Athanasios Saragliadis

• Saragliadis, Athanasios; Hatlem, Daniel & Linke, Dirk (2026). Purification of Bacterial Lipopolysaccharide, and How to Study Its Interactions with Proteins. Methods in molecular biology. ISSN 1064-3745. 3001, s. 117–132. doi: https:/link.springer.com/protocol/10.1007/978-1-0716-5054-7_6. Fulltekst i vitenarkiv
• Reese, Jan-Ole; Lund, Ingrid Maria Castro; Haugen, Håvard Jostein; Saragliadis, Athanasios; Lyngstadaas, Ståle Petter & Linke, Dirk (2026). A dual-loop chemostat to investigate multi-species biofilms on implant surfaces under adjustable flow conditions. Frontiers in Microbiology. 17. doi: 10.3389/fmicb.2026.1751315. Fulltekst i vitenarkiv Vis sammendrag

  Natural biofilms are typically composed of a mix of different microbial species and are often exposed to strong shear forces resulting from liquid flow. Simple biofilm models that attempt to study biofilms are based on a single species and on static growth conditions. To overcome these limitations, we developed a modular dual-loop reactor that decouples bacterial cultivation from hydrodynamic exposure, enabling independent control of nutrient availability (and thus, cell density) and flow rate (and thus, shear stress). Importantly, the system allows for testing different surface materials in a systematic manner. To validate our setup, we used a community of six keystone members of oral biofilms in conjunction with titanium materials of defined roughness that mimic dental implant surfaces. We found that biofilm mass, robustness, and species distribution not only differ significantly between static and dynamic growth conditions, but also vary strongly with different flow velocities. The biofilms formed under flow could be separated into two fractions, one that washed away very easily, and a more robust, basal layer. At low shear forces, overall biofilm mass was the highest, but at the expense of biofilm robustness. At medium shear forces, the robust fraction of the biofilm had the highest relative content of extracellular matrix. At the highest flow rates, the biofilm mass was low, but late colonizers (represented by the oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans ) had the lowest relative abundance. This is in accordance with the concept that high flow of saliva reduces the risk of oral disease. Future applications of our system will include the systematic testing of antimicrobial coatings or surface design effects under defined flow regimes, opening the path toward better medical implants.

• Reese, Jan-Ole; Lund, Ingrid M. Castro; Haugen, Håvard Jostein; Saragliadis, Athanasios; Lyngstadaas, Ståle Petter & Linke, Dirk (2026). A dual-loop chemostat to investigate multi-species biofilms on different materials under adjustable flow conditions. Frontiers in Microbiology. 17. Fulltekst i vitenarkiv Vis sammendrag

  Natural biofilms are typically composed of a mix of different microbial species and are often exposed to strong shear forces resulting from liquid flow. Simple biofilm models that attempt to study biofilms are based on a single species and on static growth conditions. To overcome these limitations, we developed a modular dual-loop reactor that decouples bacterial cultivation from hydrodynamic exposure, enabling independent control of nutrient availability (and thus, cell density) and flow rate (and thus, shear stress). Importantly, the system allows for testing different surface materials in a systematic manner. To validate our setup, we used a community of six keystone members of oral biofilms in conjunction with titanium materials of defined roughness that mimic dental implant surfaces. We found that biofilm mass, robustness, and species distribution not only differ significantly between static and dynamic growth conditions, but also vary strongly with different flow velocities. The biofilms formed under flow could be separated into two fractions, one that washed away very easily, and a more robust, basal layer. At low shear forces, overall biofilm mass was the highest, but at the expense of biofilm robustness. At medium shear forces, the robust fraction of the biofilm had the highest relative content of extracellular matrix. At the highest flow rates, the biofilm mass was low, but late colonisers (represented by the oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans) had the lowest relative abundance. This is in accordance with the concept that high flow of saliva reduces the risk of oral disease. Future applications of our system will include the systematic testing of antimicrobial coatings or surface design effects under defined flow regimes, opening the path towards better medical implants.

• Saragliadis, Athanasios & Linke, Dirk (2024). Bacterial Envelope Fractionation. I Raffaele, Ieva (Red.), Transmembrane β-Barrel Proteins. Humana Press. ISSN 9781071637333. s. 43–52. doi: 10.1007/978-1-0716-3734-0_3. Fulltekst i vitenarkiv
• Perilla, Ana Lucia Campana; Gomez-Bolivar, Jamie; Merroun, Mohamed; Joudeh, Nadeem; Saragliadis, Athanasios & Røyne, Anja [Vis alle 8 forfattere av denne artikkelen] (2024). Characterization of Bimetallic Pd-Fe Nanoparticles Synthesized in Escherichia coli. ACS Applied Bio Materials (AABM). 7(12), s. 8573–8589. doi: 10.1021/acsabm.4c01354. Fulltekst i vitenarkiv Vis sammendrag

  Biologically mediated nanoparticle (NP) synthesis offers a reliable and sustainable alternative route for metal NP production. Compared with conventional chemical and physical production methods that require hazardous materials and considerable energy expenditure, some microorganisms can reduce metal ions into NPs during standard metabolic processes. However, to be considered a feasible commercial option, the properties and inherent activity of bio-NPs still need to be significantly improved. In this work, we present an Escherichia coli-mediated synthesis method for catalytically active Pd–Fe NPs. The produced biogenic Pd–Fe NPs with varying Fe content were characterized using complementary analytical techniques to assess their size, composition, and structural properties. In addition, their catalytic performance was assessed by using standardized chemical reactions. We demonstrate that the combination of Pd with Fe leads to synergistic effects that enhance the catalytic performance of Pd NPs and make biogenic Pd–Fe NPs excellent potential substitutes for currently used catalysts. Briefly, the apparent rates for the model reaction of 4-nitrophenol reduction to 4-aminophenol catalyzed by Pd-based nanoparticles were as high as 0.1312 min–1 using bimetallic Pd–Fe NPs, which is far superior to the rates of monometallic Pd NPs counterparts. This study provides a feasible strategy for the synthesis of multimetallic Pd-based NPs using common microbial processes. It emphasizes the potential of biogenic Pd–Fe NPs as efficient and sustainable catalysts for hydrogenation reactions, offering an environmentally friendly synthesis for various applications, including wastewater treatment and the production of fine chemicals.

• Mertes, Verena; Saragliadis, Athanasios; Mascherin, E; Tysvær, Ellen-Beate; Roos, Norbert & Linke, Dirk [Vis alle 7 forfattere av denne artikkelen] (2024). Recombinant expression of Yersinia ruckeri outer membrane proteins in Escherichia coli extracellular vesicles. Protein Expression and Purification. ISSN 1046-5928. 215. doi: 10.1016/j.pep.2023.106409. Fulltekst i vitenarkiv Vis sammendrag

  The secretion of extracellular vesicles (EVs) is a common process in Gram-negative bacteria and can be exploited for biotechnological applications. EVs pose a self-adjuvanting, non-replicative vaccine platform, where membrane and antigens are presented to the host immune system in a non-infectious fashion. The secreted quantity of EVs varies between Gram-negative bacterial species and is comparatively high in the model bacterium E. coli. The outer membrane proteins OmpA and OmpF of the fish pathogen Y. ruckeri have been proposed as vaccine candidates to prevent enteric redmouth disease in aquaculture. In this work, Y.ruckeri OmpA or OmpF were expressed in E. coli and recombinant EVs were isolated. To avoid competition between endogenous E. coli OmpA or OmpF, Y. ruckeri OmpA and OmpF were expressed in E. coli strains lacking ompA, ompF, and in a quadruple knockout strain where the four major outer membrane protein genes ompA, ompC, ompF and lamB were removed. Y.ruckeri OmpA and OmpF were successfully expressed in EVs derived from the E. coli mutants as verified by SDS-PAGE, heat modifiability and proteomic analysis using mass-spectrometry. Transmission electron microscopy revealed the presence of EVs in all E. coli strains, and increased EV concentrations were detected when expressing Y. ruckeri OmpA or OmpF in recombinant EVs compared to empty vector controls as verified by nanoparticle tracking analysis. These results show that E. coli can be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri.

• Perilla, Ana Lucia Campana; Saragliadis, Athanasios; Mikheenko, Pavlo & Linke, Dirk (2023). Insights into the bacterial synthesis of metal nanoparticles. Frontiers in Nanotechnology. 5. doi: 10.3389/fnano.2023.1216921. Fulltekst i vitenarkiv
• Sande, Maria G.; Ferreira, Débora; Rodrigues, Joana L.; Melo, Luís D. R.; Saragliadis, Athanasios & Linke, Dirk [Vis alle 9 forfattere av denne artikkelen] (2023). Aptasensor for the Detection of Moraxella catarrhalis Adhesin UspA2. Bioengineering. 10(2), s. 1–20. doi: 10.3390/bioengineering10020178. Fulltekst i vitenarkiv Vis sammendrag

  Innovative point-of-care (PoC) diagnostic platforms are desirable to surpass the deficiencies of conventional laboratory diagnostic methods for bacterial infections and to tackle the growing antimicrobial resistance crisis. In this study, a workflow was implemented, comprising the identification of new aptamers with high affinity for the ubiquitous surface protein A2 (UspA2) of the bacterial pathogen Moraxella catarrhalis and the development of an electrochemical biosensor functionalized with the best-performing aptamer as a bioreceptor to detect UspA2. After cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) was performed, next-generation sequencing was used to sequence the final aptamer pool. The most frequent aptamer sequences were further evaluated using bioinformatic tools. The two most promising aptamer candidates, Apt1 and Apt1_RC (Apt1 reverse complement), had Kd values of 214.4 and 3.4 nM, respectively. Finally, a simple and label-free electrochemical biosensor was functionalized with Apt1_RC. The aptasensor surface modifications were confirmed by impedance spectroscopy and cyclic voltammetry. The ability to detect UspA2 was evaluated by square wave voltammetry, exhibiting a linear detection range of 4.0 × 104–7.0 × 107 CFU mL−1, a square correlation coefficient superior to 0.99 and a limit of detection of 4.0 × 104 CFU mL−1 at pH 5.0. The workflow described has the potential to be part of a sensitive PoC diagnostic platform to detect and quantify M. catarrhalis from biological samples.

• Hermansen, Simen; Ryoo, David; Rydmark, Marcella Christine Orwick; Saragliadis, Athanasios; Gumbart, James C & Linke, Dirk (2022). The Role of Extracellular Loops in the Folding of Outer Membrane Protein X (OmpX) of Escherichia coli. Frontiers in Molecular Biosciences. 9. doi: 10.3389/fmolb.2022.918480. Fulltekst i vitenarkiv Vis sammendrag

  The outer membrane of Gram-negative bacteria acts as an additional diffusion barrier for solutes and nutrients. It is perforated by outer membrane proteins (OMPs) that function most often as diffusion pores, but sometimes also as parts of larger cellular transport complexes, structural components of the cell wall, or even as enzymes. These OMPs often have large loops that protrude into the extracellular environment, which have promise for biotechnological applications and as therapeutic targets. Thus, understanding how modifications to these loops affect OMP stability and folding is critical for their efficient application. In this work, the small outer membrane protein OmpX was used as a model system to quantify the effects of loop insertions on OMP folding and stability. The insertions were varied according to both hydrophobicity and size, and their effects were determined by assaying folding into detergent micelles in vitro by SDS-PAGE and in vivo by isolating the outer membrane of cells expressing the constructs. The different insertions were also examined in molecular dynamics simulations to resolve how they affect OmpX dynamics in its native outer membrane. The results indicate that folding of OMPs is affected by both the insert length and by its hydrophobic character. Small insertions sometimes even improved the folding efficiency of OmpX, while large hydrophilic inserts reduced it. All the constructs that were found to fold in vitro could also do so in their native environment. One construct that could not fold in vitro was transported to the OM in vivo , but remained unfolded. Our results will help to improve the design and efficiency of recombinant OMPs used for surface display.

• Joudeh, Nadeem; Saragliadis, Athanasios; Koster, Gerbrand; Mikheenko, Pavlo & Linke, Dirk (2022). Synthesis methods and applications of palladium nanoparticles: A review. Frontiers in Nanotechnology. 4, s. 1–24. doi: 10.3389/fnano.2022.1062608. Fulltekst i vitenarkiv Vis sammendrag

  Palladium (Pd) is a key component of many catalysts. Nanoparticles (NPs) offer a larger surface area than bulk materials, and with Pd cost increasing 5-fold in the last 10 years, Pd NPs are in increasing demand. Due to novel or enhanced physicochemical properties that Pd NPs exhibit at the nanoscale, Pd NPs have a wide range of applications not only in chemical catalysis, but also for example in hydrogen sensing and storage, and in medicine in photothermal, antibacterial, and anticancer therapies. Pd NPs, on the industrial scale, are currently synthesized using various chemical and physical methods. The physical methods require energy-intensive processes that include maintaining high temperatures and/or pressure. The chemical methods usually involve harmful solvents, hazardous reducing or stabilizing agents, or produce toxic pollutants and by-products. Lately, more environmentally friendly approaches for the synthesis of Pd NPs have emerged. These new approaches are based on the use of the reducing ability of phytochemicals and other biomolecules to chemically reduce Pd ions and form NPs. In this review, we describe the common physical and chemical methods used for the synthesis of Pd NPs and compare them to the plant- and bacteria-mediated biogenic synthesis methods. As size and shape determine many of the unique properties of Pd NPs on the nanoscale, special emphasis is given to the control of these parameters, clarifying how they impact current and future applications of this exciting nanomaterial.

• Thibau, Arno; Hipp, Katharina; Vaca, Diana; Chowdhury, Sounak; Malmström, Johan & Saragliadis, Athanasios [Vis alle 9 forfattere av denne artikkelen] (2022). Long-read sequencing reveals genetic adaptation of Bartonella adhesin A among different Bartonella henselae isolates. Frontiers in Microbiology. 13. doi: 10.3389/fmicb.2022.838267. Fulltekst i vitenarkiv Vis sammendrag

  Bartonella henselae is the causative agent of cat scratch disease and other clinical entities such as endocarditis and bacillary angiomatosis. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations. Human, feline, and laboratory adapted B. henselae isolates often display genomic and phenotypic differences that are related to the expression of outer membrane proteins, for example the Bartonella adhesin A (BadA). This modularly-structured trimeric autotransporter adhesin is a major virulence factor of B. henselae and is crucial for the initial binding to the host via the extracellular matrix proteins fibronectin and collagen. By using next-generation long-read sequencing we demonstrate a conserved genome among eight B. henselae isolates and identify a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes. Two of the eight tested B. henselae strains lack BadA expression because of frameshift mutations. We suggest that active recombination mechanisms, possibly via phase variation (i.e., slipped-strand mispairing and site-specific recombination) within the repetitive badA island facilitate reshuffling of homologous domain arrays. The resulting variations among the different BadA proteins might contribute to host immune evasion and enhance long-term and efficient colonisation in the differing host environments. Considering the role of BadA as a key virulence factor, it remains important to check consistently and regularly for BadA surface expression during experimental infection procedures.

• Sande, Maria; Roque, Lucia; Braga, Adelaide; Marques, Marcia; Ferreira, Debora & Saragliadis, Athanasios [Vis alle 11 forfattere av denne artikkelen] (2022). Design of new hydrolyzed collagen-modified magnetic nanoparticles to capture pathogens. Journal of Biomedical Materials Research. Part B - Applied biomaterials. ISSN 1552-4973. doi: 10.1002/jbm.b.35155. Fulltekst i vitenarkiv
• Joudeh, Nadeem; Saragliadis, Athanasios; Schulz, Christian; Voigt, Andre; Almaas, Eivind & Linke, Dirk (2021). Transcriptomic Response Analysis of Escherichia coli to Palladium Stress. Frontiers in Microbiology. 12. doi: 10.3389/fmicb.2021.741836. Fulltekst i vitenarkiv Vis sammendrag

  Palladium (Pd), due to its unique catalytic properties, is an industrially important heavy metal especially in the form of nanoparticles. It has a wide range of applications from automobile catalytic converters to the pharmaceutical production of morphine. Bacteria have been used to biologically produce Pd nanoparticles as a new environmentally friendly alternative to the currently used energy-intensive and toxic physicochemical methods. Heavy metals, including Pd, are toxic to bacterial cells and cause general and oxidative stress that hinders the use of bacteria to produce Pd nanoparticles efficiently. In this study, we show in detail the Pd stress-related effects on E. coli. Pd stress effects were measured as changes in the transcriptome through RNA-Seq after 10 min of exposure to 100 μM sodium tetrachloropalladate (II). We found that 709 out of 3,898 genes were differentially expressed, with 58% of them being up-regulated and 42% of them being down-regulated. Pd was found to induce several common heavy metal stress-related effects but interestingly, Pd causes unique effects too. Our data suggests that Pd disrupts the homeostasis of Fe, Zn, and Cu cellular pools. In addition, the expression of inorganic ion transporters in E. coli was found to be massively modulated due to Pd intoxication, with 17 out of 31 systems being affected. Moreover, the expression of several carbohydrate, amino acid, and nucleotide transport and metabolism genes was vastly changed. These results bring us one step closer to the generation of genetically engineered E. coli strains with enhanced capabilities for Pd nanoparticles synthesis.

• Beal, J.; Saragliadis, Athanasios & Linke, Dirk (2021). Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs. PLOS ONE. 16(6). doi: 10.1371/journal.pone.0252263. Fulltekst i vitenarkiv Vis sammendrag

  Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers.

• Stephan, Mareike; Broeker, Nina; Saragliadis, Athanasios; Roos, Norbert; Linke, Dirk & Barbirz, Stefanie (2020). In vitro Analysis of O-Antigen-Specific Bacteriophage P22 Inactivation by Salmonella Outer Membrane Vesicles. Frontiers in Microbiology. 11. doi: 10.3389/fmicb.2020.510638. Fulltekst i vitenarkiv Vis sammendrag

  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 Salmonella (S.) 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. In vitro, 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.

• Wróbel, Agnieszka Ewelina; Saragliadis, Athanasios; Pérez-Ortega, Jesús; Sittman, Carolin; Göttig, Stephan & Liskiewicz, Krystyna Anna [Vis alle 11 forfattere av denne artikkelen] (2020). The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence. Environmental Microbiology. ISSN 1462-2912. 22(7), s. 2939–2955. doi: 10.1111/1462-2920.15051. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Beal, J; Saragliadis, Athanasios & Linke, Dirk (2020). Robust estimation of bacterial cell count from optical density. Communications Biology. 3(1). doi: 10.1038/s42003-020-01127-5. Fulltekst i vitenarkiv Vis sammendrag

  Abstract Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli . Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals  <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.

• Saragliadis, Athanasios & Linke, Dirk (2019). Assay development for the discovery of small-molecule inhibitors of YadA adhesion to collagen. The Cell Surface. 5, s. 1–11. doi: 10.1016/j.tcsw.2019.100025. Fulltekst i vitenarkiv Vis sammendrag

  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 Yersinia enterocolitica 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.

• Meuskens, Ina; Saragliadis, Athanasios; Leo, Jack Christopher & Linke, Dirk (2019). Type V secretion systems: An overview of passenger domain functions. Frontiers in Microbiology. 10, s. 1–19. doi: 10.3389/fmicb.2019.01163. Fulltekst i vitenarkiv Vis sammendrag

  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.

• Finke, Sarah; Fagerlund, Annette; Smith, Veronika; Krogstad, Veronica; Zhang, Mimmi Jingxi & Saragliadis, Athanasios [Vis alle 9 forfattere av denne artikkelen] (2019). Bacillus thuringiensis CbpA is a collagen binding cell surface protein under c-di-GMP control. The Cell Surface. 5, s. 1–12. doi: 10.1016/j.tcsw.2019.100032. Fulltekst i vitenarkiv Vis sammendrag

  Cyclic diguanylate (c-di-GMP) signalling affects several cellular processes in Bacillus cereus group bacteria including biofilm formation and motility, and CdgF was previously identified as a diguanylate cyclase promoting biofilm formation in B. thuringiensis. 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 cbpA in various B. cereus 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 B. cereus group c-di-GMP regulatory network. By global gene expression profiling cbpA was found to be down-regulated in a cdgF deletion mutant, and cbpA exhibited maximum expression in early exponential growth. Contrary to the wild type, a ΔcbpA 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.

• Saragliadis, Athanasios; Trunk, Thomas & Leo, Jack Christopher (2018). Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. Journal of Visualized Experiments. 2018(139). doi: 10.3791/58267. Fulltekst i vitenarkiv Vis sammendrag

  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.

Se alle arbeider i NVA

• Johansen, Emma Marinelli; Saragliadis, Athanasios & Linke, Dirk (2025). Inhibiting Collagen-Binding Trimeric Autotransporter Adhesins from Clinically Relevant Pathogens. Fulltekst i vitenarkiv
• Lund, Ingrid Maria Castro; Reese, Jan-Ole; Saragliadis, Athanasios; Lyngstadaas, Ståle Petter; Haugen, Håvard Jostein & Linke, Dirk (2025). Discovering the species-specific dynamics within a model biofilm system for peri-implantitis. Fulltekst i vitenarkiv
• Hatlem, Daniel; Saragliadis, Athanasios & Linke, Dirk (2025). An LPS-binding peptide with extreme affinity can be used for novel endotoxin detection and removal applications. Fulltekst i vitenarkiv
• Perilla, Ana Lucia Campana; Saragliadis, Athanasios; Merroun, Mohamed; Rachel, Reinhard; Mikheenko, Pavlo & Linke, Dirk (2025). Microbial strategies for tailored metal nanoparticle production. Fulltekst i vitenarkiv
• Saragliadis, Athanasios; Winther, Anja Ruud; Leo, Jack Christopher & Linke, Dirk (2025). Fluorescent labeling of bacterial cell envelope and its application in membrane protein enrichment. Fulltekst i vitenarkiv
• Lislerud, Anna; Saragliadis, Athanasios; Nakatani, Hajime; Hori, Katsutoshi & Linke, Dirk (2025). Investigating the role of the YrInv and YrIlm inverse autotransporter invasins in Y. ruckeri pathogenesis. Fulltekst i vitenarkiv
• Reese, Jan-Ole; Lund, Ingrid Maria Castro; Haugen, Håvard Jostein; Saragliadis, Athanasios; Lyngstadaas, Ståle Petter & Linke, Dirk (2025). Development of a flow-controlled biofilm model to facilitate the testing of debridement treatments of dental implant surfaces. Fulltekst i vitenarkiv
• Reese, Jan-Ole; Saragliadis, Athanasios; Haugen, Håvard Jostein & Linke, Dirk (2024). Small-scale bioreactor setup to create a multi-species oral biofilm model for evaluating medical approaches in dentistry. Fulltekst i vitenarkiv
• Mertes, Verena; Linke, Dirk; Saragliadis, Athanasios; Roos, Norbert; Tysvær, Ellen-Beate & Mascherin, Elisa (2024). Recombinant extracellular vesicles as vaccines within animal health. Fulltekst i vitenarkiv Vis sammendrag

  Introduction: Secretion of extracellular vesicles (EVs) from the bacterial body is associated with a range of phenotypes including cell-cell communication, host-pathogen interactions and antimicrobial resistance development. EV may also elicit a humoral and antibody mediated immune response after immunization of a host making them an interesting vaccine candidate against an infectious agent. We have investigated the use of EV as a potential vaccine against several intracellular bacterial pathogens creating problems in farmed animals including fish. Immunization with EV revealed protection against some diseases, but not in others where we hypothesized that the EV was part of the microbial pathogenesis of the disease. To circumvent possible negative side effects of natural BEV with the host, we have explored the immunogenic properties of recombinant BEV (rEV) isolated from E. coli expressing antigens from the infectious bacterial species. Methods: We have expressed antigens from the fish bacterial species Yersinia ruckeri in E. coli. The rEV were isolated from E. coli using tangential flow and ultracentrifugation and compared to BEV isolated from the native E. coli strain. The rEVs were analyzed by transmission electron microscopy (TEM) and Nano-particle tracking analysis (NTA), and the antigen expression were confirmed by SDS-PAGE and mass spectrometry. The rEVs were exposed on fish cell cultures and the immune modulating properties were evaluated by qPCR. Results: Transmission electron microscopy revealed the presence of rEVs in the E. coli with similar shape to native E. coli EVs. Y. ruckeri antigens were successfully expressed in the rEVs as verified by SDS-PAGE and proteomic analysis using mass-spectrometry. Increased rEV concentrations were detected E. coli when expressing Y. ruckeri antigens compared to empty vector controls as verified by nanoparticle tracking analysis. Summary/conclusion: These results show that E. coli has the potential to be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri and probably antigens from other bacterial species. These rEVs will hopefully circumvent the negative side effects of native EVs isolated from the pathogenic bacteria when used as vaccines and may enhance the repertoire of EVs based vaccines within animal health.

• Mertes, Verena; Linke, Dirk; Saragliadis, Athanasios; Roos, Norbert; Mascherin, Elisa & Tysvær, Ellen-Beate (2024). Recombinant extracellular vesicles as vaccines within animal health. Fulltekst i vitenarkiv Vis sammendrag

  Introduction: Secretion of extracellular vesicles (EVs) from the bacterial body is associated with a range of phenotypes including cell-cell communication, host-pathogen interactions and antimicrobial resistance development. EV may also elicit a humoral and antibody mediated immune response after immunization of a host making them an interesting vaccine candidate against an infectious agent. We have investigated the use of EV as a potential vaccine against several intracellular bacterial pathogens creating problems in farmed animals including fish. Immunization with EV revealed protection against some diseases, but not in others where we hypothesized that the EV was part of the microbial pathogenesis of the disease. To circumvent possible negative side effects of natural BEV with the host, we have explored the immunogenic properties of recombinant BEV (rEV) isolated from E. coli expressing antigens from the infectious bacterial species. Methods: We have expressed antigens from the fish bacterial species Yersinia ruckeri in E. coli. The rEV were isolated from E. coli using tangential flow and ultracentrifugation and compared to BEV isolated from the native E. coli strain. The rEVs were analyzed by transmission electron microscopy (TEM) and Nano-particle tracking analysis (NTA), and the antigen expression were confirmed by SDS-PAGE and mass spectrometry. The rEVs were exposed on fish cell cultures and the immune modulating properties were evaluated by qPCR. Results: Transmission electron microscopy revealed the presence of rEVs in the E. coli with similar shape to native E. coli EVs. Y. ruckeri antigens were successfully expressed in the rEVs as verified by SDS-PAGE and proteomic analysis using mass-spectrometry. Increased rEV concentrations were detected E. coli when expressing Y. ruckeri antigens compared to empty vector controls as verified by nanoparticle tracking analysis. Summary/conclusion: These results show that E. coli has the potential to be utilized as a vector for production of EVs expressing outer membrane antigens from Y. ruckeri and probably antigens from other bacterial species. These rEVs will hopefully circumvent the negative side effects of native EVs isolated from the pathogenic bacteria when used as vaccines and may enhance the repertoire of EVs based vaccines within animal health.

• Winther-Larsen, Hanne Cecilie; Mertes, Verena; Linke, Dirk; Saragliadis, Athanasios; Roos, Norbert & Mascherin, Elisa [Vis alle 7 forfattere av denne artikkelen] (2024). Recombinant extracellular vesicles as vaccines within animal health. Fulltekst i vitenarkiv Vis sammendrag

  Secretion of extracellular vesicles (EVs) from the bacterial body is associated with a range of phenotypes including cell-cell communication, host-pathogen interactions and antimicrobial resistance development. EV also elicit an immune response after immunization of a host making them an interesting vaccine candidate. We have investigated the use of EV as a potential vaccine against several bacterial pathogens creating problems in farmed animals including fish. Immunization with EV revealed protection against some diseases, but not in others where we hypothesized that the EV was part of the microbial pathogenesis. To circumvent possible negative side effects of natural EV, we have explored the recombinant EV (rEV) isolated from E. coli expressing antigens from other infectious agents. We have expressed antigens from the fish bacterial species Yersinia ruckeri in E. coli. The rEV was isolated using tangential flow and ultracentrifugation before analysis by transmission electron microscopy (TEM) and Nano-particle tracking analysis (NTA). The antigen expression was confirmed by SDS-PAGE and proteomics analysis. The rEVs did not elicit any toxicity when exposed to fish cells in culture. These results show that E. coli has the potential to be utilized as a vector for production of EVs expressing antigens from other bacterial species. These rEVs will hopefully circumvent the negative side effects of native EVs isolated from the pathogenic bacteria when used as vaccines and may enhance the repertoire of EVs based vaccines within animal health.

• Johansen, Emma; Saragliadis, Athanasios & Linke, Dirk (2024). Inhibiting bacterial adhesins. Fulltekst i vitenarkiv
• Lislerud, Anna; Saragliadis, Athanasios & Linke, Dirk (2024). Adhesins of Yersinia ruckeri. Fulltekst i vitenarkiv
• Crnic, Irene Fausk; Saragliadis, Athanasios; Cortés, Natalia Mojica; Kristiansen, Per Eugen & Krengel, Ute (2024). Molecular interactions between LPS and bacterial toxin. Fulltekst i vitenarkiv
• Perilla, Ana Lucia Campana; Saragliadis, Athanasios; Merroun, Mohamed; Mikheenko, Pavlo & Linke, Dirk (2024). Microbial-metal interactions for biosynthesis of tailored metal nanoparticles. Fulltekst i vitenarkiv
• Saragliadis, Athanasios & Linke, Dirk (2023). Assay development for novel antimicrobials: screening for small-molecule inhibitors of YadA adhesion to collagen. Fulltekst i vitenarkiv
• Reese, Jan-Ole; Saragliadis, Athanasios; Haugen, Håvard Jostein & Linke, Dirk (2023). Development of multi-species biofilm models for testing biotechnological approaches in dentistry. Fulltekst i vitenarkiv
• Lislerud, Anna; Nakatani, Hajime; Hori, Katsutoshi; Spence, Maria Helle; Saragliadis, Athanasios & Linke, Dirk (2023). Characterization of adhesion factors from the microbial fish pathogen Yersinia ruckeri and their association with host receptors. Fulltekst i vitenarkiv
• Mertes, Verena; Saragliadis, Athanasios; Mascherin, E; Antenucci, Fabio; Bojesen, Anders Miki & Colquhoun, Duncan John [Vis alle 8 forfattere av denne artikkelen] (2023). Characterization of bacterial extracellular vesicles A versatile tool for potential vaccine application in aquaculture. Fulltekst i vitenarkiv
• Saragliadis, Athanasios & Linke, Dirk (2023). Development of assays for novel antimicrobials: screening for small-molecule inhibitors of YadA adhesion to collagen. Fulltekst i vitenarkiv
• Reese, Jan-Ole; Haugen, Håvard Jostein; Saragliadis, Athanasios & Linke, Dirk (2023). Development of multi-species biofilm models for evaluating medical approaches in dentistry. Fulltekst i vitenarkiv
• Lislerud, Anna; Nakatani, Hajime; Spence, Maria Helle; Saragliadis, Athanasios; Hori, Katsutoshi & Linke, Dirk (2023). Adhesins of Yersinia ruckeri. Fulltekst i vitenarkiv
• Mertes, Verena; Saragliadis, Athanasios; Ciani, Elia; Colquhoun, Duncan John; Linke, Dirk & Winther-Larsen, Hanne Cecilie (2023). Characterization of extracellular membrane vesicles secreted from the Gram-negative bacterial fish pathogen Yersinia ruckeri. Fulltekst i vitenarkiv
• Reese, Jan-Ole; Saragliadis, Athanasios; Haugen, Håvard Jostein & Linke, Dirk (2023). Development of multi-species biofilm models for evaluating medical approaches in dentistry. Fulltekst i vitenarkiv
• Lislerud, Anna; Nakatani, Hajime; Spence, Maria Helle; Saragliadis, Athanasios; Hori, Katsutoshi & Linke, Dirk (2023). Adhesins of Yersinia ruckeri - Characterization of adhesion factors from the microbial fish pathogen Yersinia ruckeri and their association with host receptors. Fulltekst i vitenarkiv
• Reese, Jan-Ole; Saragliadis, Athanasios; Haugen, Håvard Jostein & Linke, Dirk (2022). Development of multi-species biofilm models for testing biotechnological approaches in dentistry. Fulltekst i vitenarkiv
• Saragliadis, Athanasios & Linke, Dirk (2022). Structure-function relationship in bacterial adhesins. Fulltekst i vitenarkiv
• Mertes, Verena; Saragliadis, Athanasios; Attenucci, Fabio; Wrobel, Agnieszka; Ciani, Elia & Linke, Dirk [Vis alle 8 forfattere av denne artikkelen] (2022). Characterization of bacterial extracellular vesicles from a fish pathogen, their role in virulence and possible function as vaccine in aquaculture. Fulltekst i vitenarkiv
• Mertes, Verena; Saragliadis, Athanasios; Ciani, Elia; Linke, Dirk; Colquhoun, Duncan J & Winther-Larsen, Hanne Cecilie (2021). Characterization of membrane vesicles from fish pathogens, their role in virulence and possible function as a vaccine in aquaculture. Fulltekst i vitenarkiv
• Saragliadis, Athanasios & Linke, Dirk (2019). Assay development for the discovery of small-molecule inhibitors of YadA adhesion to collagen. Fulltekst i vitenarkiv
• Linke, Dirk & Saragliadis, Athanasios (2019). Bacterial adhesion to living and non-living surfaces. Fulltekst i vitenarkiv
• Saragliadis, Athanasios & Linke, Dirk (2019). Screening for small-molecule inhibitors of YadA adhesion to collagen. Fulltekst i vitenarkiv
• Linke, Dirk & Saragliadis, Athanasios (2018). Trimeric Autotransporter Adhesins - understanding adhesive function and screening for inhibitors. Fulltekst i vitenarkiv
• Wrobel, Agnieszka; Saragliadis, Athanasios; Leo, Jack Christopher; Ottoni, Claudio & Linke, Dirk (2018). Type Ve autotransporters in Yersinia ruckeri. Fulltekst i vitenarkiv
• Linke, Dirk & Saragliadis, Athanasios (2018). Trimeric Autotransporter Adhesins - Understanding adhesive function and screening for inhibitors. Fulltekst i vitenarkiv
• Hatlem, Daniel; Leo, Jack Christopher; Saragliadis, Athanasios; Krengel, Ute & Linke, Dirk (2017). Bacterial adhesion: Characterization of trimeric autotransporter binding. Fulltekst i vitenarkiv
• Saragliadis, Athanasios & Linke, Dirk (2017). STRUCTURE-FUNCTION RELATIONSHIP IN BACTERIAL ADHESINS. Fulltekst i vitenarkiv
• Schneider, Kenneth; Saragliadis, Athanasios; Haugen, Håvard Jostein & Linke, Dirk (2016). Aggregatibacter Adhesins and their interaction with implant materials. Fulltekst i vitenarkiv
• Hatlem, Daniel; Saragliadis, Athanasios; Leo, Jack Christopher; Shahid, Shakeel; Rossum, Barth van & Linke, Dirk (2015). TAAs: fibrous adhesins on the bacterial cell surface. Fulltekst i vitenarkiv
• Bergvoll, Helga; Linke, Dirk & Saragliadis, Athanasios (2022). Secreted toxin components in the fish pathogen Tenacibaculum maritimum. Universitetet i Oslo. Fulltekst i vitenarkiv
• Hovland, Hege; Linke, Dirk & Saragliadis, Athanasios (2022). Palladium nanoparticles in copper transport mutants of Escherichia coli. Universitetet i Oslo. Fulltekst i vitenarkiv
• Spence, Maria Helle; Linke, Dirk & Saragliadis, Athanasios (2021). Yersinia adhesins: Investigating the role of autotransporter adhesins in fish infection by Yersinia ruckeri. Universitetet i Oslo. Fulltekst i vitenarkiv

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