Publications by authors named "Athanasios Saragliadis"

12 Publications

  • Page 1 of 1

Analysis of O-Antigen-Specific Bacteriophage P22 Inactivation by Outer Membrane Vesicles.

Front Microbiol 2020 24;11:510638. Epub 2020 Sep 24.

Physical Biochemistry, Department for Biochemistry and Biology, University of Potsdam, Potsdam, Germany.

Bacteriophages use a large number of different bacterial cell envelope structures as receptors for surface attachment. As a consequence, bacterial surfaces represent a major control point for the defense against phage attack. One strategy for phage population control is the production of outer membrane vesicles (OMVs). In Gram-negative host bacteria, O-antigen-specific bacteriophages address lipopolysaccharide (LPS) to initiate infection, thus relying on an essential outer membrane glycan building block as receptor that is constantly present also in OMVs. In this work, we have analyzed interactions of (.) bacteriophage P22 with OMVs. For this, we isolated OMVs that were formed in large amounts during mechanical cell lysis of the P22 S. Typhimurium host. , these OMVs could efficiently reduce the number of infective phage particles. Fluorescence spectroscopy showed that upon interaction with OMVs, bacteriophage P22 released its DNA into the vesicle lumen. However, only about one third of the phage P22 particles actively ejected their genome. For the larger part, no genome release was observed, albeit the majority of phages in the system had lost infectivity towards their host. With OMVs, P22 ejected its DNA more rapidly and could release more DNA against elevated osmotic pressures compared to DNA release triggered with protein-free LPS aggregates. This emphasizes that OMV composition is a key feature for the regulation of infective bacteriophage particles in the system.
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http://dx.doi.org/10.3389/fmicb.2020.510638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541932PMC
September 2020

CbpA is a collagen binding cell surface protein under c-di-GMP control.

Cell Surf 2019 Dec 23;5:100032. Epub 2019 Aug 23.

Centre for Integrative Microbial Evolution and Section for Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Norway.

Cyclic diguanylate (c-di-GMP) signalling affects several cellular processes in group bacteria including biofilm formation and motility, and CdgF was previously identified as a diguanylate cyclase promoting biofilm formation in C-di-GMP can exert its function as a second messenger via riboswitch binding, and a functional c-di-GMP-responsive riboswitch has been found upstream of in various group strains. Protein signature recognition predicted CbpA to be a cell wall-anchored surface protein with a fibrinogen or collagen binding domain. The aim of this study was to identify the binding ligand of CbpA and the function of CbpA in cellular processes that are part of the group c-di-GMP regulatory network. By global gene expression profiling was found to be down-regulated in a deletion mutant, and exhibited maximum expression in early exponential growth. Contrary to the wild type, a deletion mutant showed no binding to collagen in a cell adhesion assay, while a CbpA overexpression strain exhibited slightly increased collagen binding compared to the control. For both fibrinogen and fibronectin there was however no change in binding activity compared to controls, and CbpA did not appear to contribute to binding to abiotic surfaces (polystyrene, glass, steel). Also, the CbpA overexpression strain appeared to be less motile and showed a decrease in biofilm formation compared to the control. This study provides the first experimental proof that the binding ligand of the c-di-GMP regulated adhesin CbpA is collagen.
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http://dx.doi.org/10.1016/j.tcsw.2019.100032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423583PMC
December 2019

The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence.

Environ Microbiol 2020 07 17;22(7):2939-2955. Epub 2020 May 17.

Department of Biosciences, University of Oslo, 0316, Oslo, Norway.

Yersinia ruckeri causes enteric redmouth disease (ERM) that mainly affects salmonid fishes and leads to significant economic losses in the aquaculture industry. An increasing number of outbreaks and the lack of effective vaccines against some serotypes necessitates novel measures to control ERM. Importantly, Y. ruckeri survives in the environment for long periods, presumably by forming biofilms. How the pathogen forms biofilms and which molecular factors are involved in this process, remains unclear. Yersinia ruckeri produces two surface-exposed adhesins, belonging to the inverse autotransporters (IATs), called Y. ruckeri invasin (YrInv) and Y. ruckeri invasin-like molecule (YrIlm). Here, we investigated whether YrInv and YrIlm play a role in biofilm formation and virulence. Functional assays revealed that YrInv and YrIlm promote biofilm formation on different abiotic substrates. Confocal microscopy revealed that they are involved in microcolony interaction and formation, respectively. The effect of both IATs on biofilm formation correlated with the presence of different biopolymers in the biofilm matrix, including extracellular DNA, RNA and proteins. Moreover, YrInv and YrIlm contributed to virulence in the Galleria mellonella infection model. Taken together, we propose that both IATs are possible targets for the development of novel diagnostic and preventative strategies to control ERM.
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http://dx.doi.org/10.1111/1462-2920.15051DOI Listing
July 2020

Type V Secretion Systems: An Overview of Passenger Domain Functions.

Front Microbiol 2019 31;10:1163. Epub 2019 May 31.

Department of Biosciences, Section for Genetics and Evolutionary Biology, University of Oslo, Oslo, Norway.

Bacteria secrete proteins for different purposes such as communication, virulence functions, adhesion to surfaces, nutrient acquisition, or growth inhibition of competing bacteria. For secretion of proteins, Gram-negative bacteria have evolved different secretion systems, classified as secretion systems I through IX to date. While some of these systems consist of multiple proteins building a complex spanning the cell envelope, the type V secretion system, the subject of this review, is rather minimal. Proteins of the Type V secretion system are often called autotransporters (ATs). In the simplest case, a type V secretion system consists of only one polypeptide chain with a β-barrel translocator domain in the membrane, and an extracellular passenger or effector region. Depending on the exact domain architecture of the protein, type V secretion systems can be further separated into sub-groups termed type Va through e, and possibly another recently identified subtype termed Vf. While this classification works well when it comes to the architecture of the proteins, this is not the case for the function(s) of the secreted passenger. In this review, we will give an overview of the functions of the passengers of the different AT classes, shedding more light on the variety of functions carried out by type V secretion systems.
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http://dx.doi.org/10.3389/fmicb.2019.01163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555100PMC
May 2019

Assay development for the discovery of small-molecule inhibitors of YadA adhesion to collagen.

Cell Surf 2019 Dec 23;5:100025. Epub 2019 May 23.

Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo, Norway.

We set out to develop scalable assays to measure bacterial adhesion to mammalian extracellular matrix proteins, with the aim to perform high-throughput screening for inhibitors. Our model system is the trimeric autotransporter adhesin YadA from that binds to collagen. Using bacterial cells expressing GFP under an inducible promotor, and co-expressing the adhesin of choice, we were able to establish a 384-well plate-based assay that allowed us to screen 28,000 compounds in 8 days (3520 compounds per day). We have collected all parameters that were essential in assay development, and describe how they can be tuned for improved performance. Out of 28,000 compounds, 5 compounds showed significant inhibitory activity, measured as loss of fluorescence compared to control wells. Our assay is easy to scale up, and can be adopted to different ECM component/Adhesin combinations. Alternatively, bacterial pathogens (harboring deletion mutants of adhesins compared to wildtype) could be used directly in the same assay if they express GFP as a reporter at high levels.
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http://dx.doi.org/10.1016/j.tcsw.2019.100025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388967PMC
December 2019

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes.

J Vis Exp 2018 09 1(139). Epub 2018 Sep 1.

Evolution and Genetics, Department of Biosciences, University of Oslo;

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.
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http://dx.doi.org/10.3791/58267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235078PMC
September 2018

Ribozyme-based transfer RNA switches for post-transcriptional control of amino acid identity in protein synthesis.

J Am Chem Soc 2013 Jun 21;135(22):8222-6. Epub 2013 May 21.

Department of Chemistry and Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz , Universitätsstr. 10, 78457 Konstanz, Germany.

Protein mutants are studied in a variety of contexts in the life sciences. However, individual mutations need to be generated in order to transcribe and translate the respective protein variants. Here, we introduce a novel strategy for controlling the incorporation of different amino acids in response to an amber stop codon by utilizing switchable designer transfer RNAs in Escherichia coli .
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http://dx.doi.org/10.1021/ja311107pDOI Listing
June 2013

Thermozymes: Synthetic RNA thermometers based on ribozyme activity.

RNA Biol 2013 Jun 1;10(6):1010-6. Epub 2013 Apr 1.

Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.

Synthetic biology approaches often combine natural building blocks to generate new cellular activities. Here, we make use of two RNA elements to design a regulatory device with novel functionality. The system is based on a hammerhead ribozyme (HHR) that cleaves itself to generate a liberated ribosome-binding site and, thus, permits expression of a downstream gene. We connected a temperature-responsive RNA hairpin to the HHR and, thus, generated a temperature-controlled ribozyme that we call thermozyme. Specifically, a Salmonella RNA thermometer (RNAT) known to modulate small heat shock gene expression by temperature-controlled base-pairing and melting was fused to the ribozyme. Following an in vivo screening approach, we isolated two functional thermozymes. In vivo expression studies and in vitro structure probing experiments support a mechanism in which rising temperatures melt the thermometer structure impairing the self-cleavage reaction of the ribozyme. Since RNA cleavage is necessary to liberate the RBS, these engineered thermozymes shut off gene expression in response to a temperature increase and, thus, act in a reverse manner as the natural RNAT. Our results clearly emphasize the highly modular nature and biotechnological potential of ribozyme-based RNA thermometers.
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http://dx.doi.org/10.4161/rna.24482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111729PMC
June 2013

Post-transcriptional Boolean computation by combining aptazymes controlling mRNA translation initiation and tRNA activation.

Mol Biosyst 2012 Sep 9;8(9):2242-8. Epub 2012 Jul 9.

Department of Chemistry, University of Konstanz, Universitätsstr. 10, 78457 Konstanz, Germany.

In cellular systems environmental and metabolic signals are integrated for the conditional control of gene expression. On the other hand, artificial manipulation of gene expression is of high interest for metabolic and genetic engineering. Especially the reprogramming of gene expression patterns to orchestrate cellular responses in a predictable fashion is considered to be of great importance. Here we introduce a highly modular RNA-based system for performing Boolean logic computation at a post-transcriptional level in Escherichia coli. We have previously shown that artificial riboswitches can be constructed by utilizing ligand-dependent Hammerhead ribozymes (aptazymes). Employing RNA self-cleavage as the expression platform-mechanism of an artificial riboswitch has the advantage that it can be applied to control several classes of RNAs such as mRNAs, tRNAs, and rRNAs. Due to the highly modular and orthogonal nature of these switches it is possible to combine aptazyme regulation of activating a suppressor tRNA with the regulation of mRNA translation initiation. The different RNA classes can be controlled individually by using distinct aptamers for individual RNA switches. Boolean logic devices are assembled by combining such switches in order to act on the expression of a single mRNA. In order to demonstrate the high modularity, a series of two-input Boolean logic operators were constructed. For this purpose, we expanded our aptazyme toolbox with switches comprising novel behaviours with respect to the small molecule triggers thiamine pyrophosphate (TPP) and theophylline. Then, individual switches were combined to yield AND, NOR, and ANDNOT gates. This study demonstrates that post-transcriptional aptazyme-based switches represent versatile tools for engineering advanced genetic devices and circuits without the need for regulatory protein cofactors.
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http://dx.doi.org/10.1039/c2mb25091hDOI Listing
September 2012

In vivo screening of ligand-dependent hammerhead ribozymes.

Methods Mol Biol 2012 ;848:455-63

Department of Chemistry, University of Konstanz, Konstanz, Germany.

The development of artificial switches of gene expression is of high importance for future applications in biotechnology and synthetic biology. We have developed a powerful RNA-based system which allows for the ligand-dependent and reprogrammable control of gene expression in Escherichia coli. Our system makes use of the hammerhead ribozyme (HHR) which acts as molecular scaffold for the sequestration of the ribosome binding site (RBS), mimicking expression platforms in naturally occurring riboswitches. Aptamer domains can be attached to the ribozyme as exchangeable ligand-sensing modules. Addition of ligands to the bacterial growth medium changes the activity of the ligand-dependent self-cleaving ribozyme which in turn switches gene expression. In this chapter, we describe the in vivo screening procedure allowing for reprogramming the ligand-specificity of our system.
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http://dx.doi.org/10.1007/978-1-61779-545-9_28DOI Listing
May 2012

Receptor-mediated targeting of cathepsins in professional antigen presenting cells.

Angew Chem Int Ed Engl 2009 ;48(9):1629-32

Leiden Institute of Chemistry, Leiden University, P.O. Box 9052, 2300 RA Leiden, The Netherlands.

Tag for professionals: A fluorescently tagged clustered mannoside DCG-04 analogue (see structure) is designed and synthesized using a modular approach. Uptake of the probe in professional antigen presenting cells and subsequent labeling of cathepsins proceeded in a mannose-receptor dependent manner.
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http://dx.doi.org/10.1002/anie.200805529DOI Listing
March 2009

Design, synthesis, and evaluation of a lanthanide chelating protein probe: CLaNP-5 yields predictable paramagnetic effects independent of environment.

J Am Chem Soc 2008 Nov 1;130(44):14802-12. Epub 2008 Oct 1.

Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Post Office Box 9502, 2300 RA Leiden, The Netherlands.

Immobilized lanthanide ions offer the opportunity to refine structures of proteins and the complexes they form by using restraints obtained from paramagnetic NMR experiments. We report the design, synthesis, and spectroscopic evaluation of the lanthanide chelator, Caged Lanthanide NMR Probe 5 (CLaNP-5) readily attachable to a protein surface via two cysteine residues. The probe causes tunable pseudocontact shifts, alignment, paramagnetic relaxation enhancement, and luminescence, by chelating it to the appropriate lanthanide ion. The observation of single shifts and the finding that the magnetic susceptibility tensors obtained from shifts and alignment analyses are highly similar strongly indicate that the probe is rigid with respect to the protein backbone. By placing the probe at various positions on a model protein it is demonstrated that the size and orientation of the magnetic susceptibility tensor of the probe are independent of the local protein environment. Consequently, the effects of the probe are readily predictable using a protein structure only. These findings designate CLaNP-5 as a protein probe to deliver unambiguous high quality structural restraints in studies on protein-protein and protein-ligand interactions.
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http://dx.doi.org/10.1021/ja8054832DOI Listing
November 2008
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