]

Defining direct targets of transcription factors and regulatory pathways is key to understanding their roles in physiology and disease. We combined SLAM-seq [thiol(SH)-linked alkylation for the metabolic sequencing of RNA], a method for direct quantification of newly synthesized messenger RNAs (mRNAs), with pharmacological and chemical-genetic perturbation in order to define regulatory functions of two transcriptional hubs in cancer, BRD4 and MYC, and to interrogate direct responses to BET bromodomain inhibitors (BETis). We found that BRD4 acts as general coactivator of RNA polymerase II-dependent transcription, which is broadly repressed upon high-dose BETi treatment. At doses triggering selective effects in leukemia, BETis deregulate a small set of hypersensitive targets including MYC. In contrast to BRD4, MYC primarily acts as a selective transcriptional activator controlling metabolic processes such as ribosome biogenesis and de novo purine synthesis. Our study establishes a simple and scalable strategy to identify direct transcriptional targets of any gene or pathway.

National Category
Medical and Health Sciences
Research subject
molecular medicine (medical sciences)
Identifiers
urn:nbn:se:umu:diva-156761 (URN)10.1126/science.aao2793 (DOI)29622725 (PubMedID)
Available from: 2019-02-26 Created: 2019-02-26 Last updated: 2019-03-05Bibliographically approved
Wutz, G., Várnai, C., Nagasaka, K., Cisneros, D. A., Stocsits, R. R., Tang, W., . . . Peters, J.-M. (2017). Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins. EMBO Journal, 36(24), 3573-3599
Open this publication in new window or tab >>Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins
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2017 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 36, no 24, p. 3573-3599Article in journal (Refereed) Published
Abstract [en]

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome-wide function in mediating long-range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.

Place, publisher, year, edition, pages
Springer Nature, 2017
Keywords
chromatin condensation, chromatin structure, genome organization, loop extrusion, vermicelli
National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-156764 (URN)10.15252/embj.201798004 (DOI)000418230000004 ()29217591 (PubMedID)2-s2.0-85039561570 (Scopus ID)
Available from: 2019-02-26 Created: 2019-02-26 Last updated: 2024-09-18Bibliographically approved
Davidson, I. F., Goetz, D., Zaczek, M. P., Molodtsov, M. I., in't Veld, P. J. H., Weissmann, F., . . . Peters, J.-M. (2016). Rapid movement and transcriptional re-localization of human cohesin on DNA. EMBO Journal, 35(24), 2671-2685
Open this publication in new window or tab >>Rapid movement and transcriptional re-localization of human cohesin on DNA
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2016 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 35, no 24, p. 2671-2685Article in journal (Refereed) Published
Abstract [en]

The spatial organization, correct expression, repair, and segregation of eukaryotic genomes depend on cohesin, ring-shaped protein complexes that are thought to function by entrapping DNA. It has been proposed that cohesin is recruited to specific genomic locations from distal loading sites by an unknown mechanism, which depends on transcription, and it has been speculated that cohesin movements along DNA could create three-dimensional genomic organization by loop extrusion. However, whether cohesin can translocate along DNA is unknown. Here, we used single-molecule imaging to show that cohesin can diffuse rapidly on DNA in a manner consistent with topological entrapment and can pass over some DNA-bound proteins and nucleosomes but is constrained in its movement by transcription and DNA-bound CCCTC-binding factor (CTCF). These results indicate that cohesin can be positioned in the genome by moving along DNA, that transcription can provide directionality to these movements, that CTCF functions as a boundary element for moving cohesin, and they are consistent with the hypothesis that cohesin spatially organizes the genome via loop extrusion.

Keywords
cell cycle, cohesin, genome organization, single-molecule TIRF microscopy, transcription
National Category
Biochemistry Molecular Biology
Identifiers
urn:nbn:se:umu:diva-130454 (URN)10.15252/embj.201695402 (DOI)000390948700008 ()27799150 (PubMedID)2-s2.0-84997281764 (Scopus ID)
Available from: 2017-01-24 Created: 2017-01-20 Last updated: 2025-02-20Bibliographically approved
Ladurner, R., Kreidl, E., Ivanov, M. P., Ekker, H., Idarraga-Amado, M. H., Busslinger, G. A., . . . Peters, J.-M. (2016). Sororin actively maintains sister chromatid cohesion. EMBO Journal, 35(6), 635-653
Open this publication in new window or tab >>Sororin actively maintains sister chromatid cohesion
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2016 (English)In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 35, no 6, p. 635-653Article in journal (Refereed) Published
Abstract [en]

Cohesion between sister chromatids is established during DNA replication but needs to be maintained to enable proper chromosome-spindle attachments in mitosis or meiosis. Cohesion is mediated by cohesin, but also depends on cohesin acetylation and sororin. Sororin contributes to cohesion by stabilizing cohesin on DNA. Sororin achieves this by inhibiting WAPL, which otherwise releases cohesin from DNA and destroys cohesion. Here we describe mouse models which enable the controlled depletion of sororin by gene deletion or auxin-induced degradation. We show that sororin is essential for embryonic development, cohesion maintenance, and proper chromosome segregation. We further show that the acetyltransferases ESCO1 and ESCO2 are essential for stabilizing cohesin on chromatin, that their only function in this process is to acetylate cohesin's SMC3 subunit, and that DNA replication is also required for stable cohesin-chromatin interactions. Unexpectedly, we find that sororin interacts dynamically with the cohesin complexes it stabilizes. This implies that sororin recruitment to cohesin does not depend on the DNA replication machinery or process itself, but on a property that cohesin acquires during cohesion establishment.

Keywords
cell cycle, cohesin acetylation, mitosis, sister chromatid cohesion
National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-156766 (URN)10.15252/embj.201592532 (DOI)26903600 (PubMedID)
Available from: 2019-02-26 Created: 2019-02-26 Last updated: 2019-03-08Bibliographically approved
Amorim, G. C., Cisneros, D. A., Delepierre, M., Francetic, O. & Izadi-Pruneyre, N. (2014). ¹H, ¹⁵N and ¹³C resonance assignments of PpdD, a type IV pilin from enterohemorrhagic Escherichia coli. Biomolecular NMR Assignments, 8(1), 43-46
Open this publication in new window or tab >>¹H, ¹⁵N and ¹³C resonance assignments of PpdD, a type IV pilin from enterohemorrhagic Escherichia coli
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2014 (English)In: Biomolecular NMR Assignments, ISSN 1874-2718, E-ISSN 1874-270X, Vol. 8, no 1, p. 43-46Article in journal (Refereed) Published
Abstract [en]

Bacterial type 4 pili (T4P) are long flexible fibers involved in adhesion, DNA uptake, phage transduction, aggregation and a flagella-independent movement called "twitching motility". T4P comprise thousands of copies of the major pilin subunit, which is initially inserted in the plasma membrane, processed and assembled into dynamic helical filaments. T4P are crucial for host colonization and virulence of many Gram-negative bacteria. In enterohemorrhagic Escherichia coli the T4P, called hemorrhagic coli pili (HCP) promote cell adhesion, motility, biofilm formation and signaling. To understand the mechanism of HCP assembly and function, we analyzed the structure of the major subunit prepilin peptidase-dependent protein D (PpdD) (also called HcpA), a 15 kDa pilin with two potential disulfide bonds. Here we present the (1)H, (15)N and (13)C backbone and side chain resonance assignments of the C-terminal globular domain of PpdD as a first step to its structural determination.

National Category
Infectious Medicine Microbiology
Identifiers
urn:nbn:se:umu:diva-156769 (URN)10.1007/s12104-012-9449-z (DOI)23242787 (PubMedID)
Available from: 2019-02-26 Created: 2019-02-26 Last updated: 2019-03-08Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-9919-0075

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