Dirk Linke

• 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
• Adélaïde, Morgane; Yarava, Jayasubba Reddy; Lindemann, Florian; Abbas, Massilia; Cremer, Nils & Gerland, Lisa [Vis alle 14 forfattere av denne artikkelen] (2026). Structure and Dynamics of Porin Loops in Asymmetric Outer Membranes of E. coli. Chemistry - A European Journal. ISSN 0947-6539. doi: 10.1002/chem.71575. Fulltekst i vitenarkiv Vis sammendrag

  Biological membranes are usually asymmetric lipid bilayers that possess an oligosaccharide coat on the outside into which loops of transmembrane proteins extend. Structurally, the protein-bilayer interface is not well explored. As a paradigmatic example, we examine the structure of the bacterial outer membrane protein G (OmpG) expressed into E. coli outer membranes (OM). We investigate two strands of the β-sheet and loops 6 and 7 that extend into the lipopolysaccharide (LPS) layer and which are involved in pore opening (pH > 6.0) and closing (pH < 6.0). Previous x-ray crystallography studies described these loops as well-ordered, whereas solid-state NMR described them as fully disordered, largely due to a lack of signal. Here, solid-state NMR measurements of OmpG in OM vesicles at 1.2 GHz 1H frequency now yield extended sets of cross peaks for His and Trp residues. Two alternative conformations are observed for two His residues, which are likely H231 and H261. Trp sidechain cross peak intensities in cross polarization spectra are in agreement with an increase of conformational freedom toward the surface. The oligosaccharide layer appears to protect the loops while promoting flexibility and multiple conformations. Our investigation highlights the importance of structural investigations in a quasi-native environment.

• Lauretis, Angela De; Santacroce, Marco; Wang, Qiang; Ramirez, Manuel; Ma, Qianli & Lyngstadaas, Ståle Petter [Vis alle 9 forfattere av denne artikkelen] (2026). Combined Chemical and Mechanical Debridement Enhances Salivary Protein Removal from Titanium while Maintaining Biological Properties. ACS Biomaterials Science and Engineering. doi: 10.1021/acsbiomaterials.5c01973. Fulltekst i vitenarkiv Vis sammendrag

  Effective decontamination of dental implants is essential for managing peri-implant diseases, yet its impact on the salivary pellicle and cell response remains unclear. As the pellicle mediates bacterial and host cell interactions, investigating how decontamination alters its composition is key to understanding the effects on biological outcomes. Accordingly, we examined how a Poloxamer 407 (P407) hydrogel, a P407 + hydrogen peroxide (H2O2) hydrogel, a titanium brush, and their combinations influence the adsorbed salivary proteins and cell adhesion on OsseoSpeed-like titanium surfaces. Surfaces with salivary proteins were treated with different decontamination methods and re-exposed to saliva. Protein quantity was measured using the BCA assay, while the proteomic composition and molecular functions of the adsorbed proteins were determined by liquid chromatography-mass spectrometry and gene ontology analysis. Human bone marrow mesenchymal stem cell (hBMMSC) adhesion was assessed on the surfaces, and cytotoxicity was evaluated using an LDH assay. Protein-coated and protein-free surfaces served as controls. Although protein levels were consistent across groups after either decontamination or recontamination, each treatment produced a different proteomic profile. Mechanical decontamination, alone or combined with chemicals, enhanced protein removal efficiency. Combined treatments yielded reduced protein diversity following recontamination and enriched adhesion-related proteins. None of the treatments compromised hBMMSC adhesion nor made the surface cytotoxic. Decontamination methods selectively modify the proteomic profile. Combined chemical and mechanical decontamination promotes the removal of salivary proteins and limits their readsorption while preserving cytocompatibility, supporting their relevance to peri-implant health.

• 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.

• Wang, Qiang; Haugen, Håvard Jostein; Linke, Dirk; Lyngstadaas, Ståle Petter & Ma, Qianli (2026). Residuals of Chemical Cleaning Agents Impair Peri-Implant Cell Viability: An in Vitro Study. ACS Biomaterials Science and Engineering. doi: 10.1021/acsbiomaterials.5c01777. Fulltekst i vitenarkiv Vis sammendrag

  Background: Chemical debridement agents are commonly used during the cleaning of implants for peri-implantitis treatment; however, how these agents affect lesion healing remains unclear. In addition, the dose- and time-dependent effects of these residuals on implant biocompatibility remain poorly understood. Materials and methods: We evaluated the effects of active compounds in commercial products-3% hydrogen peroxide (H2O2), 0.43% sodium hypochlorite (NaClO), and 0.12% chlorhexidine with 0.05% cetylpyridinium chloride (CHX-CPC) at graded dilutions on murine osteoblastic cells (MC3T3-E1), human gingival fibroblasts (HGFs), and human bone marrow mesenchymal stromal cells (hBMSCs). Cells were cultured for 24 h, then exposed to the agents for 2, 12, or 24 h. Cytotoxicity and viability were assessed using lactate dehydrogenase (LDH) release and CCK-8 assays, while cell morphology was examined by scanning electron microscopy (SEM). Apoptotic gene expression (BCL2, MCL1, BAX) was analyzed after 2 h using quantitative PCR. Results: At high concentrations, H2O2 and NaClO significantly reduced LDH activity in supernatant, likely due to oxidant-induced enzyme inactivation. All three agents inhibited cell viability in a dose- and time-dependent manner, accompanied by cell shrinkage and deformation. Among the tested cell types, hBMSCs displayed greater resistance to H2O2, maintaining proliferative viability at 0.15% (1:20 dilution). Gene expression analysis revealed that concentrated H2O2 and CHX-CPC downregulated BCL2 and MCL1 expression in MC3T3-E1 cells, with broader suppression of these genes observed in HGFs across all agents. In hBMSCs, high concentrations of the agents did not significantly reduce BCL2 and MCL1 levels. Conclusion: Residual chemical debridement agents, when inadequately removed, compromise the viability of cells in peri-implant tissues in a dose- and time-dependent manner. hBMSCs exhibited greater resistance to apoptosis than MC3T3-E1 cells and HGFs. Thorough removal of residual chemical cleaning agents after peri-implant debridement is therefore crucial to preserve the biocompatibility of the implant and the healing

• Lauretis, Angela De; Wang, Qiang; Santacroce, Marco; Thiede, Bernd; Ma, Qianli & Lyngstadaas, Ståle Petter [Vis alle 9 forfattere av denne artikkelen] (2025). A Comparative Study of the Impact of Chemical Debridement Products on the Proteomic Profile of the Salivary Pellicle and Cell Adhesion on OsseoSpeed Titanium Dental Implant Surfaces. ACS Applied Materials & Interfaces. ISSN 1944-8244. 17(39), s. 54592–54592. doi: 10.1021/acsami.5c14570. Fulltekst i vitenarkiv
• Lauretis, Angela de; Wang, Qiang; Santacroce, Marco; Thiede, Bernd; Ma, Qianli & Lyngstadaas, Ståle Petter [Vis alle 9 forfattere av denne artikkelen] (2025). A Comparative Study of the Impact of Chemical Debridement Products on the Proteomic Profile of the Salivary Pellicle and Cell Adhesion on OsseoSpeed Titanium Dental Implant Surfaces. ACS Applied Materials & Interfaces. ISSN 1944-8244. 17, s. 54579–54592. doi: 10.1021/acsami.5c14570. Fulltekst i vitenarkiv Vis sammendrag

  Background and objectives: Dental implants are commonly used for tooth replacement, with excellent success rates over 10 years. However, exposure to oral cavity bacteria makes them susceptible to biofilm accumulation, which can lead to peri-implant diseases. The salivary pellicle, a protein-rich layer formed on implant surfaces, mediates bacterial adhesion and cell attachment. Effective decontamination is essential for managing bacterial infections, yet no consensus exists on the best chemical agent. Understanding how chemical agents affect the pellicle proteomic profile may guide the selection of optimal treatments. Methods: We investigate the impact of chemical decontamination agents on the salivary pellicle on OsseoSpeed-like titanium dental implant surfaces using a BCA assay and proteomics, and on cell adhesion using human gingival fibroblasts (HGF) and human bone marrow mesenchymal stem cells (hBMSC). We compare the effects of hydrogen peroxide (H2O2), Poloxamer 407 (P407), P407 + H2O2 and sodium hypochlorite + amino acids (NaOCl + AA) using the salivary pellicle and a clean titanium surface as controls. By quantifying and identifying proteins on titanium surfaces after decontamination, this study reveals how chemical agents affect the salivary pellicle. Results: All products reduced the total protein on the surfaces, with the P407 + H2O2 hydrogel demonstrating lower protein variety and superior effectiveness in preventing surface recontamination. Not all proteins were removed during decontamination procedures, and unique proteins were detected in each experimental condition, suggesting protein readsorption depends on surface chemistry. P407 + H2O2 shows the highest enrichment in integrin- and cadherin-binding proteins upon recontamination. P407 and P407 + H2O2 promoted HGFs and hBMSCs attachment. Conversely, NaOCl + AA and H2O2 showed lower HGFs and hBMSCs counts. Conclusion: The P407 + H2O2 hydrogel had the strongest decontamination effect and limited surface recontamination. Each chemical agent produced a distinct proteomic profile. P407 and P407 + H2O2 promoted initial cell adhesion.

• Yarava, Jayasubba Reddy; Rydmark, Marcella Christine Orwick; Ryoo, David; Hofstetter, Albert; Gumbart, James C. & Habeck, Michael [Vis alle 9 forfattere av denne artikkelen] (2025). Probing the Dynamics of Yersinia Adhesin A (YadA) in Outer Membranes Hints at Requirements for β-Barrel Membrane Insertion. Journal of the American Chemical Society. ISSN 0002-7863. 147(10), s. 8618–8628. doi: 10.1021/jacs.4c17726. Fulltekst i vitenarkiv Vis sammendrag

  The vast majority of cells are protected and functionalized by a dense surface layer of glycans, proteoglycans, and glycolipids. This surface represents an underexplored space in structural biology that is exceedingly challenging to recreate in vitro. Here, we investigate β-barrel protein dynamics within an asymmetric outer membrane environment, with the trimeric autotransporter Yersinia adhesin A (YadA) as an example. Magic-angle spinning NMR relaxation data and a model-free approach reveal increased mobility in the second half of strand β2 after the conserved G72, which is responsible for membrane insertion and autotransport, and in the subsequent loop toward β3. In contrast, the protomer–protomer interaction sites (β1i–β4i–1) are rigid. Intriguingly, the mobility in the β-strand section following G72 is substantially elevated in the outer membrane and less so in the detergent environment of microcrystals. A possible source is revealed by molecular dynamics simulations that show the formation of a salt bridge involving E79 and R76 in competition with a dynamic interplay of calcium binding by E79 and the phosphate groups of the lipids. An estimation of overall barrel motion in the outer membrane and detergent-containing crystals yields values of around 41 ns for both. The global motion of YadA in the outer membrane has a stronger rotational component orthogonal to the symmetry axis of the trimeric porin than in the detergent-containing crystal. In summary, our investigation shows that the mobility in the second half of β2 and the loop to β3 required for membrane insertion and autotransport is maintained in the final folded form of YadA.

• Lauretis, Angela de; Agger, Anne Eriksson; Pal, Antara; Pedersen, Jan Skov; Szostak, Szymon Mikolaj & Lund, Reidar [Vis alle 10 forfattere av denne artikkelen] (2025). Balancing sterilization and functional properties in Poloxamer 407 hydrogels: comparing heat and radiation techniques. Regenerative Biomaterials. 12. doi: 10.1093/rb/rbaf005. Fulltekst i vitenarkiv Vis sammendrag

  Poloxamer 407, also known as Pluronic® F127, is gaining interest in the cosmetic, biomedical, and pharmaceutical fields for its biocompatibility, safety, and thermosensitive properties. Ensuring sterility is critical in clinical applications, and sterilization is often preferred over aseptic processing. However, sterilization can impact the hydrogel's functional properties. In this study, we investigate the effects of steam heat (121°C, 20 min), dry heat (160°C, 1 h), gamma irradiation (25 kGy), and electron beam (e-beam) irradiation (15 kGy and 25 kGy) on a 30%w/v Poloxamer 407 hydrogel formulation. Our analysis encompasses gelling properties, pH, Fourier-transform infrared (FTIR) spectroscopy, Gel Permeation Chromatography (GPC), Small-angle X-ray scattering (SAXS), rheology, swelling, degradation by-products, and lactate dehydrogenase (LDH) release of the sterilised hydrogels, comparing them to a non-sterile counterpart. We demonstrated that heat sterilisation alters the hydrogel’s gelling and structural properties due to water evaporation and oxidation under harsh temperature conditions, especially when applying the dry heat method. Gamma irradiation proved unsuitable, resulting in an acidic and cytotoxic hydrogel due to oxidative degradation. In contrast, e-beam irradiation preserves the hydrogel's elasticity, gelling, and structural properties while enhancing mechanical resilience and moderating swelling. Therefore, e-beam irradiation within the 15 – 25 kGy range appears to be the most suitable method for sterilizing a 30%w/v Poloxamer 407 hydrogel.

• Wang, Qiang; Haugen, Håvard Jostein; Linke, Dirk; Lyngstadaas, Ståle Petter; Sigurjonsson, Olafur & Ma, Qianli (2025). Impact of different chemical debridement agents on early cellular responses to Titanium dental implants: a transcriptome-based In Vitro Study on Peri-implant tissue regeneration. Colloids and Surfaces B: Biointerfaces. ISSN 0927-7765. 253. doi: 10.1016/j.colsurfb.2025.114727. Fulltekst i vitenarkiv Vis sammendrag

  Background Poor peri-implant health leads to biofilm accumulation, peri-implantitis, and bone loss. Chemical debridement may help maintain peri-implant health, but its effects on peri-implant cells remain unclear. Methods Five cleaning agents—hydrogen peroxide (H2O2), Poloxamer, H2O2 +Poloxamer, Perisolv, and Paroex—were applied on titanium (Ti) surfaces. Mouse pre-osteoblasts (MC3T3-E1), human gingival fibroblasts (HGF), and human bone marrow stromal cells (hBMSC) were cultured on agent-treated Ti surfaces for up to 120 minutes to assess morphology, cytotoxicity, adhesion, and proliferation. RNA sequencing was performed on hBMSC. Results Except for Poloxamer, all treatments inhibited cellular spreading. Paroex increased cytotoxicity and inhibited proliferation. Perisolv impaired hBMSC adhesion and variably affected proliferation. H2O2, alone or with Poloxamer, elevated cytotoxicity and inhibited adhesion in hBMSCs but not MC3T3-E1 or HGF. In contrast, Poloxamer-treated Ti surfaces enhanced adhesion and proliferation across all cell types. RNA sequencing revealed that oxidant-based treatments (H2O2, H2O2 +Poloxamer, Perisolv) suppressed key genes for proliferation (HMGA2, JAG1, NOTCH1, YAP1, TBX3), anti-apoptosis (MCL1, BCL2L2), and adhesion (ITGA2, ITGB3, SPP1), while inhibiting MAPK, PI3K-Akt, and pluripotency pathways. Conclusion Commercial agents like Perisolv and Paroex impair hBMSC function, with Paroex demonstrating significant cytotoxicity. H2O2 exhibits toxicity, particularly to hBMSCs. Poloxamer improves cell attachment and growth. Given these findings, careful selection of debridement agents is critical to balance cleaning efficacy and cytocompatibility. The adverse effects on hBMSCs necessitate prompt removal postapplication. Further research on biomaterials supporting tissue regeneration postdebridement is needed to restore peri-implant health.

• Lauretis, Angela de; Santacroce, Marco; Ellingsen, Jan Eirik; Lyngstadaas, Ståle Petter; Linke, Dirk & Haugen, Håvard Jostein (2025). Surface-specific outcomes of mechanical debridement with titanium, chitosan and nitinol brushes on titanium dental implant surfaces. Journal of Dentistry. ISSN 0300-5712. doi: 10.1016/j.jdent.2025.106010. Fulltekst i vitenarkiv Vis sammendrag

  Objectives Dental implants are susceptible to peri‑implant inflammation caused by bacterial biofilms, with surface properties playing a key role in bacterial adhesion and osseointegration. While mechanical debridement is essential for biofilm removal, consensus on the optimal tool remains limited. In this study, we evaluate the impact of a titanium, a chitosan and a nitinol brush on the surface properties of four titanium dental implant surfaces: OsseoSpeed®-like, SLActive®-like, machined, and polished. Methods Profilometry, contact angle measurements, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS) were used to assess the surface properties and chemistry. Results SEM and profilometry revealed that the titanium and nitinol brushes altered the morphology and topography of the rough OsseoSpeed®-like and SLActive®-like surfaces, while these effects were less pronounced on machined surfaces. Conversely, the chitosan brush caused minimal changes, but its bristles wore down quickly by physical abrasion, increasing surface carbon as detected by EDX and identified by XPS as organic material. Conversely, XPS confirmed that carbon contamination in samples treated with metal brushes was primarily environmental. The increased hydrophobicity observed across all groups was linked to carbon contamination. Polished surfaces remained unaffected by mechanical debridement. Conclusions The chitosan brush showed minimal surface alterations, but the rapid bristle wear left organic residues that increased hydrophobicity. The titanium and nitinol brushes caused more pronounced surface changes, while contributing to increased hydrophobicity through environmental carbon contamination during debridement. Clinical significance Dental implant surface properties, including topography, morphology, wettability, and surface chemistry, play a critical role in bacterial adhesion and osseointegration. Selecting an optimal mechanical debridement instrument requires balancing the effective removal of biofilm with the impact on the implant surface properties.

• 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
• Duodu, Rachael; Linke, Dirk & Leo, Jack Christopher (2024). Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System. I Raffaele, Ieva (Red.), Transmembrane β-Barrel Proteins. Humana Press. ISSN 9781071637333. s. 53–63. doi: 10.1007/978-1-0716-3734-0_4. Fulltekst i vitenarkiv
• Meuskens, Ina; Leo, Jack Christopher & Linke, Dirk (2024). Recombinant Expression and Overproduction of Transmembrane β-Barrel Proteins. I Raffaele, Ieva (Red.), Transmembrane β-Barrel Proteins. Humana Press. ISSN 9781071637333. s. 31–41. doi: 10.1007/978-1-0716-3734-0_2. 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.

• Vogelaar, Thomas Daniel; Agger, Anne Eriksson; Reseland, Janne Elin; Linke, Dirk; Jenssen, Håvard & Lund, Reidar (2024). Crafting Stable Antibiotic Nanoparticles via Complex Coacervation of Colistin with Block Copolymers. Biomacromolecules. ISSN 1525-7797. 25(7), s. 4267–4280. doi: 10.1021/acs.biomac.4c00337. Fulltekst i vitenarkiv Vis sammendrag

  To combat the ever-growing increase of multidrug-resistant (MDR) bacteria, action must be taken in the development of antibiotic formulations. Colistin, an effective antibiotic, was found to be nephrotoxic and neurotoxic, consequently leading to a ban on its use in the 1980s. A decade later, colistin use was revived and nowadays used as a last-resort treatment against Gram-negative bacterial infections, although highly regulated. If cytotoxicity issues can be resolved, colistin could be an effective option to combat MDR bacteria. Herein, we investigate the complexation of colistin with poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMAA) block copolymers to form complex coacervate core micelles (C3Ms) to ultimately improve colistin use in therapeutics while maintaining its effectiveness. We show that well-defined and stable micelles can be formed in which the cationic colistin and anionic PMAA form the core while PEO forms a protecting shell. The resulting C3Ms are in a kinetically arrested and stable state, yet they can be made reproducibly using an appropriate experimental protocol. By characterization through dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS), we found that the best C3M formulation, based on long-term stability and complexation efficiency, is at charge-matching conditions. This nanoparticle formulation was compared to noncomplexed colistin on its antimicrobial properties, enzymatic degradation, serum protein binding, and cytotoxicity. The studies indicate that the antimicrobial properties and cytotoxicity of the colistin-C3Ms were maintained while protein binding was limited, and enzymatic degradation decreased after complexation. Since colistin-C3Ms were found to have an equal effectivity but with increased cargo protection, such nanoparticles are promising components for the antibiotic formulation toolbox.

• Yoshimoto, Shogo; Ishii, Satoshi; Kawashiri, Ayane; Matsushita, Taishi; Linke, Dirk & Göttig, Stephan [Vis alle 9 forfattere av denne artikkelen] (2024). Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5. Frontiers in Bioengineering and Biotechnology. 12. doi: 10.3389/fbioe.2024.1342418. Fulltekst i vitenarkiv Vis sammendrag

  Gram-negative bacterium Acinetobacter sp. Tol 5 exhibits high adhesiveness to various surfaces of general materials, from hydrophobic plastics to hydrophilic glass and metals, via AtaA, an Acinetobacter trimeric autotransporter adhesin Although the adhesion of Tol 5 is nonspecific, Tol 5 cells may have prefer materials for adhesion. Here, we examined the adhesion of Tol 5 and other bacteria expressing different TAAs to various materials, including antiadhesive surfaces. The results highlighted the stickiness of Tol 5 through the action of AtaA, which enabled Tol 5 cells to adhere even to antiadhesive materials, including polytetrafluoroethylene with a low surface free energy, a hydrophilic polymer brush with steric hindrance, and mica with an ultrasmooth surface. Single-cell force spectroscopy as an atomic force microscopy technique revealed the strong cell adhesion force of Tol 5 to these antiadhesive materials. Nevertheless, Tol 5 cells showed a weak adhesion force toward a zwitterionic 2-methacryloyloxyethyl-phosphorylcholine (MPC) polymer-coated surface. Dynamic flow chamber experiments revealed that Tol 5 cells, once attached to the MPC polymer-coated surface, were exfoliated by weak shear stress. The underlying adhesive mechanism was presumed to involve exchangeable, weakly bound water molecules. Our results will contribute to the understanding and control of cell adhesion of Tol 5 for immobilized bioprocess applications and other TAA-expressing pathogenic bacteria of medical importance.

• 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.

• Yoshimoto, Shogo; Aoki, Sota; Ohara, Yuki; Ishikawa, Masahito; Suzuki, Atsuo & Linke, Dirk [Vis alle 8 forfattere av denne artikkelen] (2023). Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli. Frontiers in Bioengineering and Biotechnology. 10. doi: 10.3389/fbioe.2022.1095057. Fulltekst i vitenarkiv Vis sammendrag

  Cell immobilization is an important technique for efficiently utilizing whole-cell biocatalysts. We previously invented a method for bacterial cell immobilization using AtaA, a trimeric autotransporter adhesin from the highly sticky bacterium Acinetobacter sp. Tol 5. However, except for Acinetobacter species, only one bacterium has been successfully immobilized using AtaA. This is probably because the heterologous expression of large AtaA (1 MDa), that is a homotrimer of polypeptide chains composed of 3,630 amino acids, is difficult. In this study, we identified the adhesive domain of AtaA and constructed a miniaturized AtaA (mini-AtaA) to improve the heterologous expression of ataA. In-frame deletion mutants were used to perform functional mapping, revealing that the N-terminal head domain is essential for the adhesive feature of AtaA. The mini-AtaA, which contains a homotrimer of polypeptide chains from 775 amino acids and lacks the unnecessary part for its adhesion, was properly expressed in E. coli, and a larger amount of molecules was displayed on the cell surface than that of full-length AtaA (FL-AtaA). The immobilization ratio of E. coli cells expressing mini-AtaA on a polyurethane foam support was significantly higher compared to the cells with or without FL-AtaA expression, respectively. The expression of mini-AtaA in E. coli had little effect on the cell growth and the activity of another enzyme reflecting the production level, and the immobilized E. coli cells could be used for repetitive enzymatic reactions as a whole-cell catalyst

• 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
• Hatlem, Daniel; Christensen, Mikkel; Broeker, Nina; Kristiansen, Per Eugen; Lund, Reidar & Barbirz, Stefanie [Vis alle 7 forfattere av denne artikkelen] (2023). A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity. Frontiers in Cellular and Infection Microbiology. 13. doi: 10.3389/fcimb.2023.1125482. Fulltekst i vitenarkiv Vis sammendrag

  α-helical coiled-coils are ubiquitous protein structures in all living organisms. For decades, modified coiled-coils sequences have been used in biotechnology, vaccine development, and biochemical research to induce protein oligomerization, and form self-assembled protein scaffolds. A prominent model for the versatility of coiled-coil sequences is a peptide derived from the yeast transcription factor, GCN4. In this work, we show that its trimeric variant, GCN4-pII, binds bacterial lipopolysaccharides (LPS) from different bacterial species with picomolar affinity. LPS molecules are highly immunogenic, toxic glycolipids that comprise the outer leaflet of the outer membrane of Gram-negative bacteria. Using scattering techniques and electron microscopy, we show how GCN4-pII breaks down LPS micelles in solution. Our findings suggest that the GCN4-pII peptide and derivatives thereof could be used for novel LPS detection and removal solutions with high relevance to the production and quality control of biopharmaceuticals and other biomedical products, where even minuscule amounts of residual LPS can be lethal.

• 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.

• Meuskens, Ina; Kristiansen, Per Eugen; Bardiaux, Benjamin; Koynarev, Vladimir Rosenov; Hatlem, Daniel & Prydz, Kristian [Vis alle 9 forfattere av denne artikkelen] (2023). A poly-proline II helix in YadA from Yersinia enterocolitica serotype O:9 facilitates heparin binding through electrostatic interactions. The FEBS Journal. ISSN 1742-464X. 291(4), s. 761–777. doi: 10.1111/febs.17001. Fulltekst i vitenarkiv Vis sammendrag

  Poly‐proline II helices are secondary structure motifs frequently found in ligand‐binding sites. They exhibit increased flexibility and solvent exposure compared to the strongly hydrogen‐bonded α‐helices or β‐strands and can therefore easily be misinterpreted as completely unstructured regions with an extremely high rotational freedom. Here, we show that the adhesin YadA of Yersinia enterocolitica serotype O:9 contains a poly‐proline II helix interaction motif in the N‐terminal region. The motif is involved in the interaction of YadA O:9 with heparin, a host glycosaminoglycan. We show that the basic residues within the N‐terminal motif of YadA are required for electrostatic interactions with the sulfate groups of heparin. Biophysical methods including CD spectroscopy, solution‐state NMR and SAXS all independently support the presence of a poly‐proline helix allowing YadA O:9 binding to the rigid heparin. Lastly, we show that host cells deficient in sulfation of heparin and heparan sulfate are not targeted by YadA O:9 ‐mediated adhesion. We speculate that the YadA O:9 –heparin interaction plays an important and highly strain‐specific role in the pathogenicity of Yersinia enterocolitica serotype O:9.

• 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.

• Meuskens, Ina; Leva-Bueno, Juan; Millner, Paul; Schütz, Monika; Peyman, Sally & Linke, Dirk (2022). The Trimeric Autotransporter Adhesin YadA of Yersinia enterocolitica Serotype O:9 Binds Glycan Moieties. Frontiers in Microbiology. 12. doi: 10.3389/fmicb.2021.738818. Fulltekst i vitenarkiv Vis sammendrag

  Yersinia adhesin A (YadA) is a key virulence factor of Yersinia enterocolitica and Yersinia pseudotuberculosis . YadA is a trimeric autotransporter adhesin, a class of adhesins that have been shown to enable many Gram-negative pathogens to adhere to/interact with the host extracellular matrix proteins such as collagen, vitronectin, and fibronectin. Here, we show for the first time that YadA of Yersinia enterocolitica serotype O:9 not only interacts with proteinaceous surface molecules but can also attach directly to glycan moieties. We show that YadA from Y. enterocolitica serotype O:9 does not interact with the vitronectin protein itself but exclusively with its N-linked glycans. We also show that YadA can target other glycan moieties as found in heparin, for example. So far, little is known about specific interactions between bacterial autotransporter adhesins and glycans. This could potentially lead to new antimicrobial treatment strategies, as well as diagnostic applications.

• Michalik, Marcin; Djahanschiri, Bardya; Leo, Jack Christopher & Linke, Dirk (2022). An Update on “Reverse Vaccinology”: The Pathway from Genomes and Epitope Predictions to Tailored, Recombinant Vaccines. I Thomas, Sunil (Red.), Vaccine Design. Methods and Protocols, Volume 3: Resources for Vaccine Development. Second Edition. Humana Press. ISSN 9781071618912. s. 45–71. doi: 10.1007/978-1-0716-1892-9. Fulltekst i vitenarkiv Vis sammendrag

  In this chapter, we review the computational approaches that have led to a new generation of vaccines in recent years. There are many alternative routes to develop vaccines based on the concept of reverse vaccinology. They all follow the same basic principles—mining available genome and proteome information for antigen candidates, and recombinantly expressing them for vaccine production. Some of the same principles have been used successfully for cancer therapy approaches. In this review, we focus on infectious diseases, describing the general workflow from bioinformatic predictions of antigens and epitopes down to examples where such predictions have been used successfully for vaccine development.

• 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.

• Leva-Bueno, Juan; Meuskens, Ina; Linke, Dirk; Millner, Paul & Peyman, Sally A. (2022). A novel, proof-of-concept electrochemical impedimetric biosensor based on extracellular matrix protein-adhesin interaction. Sensors & Diagnostics. 1(5), s. 1003–1013. doi: 10.1039/d2sd00075j. Fulltekst i vitenarkiv Vis sammendrag

  This work presents a novel perspective for electrochemical biosensors based on the detection of the interaction between extracellular matrix proteins and bacterial adhesins. Two types of impedimetric biosensors using collagen as a bioreceptor were investigated for their ability to detect pathogen adhesion using a well-characterised system of collagen-binding to recombinant E. coli expressing adhesin. First, a collagen–polymer-matrix-based biosensor was shown to detect whole bacteria in buffer media over the range 8 × 105 cfu to 8 × 107 cfu in a 10 μL sample. Second, a step-by-step full optimised biosensor based on direct collagen attachment showed bacterial detection over the range of 8 × 104 cfu to 8 × 107 cfu in a 10 μL sample. Electrochemical impedance spectroscopy and cyclic voltammetry were employed to assess the binding, which was corroborated by fluorescence binding assays. Up to now, a wide range of bioreceptors have been used in biosensor fabrication, including antibodies, oligonucleotides, phages and molecularly imprinted polymers. In this study, we show that extracellular matrix proteins can also be used for whole pathogen recognition by binding to adhesins.

Se alle arbeider i NVA

• Linke, Dirk (2025). Trimeric Autotransporter Adhesins: structure, function, innovation potential. Fulltekst i vitenarkiv
• Linke, Dirk (2025). AlphaFold user errors. Fulltekst i vitenarkiv
• Linke, Dirk (2025). Bacterial Autotransporter Adhesins: structure, function, and surprising innovation potential. Fulltekst i vitenarkiv
• Linke, Dirk (2025). Bacterial Autotransporter Adhesins: structure, function, and surprising innovation potential. Fulltekst i vitenarkiv
• Linke, Dirk (2025). A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity. Fulltekst i vitenarkiv
• Linke, Dirk (2025). Bacterial Autotransporter Adhesins: structure, function, and surprising innovation potential. Fulltekst i vitenarkiv
• 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
• Mascherin, Elisa; Karlsen, Markus; Wohlmann, Jens; Reseland, Janne Elin & Linke, Dirk (2025). In vitro toxicology and antimicrobial efficacy of Q-FIELD™, an innovative photocatalytic additive for paints. 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
• Lauretis, Angela de; Santacroce, Marco; Hussain, Badra; Ma, Qianli; Ellingsen, Jan Eirik & Lyngstadaas, Ståle Petter [Vis alle 8 forfattere av denne artikkelen] (2025). A poloxamer-peroxide forumulation effectively removes pellicle and limits recontamination. 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
• Linke, Dirk (2024). A disordered loop in a bacterial adhesin can form a poly-proline II helix upon binding to heparin. Fulltekst i vitenarkiv
• Linke, Dirk & Falk, Jonathan (2024). Beroliger folk etter «zombie-virus»-funn. [Internet]. VG.NO. Fulltekst i vitenarkiv
• Hatlem, Daniel; Barbirz, Stefanie & Linke, Dirk (2024). A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity - and can be used in LPS detection and removal applications. 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
• Linke, Dirk (2024). A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity. 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
• Linke, Dirk (2024). Trimeric Autotransporter Adhesins: structure, function, evolution. Fulltekst i vitenarkiv
• Nakatani, Hajime; Linke, Dirk & Hori, Katsutoshi (2023). Study on the fish epidermal bacterial flora for fish disease control. 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
• Campana, A.; Joudeh, Nadeem; Merroun, Mohamed; Gomez-Bolivar, Jamie; Linke, Dirk & Mikheenko, Pavlo (2023). Biosynthesis and characterization of Palladium-based nanoparticles produced by Escherichia coli. Fulltekst i vitenarkiv
• Fan, Enguo & Linke, Dirk (2023). Methods and assays to study protein transport in vivo. Fulltekst i vitenarkiv
• Sanz-Gaitero, Marta; Cunha, Eva S.; Linke, Dirk & Lücke, Hartmut (2023). Using Escherichia coli as a model to test Helicobacter pylori urease inhibitors. 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
• Mekasha, Sophanit; Petrikonytė, Patricija & Linke, Dirk (2023). Virulence factors of the bacterial fish pathogen Tenacibaculum maritimum. Fulltekst i vitenarkiv
• Hori, Katsutoshi & Linke, Dirk (2023). Application of a bacterial adhesin to bioprocesses. Fulltekst i vitenarkiv
• Ledo, Jesus & Linke, Dirk (2023). Biofouling on metal surfaces. 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
• Yoshimoto, Shogo; Linke, Dirk & Hori, Katsutoshi (2023). Single-cell analysis of adhesive bacteria using atomic force microscopy. Fulltekst i vitenarkiv
• Leo, Jack C.; Linke, Dirk & Morth, P (2023). Role of the extracellular 'juncture' domain in the secretion and function of inverse autotransporters. Fulltekst i vitenarkiv
• Linke, Dirk (2023). A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity. Fulltekst i vitenarkiv
• Linke, Dirk (2023). BEDPAN: Palladium nanoparticles from bacteria. 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
• Petrikonyte, Patricija; Mekasha, Sophanit & Linke, Dirk (2023). Tenacibaculum maritimum virulence factors. 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
• Linke, Dirk (2023). Tandem repeat adhesins and dead repeat genes in bacteria. Fulltekst i vitenarkiv
• Perilla, Ana Lucia Campana; Joudeh, Nadeem; Merroun, Mohamed; Gomez-Bolivar, Jamie; Linke, Dirk & Mikheenko, Pavlo (2023). Bacterial synthesis of Palladium nanoparticles. Fulltekst i vitenarkiv
• Yoshimoto, Shogo; Aoki, S.; Ohara, Y; Ishikawa, Masahito; Suzuki, A & Lupas, Andrei [Vis alle 8 forfattere av denne artikkelen] (2023). Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli. Fulltekst i vitenarkiv
• Mekasha, Sophanit; Haslene-Hox, Hanne; Sperstad, Sigrid Bakken; Tøndervik, Anne & Linke, Dirk (2023). Exploring virulence factors of Tenacibaculum maritimum. Fulltekst i vitenarkiv
• Perilla, Ana Lucia Campana; Joudeh, Nadeem; Merroun, Mohamed; Gomez-Bolivar, Jamie; Linke, Dirk & Mikheenko, Pavlo (2023). Bacterial synthesis of Palladium nanoparticles. Fulltekst i vitenarkiv
• Lislerud, Anna; Nakatani, Hajime; Spence, Maria Helle; Saragliadis, Athanasios; Hori, Katsutoshi & Linke, Dirk (2023). Adhesins of Yersinia ruckeri. Fulltekst i vitenarkiv
• Linke, Dirk (2023). Structure, Function and Evolution of Autotransporter Adhesins. Fulltekst i vitenarkiv
• Petrikonyte, Patricija; Mekasha, Sophanit & Linke, Dirk (2023). T. maritimum virulence factors. Fulltekst i vitenarkiv
• Mekasha, Sophanit; Tøndervik, Anne & Linke, Dirk (2023). Exploring virulence factors of Tenacibaculum maritimum. Fulltekst i vitenarkiv
• Linke, Dirk (2023). Trimeric Autotransporters: non-globular, repetitive virulence factors with peculiar genomic recombination mechanisms. 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
• Linke, Dirk (2023). Trimeric Autotransporters: Function, Structure, Evolution. Fulltekst i vitenarkiv
• Yoshimoto, Shogo; Aoki, S.; Ohara, Y; Ishikawa, Masahito; Suzuki, A & Linke, Dirk [Vis alle 7 forfattere av denne artikkelen] (2023). Downsizing of AtaA from Acinetobacter p. Tol5 and its application in immobilizing E.coli. Fulltekst i vitenarkiv
• Gaitero, Marta Sanz; Cunha, Eva S.; Linke, Dirk & Luecke, Hartmut (2023). Using Escherichia coli as a model to test Helicobacter pylori urease inhibitors. Fulltekst i vitenarkiv
• Linke, Dirk (2023). Trimeric Autotransporter Adhesins: Structure, Function, and Evolution. Fulltekst i vitenarkiv
• Perilla, Ana Lucia Campana; Joudeh, Nadeem; Merroun, Mohamed; Gomez-Bolivar, Jamie; Linke, Dirk & Mikheenko, Pavlo (2023). Biosynthesis and characterization of Palladium-based nanoparticles produced by Escherichia coli. 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
• Meuskens, Ina; Kristiansen, Per Eugen; Bardiaux, Benjamin; Koynarev, Vladimir Rosenov; Hatlem, Daniel & Prydz, Kristian [Vis alle 9 forfattere av denne artikkelen] (2023). Strain variations in a bacterial adhesin lead to different binding partners in the pathogen Yersinia enterocolitica. Fulltekst i vitenarkiv
• Petrikonyte, Patricija; Mekasha, Sophanit & Linke, Dirk (2023). Tenacibaculum maritimum virulence factors. 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
• Mekasha, Sophanit & Linke, Dirk (2023). Virulence factors of the bacterial fish pathogen Tenacibaculum maritimum. Fulltekst i vitenarkiv
• Linke, Dirk (2023). Type-5-secreted adhesins in human and animal disease. Fulltekst i vitenarkiv
• Linke, Dirk (2022). Bacterial adhesion and how to inhibit it. Fulltekst i vitenarkiv
• Linke, Dirk (2022). Structure, Function, and Evolution of trimeric autotransporter adhesins. Fulltekst i vitenarkiv
• Perilla, Ana Lucia Campana; Linke, Dirk; Mikheenko, Pavlo & Røyne, Anja (2024). Palladium-based nanoparticles produced by Escherichia coli: Insights into synthesis and physicochemical characterization of microbial nanoparticles. Universitetet i Oslo. Fulltekst i vitenarkiv Vis sammendrag

  Microorganisms such as bacteria can produce a wide range of metallic nanoparticles, including the highly versatile palladium nanoparticles (Pd NPs). Current industrial methods for NP production often require large amounts of energy, use toxic chemicals, and generate waste. Microbial processes offer a green alternative for NP production, and the possibility to engineer these processes using genetic tools. Pd NPs are invaluable tools across numerous exciting applications in medicine, engineering, electronics, and chemistry. Understanding and controlling the synthesis of Pd NPs is crucial as their magnetic and catalytic properties are strongly dependent on their size, shape, and on the presence of other metals such as iron (Fe) that could enhance their performance even further. Surprisingly, little is known about the production of Pd-Fe nanoparticles by bacteria, despite their promising uses. In this thesis, we employed various advanced techniques to characterize Pd and Pd-Fe NPs produced by Escherichia coli bacteria, uncovering valuable insights into their composition, size, localization, and catalytic activity. These results will enable the use of bacterial Pd-Fe NPs as efficient and sustainable catalysts for applications like wastewater treatment and fine chemical synthesis in the future. Additionally, our novel methodology for studying the magnetism of Pd-based NPs directly within bacterial cells opens doors for exciting future research.

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