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, p. 117–132. doi: https:/link.springer.com/protocol/10.1007/978-1-0716-5054-7_6. Full text in Research Archive
• Adélaïde, Morgane; Yarava, Jayasubba Reddy; Lindemann, Florian; Abbas, Massilia; Cremer, Nils & Gerland, Lisa [Show all 14 contributors for this article] (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. Full text in Research Archive Show summary

  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 [Show all 9 contributors for this article] (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. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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 [Show all 9 contributors for this article] (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), p. 54592–54592. doi: 10.1021/acsami.5c14570. Full text in Research Archive
• Lauretis, Angela de; Wang, Qiang; Santacroce, Marco; Thiede, Bernd; Ma, Qianli & Lyngstadaas, Ståle Petter [Show all 9 contributors for this article] (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, p. 54579–54592. doi: 10.1021/acsami.5c14570. Full text in Research Archive Show summary

  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 [Show all 9 contributors for this article] (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), p. 8618–8628. doi: 10.1021/jacs.4c17726. Full text in Research Archive Show summary

  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 [Show all 10 contributors for this article] (2025). Balancing sterilization and functional properties in Poloxamer 407 hydrogels: comparing heat and radiation techniques. Regenerative Biomaterials. 12. doi: 10.1093/rb/rbaf005. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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. In Raffaele, Ieva (Eds.), Transmembrane β-Barrel Proteins. Humana Press. ISSN 9781071637333. p. 43–52. doi: 10.1007/978-1-0716-3734-0_3. Full text in Research Archive
• Duodu, Rachael; Linke, Dirk & Leo, Jack Christopher (2024). Fluorescent Labeling of Outer Membrane Proteins Using the SpyCatcher-SpyTag System. In Raffaele, Ieva (Eds.), Transmembrane β-Barrel Proteins. Humana Press. ISSN 9781071637333. p. 53–63. doi: 10.1007/978-1-0716-3734-0_4. Full text in Research Archive
• Meuskens, Ina; Leo, Jack Christopher & Linke, Dirk (2024). Recombinant Expression and Overproduction of Transmembrane β-Barrel Proteins. In Raffaele, Ieva (Eds.), Transmembrane β-Barrel Proteins. Humana Press. ISSN 9781071637333. p. 31–41. doi: 10.1007/978-1-0716-3734-0_2. Full text in Research Archive
• Perilla, Ana Lucia Campana; Gomez-Bolivar, Jamie; Merroun, Mohamed; Joudeh, Nadeem; Saragliadis, Athanasios & Røyne, Anja [Show all 8 contributors for this article] (2024). Characterization of Bimetallic Pd-Fe Nanoparticles Synthesized in Escherichia coli. ACS Applied Bio Materials (AABM). 7(12), p. 8573–8589. doi: 10.1021/acsabm.4c01354. Full text in Research Archive Show summary

  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), p. 4267–4280. doi: 10.1021/acs.biomac.4c00337. Full text in Research Archive Show summary

  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 [Show all 9 contributors for this article] (2024). Adhesion preference of the sticky bacterium Acinetobacter sp. Tol 5. Frontiers in Bioengineering and Biotechnology. 12. doi: 10.3389/fbioe.2024.1342418. Full text in Research Archive Show summary

  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 [Show all 7 contributors for this article] (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. Full text in Research Archive Show summary

  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 [Show all 8 contributors for this article] (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. Full text in Research Archive Show summary

  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. Full text in Research Archive
• Hatlem, Daniel; Christensen, Mikkel; Broeker, Nina; Kristiansen, Per Eugen; Lund, Reidar & Barbirz, Stefanie [Show all 7 contributors for this article] (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. Full text in Research Archive Show summary

  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 [Show all 9 contributors for this article] (2023). Aptasensor for the Detection of Moraxella catarrhalis Adhesin UspA2. Bioengineering. 10(2), p. 1–20. doi: 10.3390/bioengineering10020178. Full text in Research Archive Show summary

  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 [Show all 9 contributors for this article] (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), p. 761–777. doi: 10.1111/febs.17001. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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. In Thomas, Sunil (Eds.), Vaccine Design. Methods and Protocols, Volume 3: Resources for Vaccine Development. Second Edition. Humana Press. ISSN 9781071618912. p. 45–71. doi: 10.1007/978-1-0716-1892-9. Full text in Research Archive Show summary

  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, p. 1–24. doi: 10.3389/fnano.2022.1062608. Full text in Research Archive Show summary

  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 [Show all 9 contributors for this article] (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. Full text in Research Archive Show summary

  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), p. 1003–1013. doi: 10.1039/d2sd00075j. Full text in Research Archive Show summary

  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.

• Thibau, Arno; Vaca, Diana J.; Bagowski, Marlene; Hipp, Katharina; Bender, Daniela & Ballhorn, Wibke [Show all 8 contributors for this article] (2022). Adhesion of Bartonella henselae to Fibronectin Is Mediated via Repetitive Motifs Present in the Stalk of Bartonella Adhesin A. Microbiology spectrum. 10(5). doi: 10.1128/spectrum.02117-22. Full text in Research Archive Show summary

  Adhesion to host cells is the first and most crucial step in infections with pathogenic Gram-negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). Bartonella henselae targets the extracellular matrix glycoprotein fibronectin (Fn) via the Bartonella adhesin A (BadA) attaching the bacteria to the host cell. The TAA BadA is characterized by a highly repetitive passenger domain consisting of 30 neck/stalk domains with various degrees of similarity. To elucidate the motif sequences mediating Fn binding, we generated 10 modified BadA constructs and verified their expression via Western blotting, confocal laser scanning, and electron microscopy. We analyzed their ability to bind human plasma Fn using quantitative whole-cell enzyme-linked immunosorbent assays (ELISAs) and fluorescence microscopy. Polyclonal antibodies targeting a 15-mer amino acid motif sequence proved to reduce Fn binding. We suggest that BadA adheres to Fn in a cumulative effort with quick saturation primarily via unpaired β-strands appearing in motifs repeatedly present throughout the neck/stalk region. In addition, we demonstrated that the length of truncated BadA constructs correlates with the immunoreactivity of human patient sera. The identification of BadA-Fn binding regions will support the development of new “antiadhesive” compounds inhibiting the initial adherence of B. henselae and other TAA-expressing pathogens to host cells.

• Sande, Maria; Ferreira, Debora; Rodrigues, Joana; Melo, Luis; Linke, Dirk & Silva, Carla [Show all 9 contributors for this article] (2022). Electrochemical Aptasensor for the Detection of the Key Virulence Factor YadA of Yersinia enterocolitica. Biosensors. ISSN 2079-6374. 12(8). doi: 10.3390/bios12080614. Full text in Research Archive Show summary

  New point-of-care (POC) diagnosis of bacterial infections are imperative to overcome the deficiencies of conventional methods, such as culture and molecular methods. In this study, we identified new aptamers that bind to the virulence factor Yersinia adhesin A (YadA) of Yersinia enterocolitica using cell-systematic evolution of ligands by exponential enrichment (cell-SELEX). Escherichia coli expressing YadA on the cell surface was used as a target cell. After eight cycles of selection, the final aptamer pool was sequenced by high throughput sequencing using the Illumina Novaseq platform. The sequencing data, analyzed using the Geneious software, was aligned, filtered and demultiplexed to obtain the key nucleotides possibly involved in the target binding. The most promising aptamer candidate, Apt1, bound specifically to YadA with a dissociation constant (Kd) of 11 nM. Apt1 was used to develop a simple electrochemical biosensor with a two-step, label-free design towards the detection of YadA. The sensor surface modifications and its ability to bind successfully and stably to YadA were confirmed by cyclic voltammetry, impedance spectroscopy and square wave voltammetry. The biosensor enabled the detection of YadA in a linear range between 7.0 × 104 and 7.0 × 107 CFU mL−1 and showed a square correlation coefficient &gt;0.99. The standard deviation and the limit of detection was ~2.5% and 7.0 × 104 CFU mL−1, respectively. Overall, the results suggest that this novel biosensor incorporating Apt1 can potentially be used as a sensitive POC detection system to aid the diagnosis of Y. enterocolitica infections. Furthermore, this simple yet innovative approach could be replicated to select aptamers for other (bacterial) targets and to develop the corresponding biosensors for their detection.

• Sande, Maria; Roque, Lucia; Braga, Adelaide; Marques, Marcia; Ferreira, Debora & Saragliadis, Athanasios [Show all 11 contributors for this article] (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. Full text in Research Archive
• Vaca, Diana; Thibau, Arno; Leisegang, Matthias; Malmström, Johan; Linke, Dirk & Eble, Johannes A. [Show all 10 contributors for this article] (2022). Interaction of Bartonella henselae with Fibronectin Represents the Molecular Basis for Adhesion to Host Cells. Microbiology spectrum. 10(3). doi: 10.1128/spectrum.00598-22. Full text in Research Archive Show summary

  Bacterial adhesion to the host is the most decisive step in infections. Trimeric autotransporter adhesins (TAA) are important pathogenicity factors of Gram-negative bacteria. The prototypic TAA Bartonella adhesin A (BadA) from human-pathogenic Bartonella henselae mediates bacterial adherence to endothelial cells (ECs) and extracellular matrix proteins. Here, we determined the interaction between BadA and fibronectin (Fn) to be essential for bacterial host cell adhesion. BadA interactions occur within the heparin-binding domains of Fn. The exact binding sites were revealed by mass spectrometry analysis of chemically cross-linked whole-cell bacteria and Fn. Specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs and these data were confirmed by BadA-deficient bacteria and CRISPR-Cas knockout Fn host cells. Interactions between TAAs and the extracellular matrix might represent the key step for adherence of human-pathogenic Gram-negative bacteria to the host.

• Hermansen, Simen; Linke, Dirk & Leo, Jack Christopher (2022). Transmembrane β-barrel proteins of bacteria: From structure to function. In Donev, Rossen (Eds.), Membrane Proteins. Academic Press. ISSN 9780323988957. p. 113–161. doi: 10.1016/bs.apcsb.2021.07.002. Full text in Research Archive
• Joudeh, Nadeem & Linke, Dirk (2022). Nanoparticle classification, physicochemical properties, characterization, and applications: a comprehensive review for biologists. Journal of Nanobiotechnology. 20(1). doi: 10.1186/s12951-022-01477-8. Full text in Research Archive Show summary

  Interest in nanomaterials and especially nanoparticles has exploded in the past decades primarily due to their novel or enhanced physical and chemical properties compared to bulk material. These extraordinary properties have created a multitude of innovative applications in the fields of medicine and pharma, electronics, agriculture, chemical catalysis, food industry, and many others. More recently, nanoparticles are also being synthesized ‘biologically’ through the use of plant- or microorganism-mediated processes, as an environmentally friendly alternative to the expensive, energy-intensive, and potentially toxic physical and chemical synthesis methods. This transdisciplinary approach to nanoparticle synthesis requires that biologists and biotechnologists understand and learn to use the complex methodology needed to properly characterize these processes. This review targets a bio-oriented audience and summarizes the physico–chemical properties of nanoparticles, and methods used for their characterization. It highlights why nanomaterials are different compared to micro- or bulk materials. We try to provide a comprehensive overview of the different classes of nanoparticles and their novel or enhanced physicochemical properties including mechanical, thermal, magnetic, electronic, optical, and catalytic properties. A comprehensive list of the common methods and techniques used for the characterization and analysis of these properties is presented together with a large list of examples for biogenic nanoparticles that have been previously synthesized and characterized, including their application in the fields of medicine, electronics, agriculture, and food production. We hope that this makes the many different methods more accessible to the readers, and to help with identifying the proper methodology for any given nanoscience problem.

• Chowdhury, Sounak; Khakzad, Hamed; Bergdahl, Gizem Ertürk; Lood, Rolf; Ekström, Simon & Linke, Dirk [Show all 9 contributors for this article] (2021). Streptococcus pyogenes Forms Serotype- and Local Environment-Dependent Interspecies Protein Complexes. mSystems. 6(5). doi: 10.1128/mSystems.00271-21. Full text in Research Archive Show summary

  Streptococcus pyogenes is known to cause both mucosal and systemic infections in humans. In this study, we used a combination of quantitative and structural mass spectrometry techniques to determine the composition and structure of the interaction network formed between human plasma proteins and the surfaces of different S. pyogenes serotypes. Quantitative network analysis revealed that S. pyogenes forms serotype-specific interaction networks that are highly dependent on the domain arrangement of the surface-attached M protein. Subsequent structural mass spectrometry analysis and computational modeling of one of the M proteins, M28, revealed that the network structure changes across different host microenvironments. We report that M28 binds secretory IgA via two separate binding sites with high affinity in saliva. During vascular leakage mimicked by increasing plasma concentrations in saliva, the binding of secretory IgA was replaced by the binding of monomeric IgA and C4b-binding protein (C4BP). This indicates that an upsurge of C4BP in the local microenvironment due to damage to the mucosal membrane drives the binding of C4BP and monomeric IgA to M28. These results suggest that S. pyogenes has evolved to form microenvironment-dependent host-pathogen protein complexes to combat human immune surveillance during both mucosal and systemic infections.

• Perilla, Ana Lucia Campana; Joudeh, Nadeem; Linke, Dirk & Mikheenko, Pavlo (2021). Magnetic decoration of Escherichia coli loaded with Palladium nanoparticles, 2021 IEEE 10th International Conference on “Nanomaterials: Applications & Properties” (NAP – 2021). IEEE (Institute of Electrical and Electronics Engineers). ISSN 9781665439077. p. NCI01-1–NCI01-5. doi: 10.1109/NAP51885.2021.9568523. Full text in Research Archive Show summary

  Palladium (Pd) is a scarce metal, which is able to catalyze a variety of important chemical reactions. In the bulk form, Pd is anomalous paramagnetic, but in the form of nanoparticles, it has been reported as ferromagnetic. Detecting the magnetism of Pd nanoparticles is, however, difficult due to their reduced dimension. In addition, the ferromagnetic nature is a size-dependent property and, for Pd, the strength of magnetism decreases with slight increments in size. A “green” methodology for the preparation of small Pd nanoparticles was followed in this study. It is based on the ability of bacteria to take up Pd 2+ ions from its surrounding solution and to enzymatically reduce it to metallic Pd 0 nanoparticles. The efficiency of the production can be determined by transmission electron microscopy and, as a new technique, magnetic force microscopy. Notwithstanding, the study of nanoparticles of the size of just a few nanometers with these techniques is still difficult. Here we present a methodology for the enhancement of the magnetic signal of biologically produced Pd-based nanoparticles through the decoration of Escherichia coli bacteria cross-sections with Fe nanoparticles. This methodology allows the visualization of bacteria that are loaded with magnetic nanoparticles even when conventional transmission electron microscopy has difficulties to resolve them inside the microorganisms.

• Rooke, Jessica; Icke, Christopher; Wells, Timothy; Rossiter, Amanda; Browning, Douglas & Morris, Faye [Show all 12 contributors for this article] (2021). BamA and BamD Are Essential for the Secretion of Trimeric Autotransporter Adhesins. Frontiers in Microbiology. 12. doi: 10.3389/fmicb.2021.628879. Full text in Research Archive Show summary

  The BAM complex in Escherichia coli is composed of five proteins, BamA-E. BamA and BamD are essential for cell viability and are required for the assembly of β-barrel outer membrane proteins. Consequently, BamA and BamD are indispensable for secretion via the classical autotransporter pathway (Type 5a secretion). In contrast, BamB, BamC, and BamE are not required for the biogenesis of classical autotransporters. Recently, we demonstrated that TamA, a homologue of BamA, and its partner protein TamB, were required for efficient secretion of proteins via the classical autotransporter pathway. The trimeric autotransporters are a subset of the Type 5-secreted proteins. Unlike the classical autotransporters, they are composed of three identical polypeptide chains which must be assembled together to allow secretion of their cognate passenger domains. In contrast to the classical autotransporters, the role of the Bam and Tam complex components in the biogenesis of the trimeric autotransporters has not been investigated fully. Here, using the Salmonella enterica trimeric autotransporter SadA and the structurally similar YadA protein of Yersinia spp., we identify the importance of BamA and BamD in the biogenesis of the trimeric autotransporters and reveal that BamB, BamC, BamE, TamA and TamB are not required for secretion of functional passenger domain on the cell surface. Importance The secretion of trimeric autotransporters (TAA’s) has yet to be fully understood. Here we show that efficient secretion of TAAs requires the BamA and D proteins, but does not require BamB, C or E. In contrast to classical autotransporter secretion, neither trimeric autotransporter tested required TamA or B proteins to be functionally secreted.

• Belkum, Alex van; Almeida, Carina; Bardiaux, Benjamin; Barrass, Sarah V.; Butcher, Sarah J. & Çaykara, Tuğçe [Show all 34 contributors for this article] (2021). Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens. Diagnostics (Basel). 11(7), p. 1–22. doi: 10.3390/diagnostics11071259. Full text in Research Archive Show summary

  Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen–surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin–ligand interaction, supported by present high-throughput “omics” technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

• Wimmi, Stephan; Balinovic, Alexander; Jeckel, Hannah; Selinger, Lisa; Lampaki, Dimitrios & Eisemann, Emma [Show all 11 contributors for this article] (2021). Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH. Nature Communications. 12(1), p. 1–14. doi: 10.1038/s41467-021-21863-4. Full text in Research Archive Show summary

  Abstract Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. Here we report that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri , cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. We show that in Yersinia enterocolitica , low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.

• Mekasha, Sophanit & Linke, Dirk (2021). Secretion Systems in Gram-Negative Bacterial Fish Pathogens. Frontiers in Microbiology. 12. doi: 10.3389/fmicb.2021.782673. Full text in Research Archive Show summary

  Bacterial fish pathogens are one of the key challenges in the aquaculture industry, one of the fast-growing industries worldwide. These pathogens rely on arsenal of virulence factors such as toxins, adhesins, effectors and enzymes to promote colonization and infection. Translocation of virulence factors across the membrane to either the extracellular environment or directly into the host cells is performed by single or multiple dedicated secretion systems. These secretion systems are often key to the infection process. They can range from simple single-protein systems to complex injection needles made from dozens of subunits. Here, we review the different types of secretion systems in Gram-negative bacterial fish pathogens and describe their putative roles in pathogenicity. We find that the available information is fragmented and often descriptive, and hope that our overview will help researchers to more systematically learn from the similarities and differences between the virulence factors and secretion systems of the fish-pathogenic species described here.

• Zhang, Zijian; Ryoo, David; Balusek, Curtis; Acharya, Atanu; Rydmark, Marcella Orwick & Linke, Dirk [Show all 7 contributors for this article] (2021). Inward-facing glycine residues create sharp turns in β-barrel membrane proteins. Biochimica et Biophysica Acta - Biomembranes. ISSN 0005-2736. 1863(10). doi: 10.1016/j.bbamem.2021.183662. Full text in Research Archive
• 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. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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.

• Claxton, James Benedict; Joudeh, Nadeem; Røyne, Anja; Linke, Dirk & Mikheenko, Pavlo (2020). Sequential magnetic mapping of bacteria loaded with Pd-Fe nanoparticles, 2020 IEEE 10th International Conference on “Nanomaterials: Applications & Properties” (NAP – 2020). IEEE (Institute of Electrical and Electronics Engineers). ISSN 9781728185064. p. 01NIC01-1–01NIC01-5. doi: 10.1109/NAP51477.2020.9309619. Full text in Research Archive Show summary

  Magnetic nanoparticles are of widespread use in nanotechnology. One of the most unusual are magnetic palladium nanoparticles that combine magnetism with high catalytic activity. These nanoparticles could be obtained biologically by exposing bacteria to a palladium salt. Due to their small size and weak magnetism, however, it is challenging to measure their magnetic properties. One of the solutions to enhance their magnetism is to incorporate a small amount of iron atoms into them. After this procedure, the nanoparticles together with bacteria can be embedded in resin and characterized by the technique of magnetic force microscopy. This technique allows imaging cross-sections of the bacteria with nanoparticles, but cannot give information from the depth of the sample. Here we report on an approach partially solving this problem. Its novelty lies in measurements of consecutive thin slices of resin, which allows mapping cross-sections of individual bacteria and different parts of the material surrounding the same bacterium. An interesting observed feature is the formation of magnetic chains of nanoparticles outside of the bacteria.

• Perilla, Ana Lucia Campaña; Joudeh, Nadeem; Høyer, Henrik; Røyne, Anja; Linke, Dirk & Mikheenko, Pavlo (2020). Probing van der Waals and magnetic forces in bacteria with magnetic nanoparticles, 2020 IEEE 10th International Conference on “Nanomaterials: Applications & Properties” (NAP – 2020). IEEE (Institute of Electrical and Electronics Engineers). ISSN 9781728185064. p. 01NSSA03-1–01NSSA03-5. doi: 10.1109/NAP51477.2020.9309722. Full text in Research Archive Show summary

  Bioinspired metal-based nanoparticles have potential uses in many applications, but before a possible commercial exploitation, it is important to clarify the pathways of their production and deposition inside the organisms, for example in bacteria. The technique of magnetic force microscopy (MFM) could be used to evaluate the nanoparticles magnetic properties, in addition to allowing tracing their location inside or outside of bacteria, which could help to understand pathways of their biosynthesis. In this work, using MFM and analyzing the interaction of magnetic tip with nanoparticles and bacteria imbedded in resin at different heights above the surface, and comparing gradients of forces recorded by magnetic and non-magnetic tips, a condition was found, which allows to measure pure magnetic response of Pd-Fe nanoparticles. For these nanoparticles, the interplay between magnetic and van der Waals forces is described at small distances to the surface. Experimental data are compared with simulations, based on the calculation of the distribution of magnetic field around a nanoparticle, which defines magnetic force acting on the MFM tip.

• Wróbel, Agnieszka Ewelina; Saragliadis, Athanasios; Pérez-Ortega, Jesús; Sittman, Carolin; Göttig, Stephan & Liskiewicz, Krystyna Anna [Show all 11 contributors for this article] (2020). The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence. Environmental Microbiology. ISSN 1462-2912. 22(7), p. 2939–2955. doi: 10.1111/1462-2920.15051. Full text in Research Archive Show summary

  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.

• Hatlem, Daniel; Trunk, Thomas; Linke, Dirk & Leo, Jack Christopher (2020). Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins. In Leitao, JH (Eds.), Microbial Virulence Factors. MDPI. ISSN 9783039369461. p. 371–400. doi: 10.3390/ijms20092129. Full text in Research Archive Show summary

  The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.

• 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. Full text in Research Archive Show summary

  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  &lt;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.

• Ryoo, David; Rydmark, Marcella Orwick; Pang, Yui Tik; Lundquist, Karl P; Linke, Dirk & Gumbart, James C (2020). BamA is required for autotransporter secretion. Biochimica et Biophysica Acta - General Subjects. ISSN 0304-4165. 1864(7), p. 1–8. doi: 10.1016/j.bbagen.2020.129581. Full text in Research Archive Show summary

  Background In Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the “passenger” domain) and a β-barrel that aids its export. While it is known that the folding and insertion of the β-barrel domain utilize the β-barrel assembly machinery (BAM) complex, how the passenger domain is secreted and folded across the membrane remains to be determined. The hairpin model states that passenger domain secretion occurs independently through the fully-formed and membrane-inserted β-barrel domain via a hairpin folding intermediate. In contrast, the BamA-assisted model states that the passenger domain is secreted through a hybrid of BamA, the essential subunit of the BAM complex, and the β-barrel domain of the autotransporter. Methods To ascertain the models' plausibility, we have used molecular dynamics to simulate passenger domain secretion for two autotransporters, EspP and YadA. Results We observed that each protein's β-barrel is unable to accommodate the secreting passenger domain in a hairpin configuration without major structural distortions. Additionally, the force required for secretion through EspP's β-barrel is more than that through the BamA β-barrel. Conclusions Secretion of autotransporters most likely occurs through an incompletely formed β-barrel domain of the autotransporter in conjunction with BamA. General significance Secretion of virulence factors is a process used by practically all pathogenic Gram-negative bacteria. Understanding this process is a necessary step towards limiting their infectious capacity.

• Gonzalez, Florencia Munoz; Sycz, Gabriela; Paiva, Ivan M Alonso; Linke, Dirk; Zorreguieta, Angeles & Baldi, Pablo C [Show all 7 contributors for this article] (2019). The BtaF Adhesin Is Necessary for Full Virulence During Respiratory Infection by Brucella suis and Is a Novel Immunogen for Nasal Vaccination Against Brucella Infection. Frontiers in Immunology. 10, p. 1–16. doi: 10.3389/fimmu.2019.01775. Full text in Research Archive Show summary

  Brucella enters their hosts mostly through mucosae from where it spreads systemically. Adhesion to extracellular matrix (ECM) components or to host cells is important for the infectious process, and is mediated by several adhesins, including the BtaF trimeric autotransporter. Although Th1 responses and gamma interferon (IFN-γ) are important for protection, antibodies able to block adhesions might also contribute to prevent Brucella infection. We evaluated the importance of BtaF for respiratory Brucella infection, and characterized the immune response and protection from mucosal challenge induced by nasal vaccination with recombinant BtaF. While lung CFU numbers did not differ at day 1 p.i. between mice intratracheally inoculated with B. suis M1330 (wild type) and those receiving a ΔbtaF mutant, they were reduced in the latter group at 7 and 30 days p.i. For vaccination studies the BtaF passenger domain was engineered and expressed as a soluble trimeric protein. Mice were immunized by the nasal route with BtaF or saline (control group) plus the mucosal adjuvant c-di-AMP. Specific anti-BtaF antibodies (IgG and IgA) were increased in serum, including a mixed IgG2a/IgG1 response. In vitro, these antibodies reduced bacterial adhesion to A549 alveolar epithelial cells. Specific IgA antibodies were also increased in several mucosae. Spleen cells from BtaF immunized mice significantly increased their IL-2, IL-5, IL-17, and IFN-γ secretion upon antigen stimulation. In cervical draining lymph nodes, antigen-experienced CD4+ T cells were maintained mainly as central memory cells. A BtaF-specific delayed-type hypersensitivity response was detected in BtaF immunized mice. Lung cells from the latter produced high levels of IFN-γ upon antigen stimulation. Although nasal immunization with BtaF did not protect mice against B. suis respiratory challenge, it conferred significant protection from intragastric challenge; the splenic load of B. suis was reduced by 3.28 log CFU in immunized mice. This study shows that nasal vaccination with BtaF+c-di-AMP protects against intragastric challenge with B. suis by inducing local and systemic antibody responses, central memory CD4+ T cells and strong Th1 responses. Therefore, although BtaF vaccination did not protect from B. suis respiratory infection, this adhesin constitutes a promising immunogen against mucosal B. suis infection.

• Wróbel, Agnieszka Ewelina; Leo, Jack Christopher & Linke, Dirk (2019). Overcoming fish defences: The virulence factors of Yersinia ruckeri. Genes. 10(9), p. 1–20. doi: 10.3390/genes10090700. Full text in Research Archive Show summary

  Yersinia ruckeri is the causative agent of enteric redmouth disease, a bacterial infection of marine and freshwater fish. The disease mainly affects salmonids, and outbreaks have significant economic impact on fish farms all over the world. Vaccination routines are in place against the major serotypes of Y. ruckeri but are not effective in all cases. Despite the economic importance of enteric redmouth disease, a detailed molecular understanding of the disease is lacking. A considerable number of mostly omics-based studies have been performed in recent years to identify genes related to Y. ruckeri virulence. This review summarizes the knowledge on Y. ruckeri virulence factors. Understanding the molecular pathogenicity of Y. ruckeri will aid in developing more efficient vaccines and antimicrobial compounds directed against enteric redmouth disease.

• Saragliadis, Athanasios & Linke, Dirk (2019). Assay development for the discovery of small-molecule inhibitors of YadA adhesion to collagen. The Cell Surface. 5, p. 1–11. doi: 10.1016/j.tcsw.2019.100025. Full text in Research Archive Show summary

  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.

• Aoki, S.; Yoshimoto, Shogo; Ishikawa, M; Linke, Dirk; Lupas, Andrei & Hori, Katsutoshi (2019). Native display of a huge homotrimeric protein fiber on the cell surface after precise domain deletion. Journal of Bioscience and Bioengineering. ISSN 1389-1723. p. 1–6. doi: 10.1016/j.jbiosc.2019.09.022. Full text in Research Archive
• Thibau, Arno; Dichter, Alexander A.; Vaca, Diana J.; Linke, Dirk; Goldman, Adrian & Kempf, Volkhard A.J. (2019). Immunogenicity of trimeric autotransporter adhesins and their potential as vaccine targets. Medical Microbiology and Immmunology. ISSN 0300-8584. 209, p. 243–263. doi: 10.1007/s00430-019-00649-y. Full text in Research Archive Show summary

  The current problem of increasing antibiotic resistance and the resurgence of numerous infections indicate the need for novel vaccination strategies more than ever. In vaccine development, the search for and the selection of adequate vaccine antigens is the first important step. In recent years, bacterial outer membrane proteins have become of major interest, as they are the main proteins interacting with the extracellular environment. Trimeric autotransporter adhesins (TAAs) are important virulence factors in many Gram-negative bacteria, are localised on the bacterial surface, and mediate the first adherence to host cells in the course of infection. One example is the Neisseria adhesin A (NadA), which is currently used as a subunit in a licensed vaccine against Neisseria meningitidis. Other TAAs that seem promising vaccine candidates are the Acinetobacter trimeric autotransporter (Ata), the Haemophilus influenzae adhesin (Hia), and TAAs of the genus Bartonella. Here, we review the suitability of various TAAs as vaccine candidates.

• Weidensdorfer, Marko; Ishikawa, Masahito; Hori, Katsutoshi; Linke, Dirk; Djahanshiri, Bardya & Iruegas, Ruben [Show all 17 contributors for this article] (2019). The Acinetobacter trimeric autotransporter adhesin Ata controls key virulence traits of Acinetobacter baumannii. Virulence. ISSN 2150-5594. 10(1), p. 68–81. doi: 10.1080/21505594.2018.1558693. Full text in Research Archive Show summary

  Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.

• Meuskens, Ina; Saragliadis, Athanasios; Leo, Jack Christopher & Linke, Dirk (2019). Type V secretion systems: An overview of passenger domain functions. Frontiers in Microbiology. 10, p. 1–19. doi: 10.3389/fmicb.2019.01163. Full text in Research Archive Show summary

  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.

• Micenková, Lenka; Bosak, Juraj; Kucera, Jiri; Hrala, Matej; Dolejsova, Tereza & Sedo, Ondrej [Show all 9 contributors for this article] (2019). Colicin Z, a structurally and functionally novel colicin type that selectively kills enteroinvasive Escherichia coli and Shigella strains. Scientific Reports. 9(1), p. 1–12. doi: 10.1038/s41598-019-47488-8. Full text in Research Archive Show summary

  Colicin production in Escherichia coli (E. coli) strains represents an important trait with regard to microbial survival and competition in the complex intestinal environment. A novel colicin type, colicin Z (26.3 kDa), was described as a product of an original producer, extraintestinal E. coli B1356 strain, isolated from the anorectal abscess of a 17 years-old man. The 4,007 bp plasmid (pColZ) was completely sequenced and colicin Z activity (cza) and colicin Z immunity (czi) genes were identified. The cza and czi genes are transcribed in opposite directions and encode for 237 and 151 amino acid-long proteins, respectively. Colicin Z shows a narrow inhibitory spectrum, being active only against enteroinvasive E. coli (EIEC) and Shigella strains via CjrC receptor recognition and CjrB- and ExbB-, ExbD-mediated colicin translocation. All tested EIEC and Shigella strains isolated between the years 1958–2010 were sensitive to colicin Z. The lethal effect of colicin Z was found to be directed against cell wall peptidoglycan (PG) resulting in PG degradation, as revealed by experiments with Remazol Brilliant Blue-stained purified peptidoglycans and with MALDI-TOF MS analyses of treated PG. Colicin Z represents a new class of colicins that is structurally and functionally distinct from previously studied colicin types.

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• Linke, Dirk (2025). Trimeric Autotransporter Adhesins: structure, function, innovation potential. Full text in Research Archive
• Linke, Dirk (2025). AlphaFold user errors. Full text in Research Archive
• Linke, Dirk (2025). Bacterial Autotransporter Adhesins: structure, function, and surprising innovation potential. Full text in Research Archive
• Linke, Dirk (2025). Bacterial Autotransporter Adhesins: structure, function, and surprising innovation potential. Full text in Research Archive
• Linke, Dirk (2025). A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity. Full text in Research Archive
• Linke, Dirk (2025). Bacterial Autotransporter Adhesins: structure, function, and surprising innovation potential. Full text in Research Archive
• Johansen, Emma Marinelli; Saragliadis, Athanasios & Linke, Dirk (2025). Inhibiting Collagen-Binding Trimeric Autotransporter Adhesins from Clinically Relevant Pathogens. Full text in Research Archive
• 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. Full text in Research Archive
• Hatlem, Daniel; Saragliadis, Athanasios & Linke, Dirk (2025). An LPS-binding peptide with extreme affinity can be used for novel endotoxin detection and removal applications. Full text in Research Archive
• 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. Full text in Research Archive
• Perilla, Ana Lucia Campana; Saragliadis, Athanasios; Merroun, Mohamed; Rachel, Reinhard; Mikheenko, Pavlo & Linke, Dirk (2025). Microbial strategies for tailored metal nanoparticle production. Full text in Research Archive
• Saragliadis, Athanasios; Winther, Anja Ruud; Leo, Jack Christopher & Linke, Dirk (2025). Fluorescent labeling of bacterial cell envelope and its application in membrane protein enrichment. Full text in Research Archive
• 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. Full text in Research Archive
• 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. Full text in Research Archive
• Lauretis, Angela de; Santacroce, Marco; Hussain, Badra; Ma, Qianli; Ellingsen, Jan Eirik & Lyngstadaas, Ståle Petter [Show all 8 contributors for this article] (2025). A poloxamer-peroxide forumulation effectively removes pellicle and limits recontamination. Full text in Research Archive
• 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. Full text in Research Archive
• Linke, Dirk (2024). A disordered loop in a bacterial adhesin can form a poly-proline II helix upon binding to heparin. Full text in Research Archive
• Linke, Dirk & Falk, Jonathan (2024). Beroliger folk etter «zombie-virus»-funn. [Internet]. VG.NO. Full text in Research Archive
• 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. Full text in Research Archive
• Mertes, Verena; Linke, Dirk; Saragliadis, Athanasios; Roos, Norbert; Tysvær, Ellen-Beate & Mascherin, Elisa (2024). Recombinant extracellular vesicles as vaccines within animal health. Full text in Research Archive Show summary

  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. Full text in Research Archive Show summary

  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 [Show all 7 contributors for this article] (2024). Recombinant extracellular vesicles as vaccines within animal health. Full text in Research Archive Show summary

  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. Full text in Research Archive
• Lislerud, Anna; Saragliadis, Athanasios & Linke, Dirk (2024). Adhesins of Yersinia ruckeri. Full text in Research Archive
• Linke, Dirk (2024). A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity. Full text in Research Archive
• Perilla, Ana Lucia Campana; Saragliadis, Athanasios; Merroun, Mohamed; Mikheenko, Pavlo & Linke, Dirk (2024). Microbial-metal interactions for biosynthesis of tailored metal nanoparticles. Full text in Research Archive
• Linke, Dirk (2024). Trimeric Autotransporter Adhesins: structure, function, evolution. Full text in Research Archive
• Nakatani, Hajime; Linke, Dirk & Hori, Katsutoshi (2023). Study on the fish epidermal bacterial flora for fish disease control. Full text in Research Archive
• Saragliadis, Athanasios & Linke, Dirk (2023). Assay development for novel antimicrobials: screening for small-molecule inhibitors of YadA adhesion to collagen. Full text in Research Archive
• 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. Full text in Research Archive
• Fan, Enguo & Linke, Dirk (2023). Methods and assays to study protein transport in vivo. Full text in Research Archive
• Sanz-Gaitero, Marta; Cunha, Eva S.; Linke, Dirk & Lücke, Hartmut (2023). Using Escherichia coli as a model to test Helicobacter pylori urease inhibitors. Full text in Research Archive
• Reese, Jan-Ole; Saragliadis, Athanasios; Haugen, Håvard Jostein & Linke, Dirk (2023). Development of multi-species biofilm models for testing biotechnological approaches in dentistry. Full text in Research Archive
• Mekasha, Sophanit; Petrikonytė, Patricija & Linke, Dirk (2023). Virulence factors of the bacterial fish pathogen Tenacibaculum maritimum. Full text in Research Archive
• Hori, Katsutoshi & Linke, Dirk (2023). Application of a bacterial adhesin to bioprocesses. Full text in Research Archive
• Ledo, Jesus & Linke, Dirk (2023). Biofouling on metal surfaces. Full text in Research Archive
• 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. Full text in Research Archive
• Yoshimoto, Shogo; Linke, Dirk & Hori, Katsutoshi (2023). Single-cell analysis of adhesive bacteria using atomic force microscopy. Full text in Research Archive
• Leo, Jack C.; Linke, Dirk & Morth, P (2023). Role of the extracellular 'juncture' domain in the secretion and function of inverse autotransporters. Full text in Research Archive
• Linke, Dirk (2023). A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity. Full text in Research Archive
• Linke, Dirk (2023). BEDPAN: Palladium nanoparticles from bacteria. Full text in Research Archive
• Mertes, Verena; Saragliadis, Athanasios; Mascherin, E; Antenucci, Fabio; Bojesen, Anders Miki & Colquhoun, Duncan John [Show all 8 contributors for this article] (2023). Characterization of bacterial extracellular vesicles A versatile tool for potential vaccine application in aquaculture. Full text in Research Archive
• Saragliadis, Athanasios & Linke, Dirk (2023). Development of assays for novel antimicrobials: screening for small-molecule inhibitors of YadA adhesion to collagen. Full text in Research Archive
• Petrikonyte, Patricija; Mekasha, Sophanit & Linke, Dirk (2023). Tenacibaculum maritimum virulence factors. Full text in Research Archive
• Reese, Jan-Ole; Haugen, Håvard Jostein; Saragliadis, Athanasios & Linke, Dirk (2023). Development of multi-species biofilm models for evaluating medical approaches in dentistry. Full text in Research Archive
• Linke, Dirk (2023). Tandem repeat adhesins and dead repeat genes in bacteria. Full text in Research Archive
• Perilla, Ana Lucia Campana; Joudeh, Nadeem; Merroun, Mohamed; Gomez-Bolivar, Jamie; Linke, Dirk & Mikheenko, Pavlo (2023). Bacterial synthesis of Palladium nanoparticles. Full text in Research Archive
• Yoshimoto, Shogo; Aoki, S.; Ohara, Y; Ishikawa, Masahito; Suzuki, A & Lupas, Andrei [Show all 8 contributors for this article] (2023). Identification of the adhesive domain of AtaA from Acinetobacter sp. Tol 5 and its application in immobilizing Escherichia coli. Full text in Research Archive
• Mekasha, Sophanit; Haslene-Hox, Hanne; Sperstad, Sigrid Bakken; Tøndervik, Anne & Linke, Dirk (2023). Exploring virulence factors of Tenacibaculum maritimum. Full text in Research Archive
• Perilla, Ana Lucia Campana; Joudeh, Nadeem; Merroun, Mohamed; Gomez-Bolivar, Jamie; Linke, Dirk & Mikheenko, Pavlo (2023). Bacterial synthesis of Palladium nanoparticles. Full text in Research Archive
• Lislerud, Anna; Nakatani, Hajime; Spence, Maria Helle; Saragliadis, Athanasios; Hori, Katsutoshi & Linke, Dirk (2023). Adhesins of Yersinia ruckeri. Full text in Research Archive
• Linke, Dirk (2023). Structure, Function and Evolution of Autotransporter Adhesins. Full text in Research Archive
• Petrikonyte, Patricija; Mekasha, Sophanit & Linke, Dirk (2023). T. maritimum virulence factors. Full text in Research Archive
• Mekasha, Sophanit; Tøndervik, Anne & Linke, Dirk (2023). Exploring virulence factors of Tenacibaculum maritimum. Full text in Research Archive
• Linke, Dirk (2023). Trimeric Autotransporters: non-globular, repetitive virulence factors with peculiar genomic recombination mechanisms. Full text in Research Archive
• 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. Full text in Research Archive
• Linke, Dirk (2023). Trimeric Autotransporters: Function, Structure, Evolution. Full text in Research Archive
• Yoshimoto, Shogo; Aoki, S.; Ohara, Y; Ishikawa, Masahito; Suzuki, A & Linke, Dirk [Show all 7 contributors for this article] (2023). Downsizing of AtaA from Acinetobacter p. Tol5 and its application in immobilizing E.coli. Full text in Research Archive
• Gaitero, Marta Sanz; Cunha, Eva S.; Linke, Dirk & Luecke, Hartmut (2023). Using Escherichia coli as a model to test Helicobacter pylori urease inhibitors. Full text in Research Archive
• Linke, Dirk (2023). Trimeric Autotransporter Adhesins: Structure, Function, and Evolution. Full text in Research Archive
• 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. Full text in Research Archive
• Reese, Jan-Ole; Saragliadis, Athanasios; Haugen, Håvard Jostein & Linke, Dirk (2023). Development of multi-species biofilm models for evaluating medical approaches in dentistry. Full text in Research Archive
• Meuskens, Ina; Kristiansen, Per Eugen; Bardiaux, Benjamin; Koynarev, Vladimir Rosenov; Hatlem, Daniel & Prydz, Kristian [Show all 9 contributors for this article] (2023). Strain variations in a bacterial adhesin lead to different binding partners in the pathogen Yersinia enterocolitica. Full text in Research Archive
• Petrikonyte, Patricija; Mekasha, Sophanit & Linke, Dirk (2023). Tenacibaculum maritimum virulence factors. Full text in Research Archive
• 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. Full text in Research Archive
• Mekasha, Sophanit & Linke, Dirk (2023). Virulence factors of the bacterial fish pathogen Tenacibaculum maritimum. Full text in Research Archive
• Linke, Dirk (2023). Type-5-secreted adhesins in human and animal disease. Full text in Research Archive
• Linke, Dirk (2022). Bacterial adhesion and how to inhibit it. Full text in Research Archive
• Linke, Dirk (2022). Structure, Function, and Evolution of trimeric autotransporter adhesins. Full text in Research Archive
• Linke, Dirk (2022). Bacterial Adhesins - Structure and Function. Full text in Research Archive
• Linke, Dirk (2022). Trimeric Autotransporter Adhesins: Structure, Function, and Evolution. Full text in Research Archive
• Linke, Dirk (2022). Bacterial adhesion by fish pathogens. Full text in Research Archive
• Linke, Dirk (2022). Bacterial adhesion - structure and evolution of autotransporters. Full text in Research Archive
• Reese, Jan-Ole; Saragliadis, Athanasios; Haugen, Håvard Jostein & Linke, Dirk (2022). Development of multi-species biofilm models for testing biotechnological approaches in dentistry. Full text in Research Archive
• Mekasha, Sophanit & Linke, Dirk (2022). Toxins and secretion systems in Gram-negative bacterial fish pathogens. Full text in Research Archive
• Saragliadis, Athanasios & Linke, Dirk (2022). Structure-function relationship in bacterial adhesins. Full text in Research Archive
• Perilla, Ana Lucia Campana; Joudeh, Nadeem; Merroun, Mohamed; Gomez-Bolivar, Jamie; Røyne, Anja & Linke, Dirk [Show all 7 contributors for this article] (2022). Biosynthesis and characterization of Palladium-Based Nanoparticles using Escherichia coli. Full text in Research Archive
• Mertes, Verena; Saragliadis, Athanasios; Attenucci, Fabio; Wrobel, Agnieszka; Ciani, Elia & Linke, Dirk [Show all 8 contributors for this article] (2022). Characterization of bacterial extracellular vesicles from a fish pathogen, their role in virulence and possible function as vaccine in aquaculture. Full text in Research Archive
• Mekasha, Sophanit & Linke, Dirk (2022). Toxins and secretion systems in Gram-negative bacterial fish pathogens. The case of T. maritimum. Full text in Research Archive
• Linke, Dirk (2022). How to apply for a PhD position... and how not to! Full text in Research Archive
• Hatlem, Daniel; Linke, Dirk Bernd Kurt & Andersen, Elisabeth K. (2022). Forskerprosjekt på antibiotika og blodforgiftning. [Internet]. Website Oslo Cancer Cluster. Full text in Research Archive
• 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. Full text in Research Archive
• Linke, Dirk; Økstad, Ole Andreas; Haugen, Håvard Jostein & Winther-Larsen, Hanne Cecilie (2021). The exchange program MISC - Options for internships at the University of Oslo (with descriptions of current projects). Full text in Research Archive
• Hori, Katsutoshi & Linke, Dirk (2021). the exchange program MISC - Options for internships at Nagoya University (with descriptions of current projects by students). Full text in Research Archive
• Fan, Enguo & Linke, Dirk (2021). the exchange program MISC - Options for internships at PMUC (with descriptions of current projects). Full text in Research Archive
• Linke, Dirk (2021). Using LPS-Binding Peptides To Detect Endotoxins. Full text in Research Archive
• Leva, Juan; Meuskens, Ina; Linke, Dirk; Peyman, Sally & Millner, Paul (2021). Novel Electrochemical Biosensor for Pathogen Detection Based on Extracellular Matrix (ECM) Protein - Adhesin Interaction. Full text in Research Archive
• Thibau, Arno; Vaca, Diana; Linke, Dirk & Kempf, Volkhard A.J. (2021). Analysing the Fibronectin Binding Properties of Bartonella henselae Adhesin A. Full text in Research Archive
• Chowdhury, Sounak; Linke, Dirk; Happonen, Lotta Johanna & Malmström, Johan (2021). Streptococcus Pyogenes Forms M Protein Specific Host-microenvironment Dependent Protein Complexes. Full text in Research Archive
• Vaca, Diana; Happonen, Lotta Johanna; Goldman, Adrian & Linke, Dirk (2021). Bacterial and Viral Adhesion; Two Sides of the Same Coin? Full text in Research Archive
• Meuskens, Ina & Linke, Dirk (2021). The Trimeric Autotransporter Adhesin YadA of Yersinia Binds to Glycan Moieties. Full text in Research Archive
• Butcher, Sarah Jane & Linke, Dirk (2021). Viral and Bacterial Adhesion - examples from current ViBrANT research. Full text in Research Archive
• Thibau, Arno; Vaca, Diana; Linke, Dirk & Kempf, Volkhard A.J. (2021). Analysing the fibronectin binding properties of B. henselae adhesin A. Full text in Research Archive
• Perilla, Ana Lucia Campana; Joudeh, Nadeem; Linke, Dirk & Mikheenko, Pavlo (2021). Magnetic decoration of Escherichia coli loaded with Palladium nanoparticles. Full text in Research Archive
• Wimmi, Stephan; Balinovic, Alexander; Jeckel, Hannah; Selinger, Lisa; Lampaki, Dimitrios & Eisemann, Emma [Show all 11 contributors for this article] (2021). Author Correction: Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH (Nature Communications, (2021), 12, 1, (1625), 10.1038/s41467-021-21863-4). Nature Communications. 12(1). doi: 10.1038/s41467-021-23080-5. Full text in Research Archive
• Linke, Dirk (2021). Developing inhibitors that block adhesin-collagen interactions. Full text in Research Archive
• Vaca, Diana; Thibau, Arno; Linke, Dirk; Happonen, Lotta Johanna; Malmström, Johan & Kempf, Volkhard A.J. (2021). Interaction between Bartonella adhesin A and fibronectin in bacterial adherence to human endothelial cells. Full text in Research Archive
• Meuskens, Ina; Leva-Bueno, Juan; Millner, Paul; Schütz, Monika; Peyman, Sally & Linke, Dirk (2021). The Trimeric Autotransporter Adhesin YadA of Yersinia binds to glycan moieties. Full text in Research Archive
• Chowdhury, Sounak; Linke, Dirk; Happonen, Lotta Johanna & Malmström, Johan (2021). Streptococcus pyogenes forms M protein specific host-environment dependent protein complexes. Full text in Research Archive
• Thibau, Arno; Linke, Dirk & Kempf, Volkhard A.J. (2021). Analysing the fibronectin binding properties and genomic variation of Bartonella henselae adhesin. Full text in Research Archive
• Leva-Bueno, Juan; Meuskens, Ina; Linke, Dirk; Millner, Paul & Peyman, Sally (2021). Impedimetric biosensor based on extracellular matrix protein-adhesin interaction. Full text in Research Archive
• Linke, Dirk (2020). Dette munnbindet skal drepe koronaviruset. [Internet]. TV2 website. Full text in Research Archive Show summary

  This interview is about the antimicrobial effect of copper particles in facemasks

• Linke, Dirk (2020). Palladium nanoparticles made by bacteria. Full text in Research Archive
• Linke, Dirk (2020). Norsk kobber inn i kampen mot koronaviruset: verdens første selvrensende munnbind. [Internet]. Nordox website. Full text in Research Archive
• Linke, Dirk (2020). LPS-Binding Peptides: New Tools for Endotoxin Detection and Removal? Full text in Research Archive
• Ryoo, David; Orwick-Rydmark, Marcella; Pang, Y; Lundqvist, Daniel; Linke, Dirk & Gumbart, JC (2020). Quantitative Comparisons of Competing Models of Autotransporter Passenger-Domain Secretion. Full text in Research Archive
• Thibau, Arno; Vaca, Diana; Linke, Dirk & Kempf, Volkhard A.J. (2020). Analysing the fibronectin binding properties of Bartonella henselae adhesin A. Full text in Research Archive
• Perilla, Ana Lucia Campaña; Joudeh, Nadeem; Høyer, Henrik; Røyne, Anja; Linke, Dirk & Mikheenko, Pavlo (2020). Probing van der Waals and magnetic forces in bacteria with magnetic nanoparticles. Full text in Research Archive Show summary

  Bioinspired metal-based nanoparticles have potential uses in many applications, but before a possible commercial exploitation, it is important to clarify the pathways of their production and deposition inside the organisms, for example in bacteria. The technique of magnetic force microscopy (MFM) could be used to evaluate the nanoparticles magnetic properties, in addition to allowing tracing their location inside or outside of bacteria, which could help to understand pathways of their biosynthesis. In this work, using MFM and analyzing the interaction of magnetic tip with nanoparticles and bacteria imbedded in resin at different heights above the surface, and comparing gradients of forces recorded by magnetic and non-magnetic tips, a condition was found, which allows to measure pure magnetic response of Pd-Fe nanoparticles. For these nanoparticles, the interplay between magnetic and van der Waals forces is described at small distances to the surface. Experimental data are compared with simulations, based on the calculation of the distribution of magnetic field around a nanoparticle, which defines magnetic force acting on the MFM tip.

• Claxton, James; Joudeh, Nadeem; Røyne, Anja; Linke, Dirk & Mikheenko, Pavlo (2020). Sequential magnetic mapping of bacteria loaded with Pd-Fe nanoparticles. Full text in Research Archive Show summary

  Magnetic nanoparticles are of widespread use in nanotechnology. One of the most unusual are magnetic palladium nanoparticles that combine magnetism with high catalytic activity. These nanoparticles could be obtained biologically by exposing bacteria to a palladium salt. Due to their small size and weak magnetism, however, it is challenging to measure their magnetic properties. One of the solutions to enhance their magnetism is to incorporate a small amount of iron atoms into them. After this procedure, the nanoparticles together with bacteria can be embedded in resin and characterized by the technique of magnetic force microscopy. This technique allows imaging cross-sections of the bacteria with nanoparticles, but cannot give information from the depth of the sample. Here we report on an approach partially solving this problem. Its novelty lies in measurements of consecutive thin slices of resin, which allows mapping cross-sections of individual bacteria and different parts of the material surrounding the same bacterium. An interesting observed feature is the formation of magnetic chains of nanoparticles outside of the bacteria.

• Meuskens, Ina; Michalik, Marcin; Chauhan, Nandini; Linke, Dirk & Leo, Jack Christopher (2020). Corrigendum: A New Strain Collection for Improved Expression of Outer Membrane Proteins (Frontiers in Cellular and Infection Microbiology, (2017), 7, (464), 10.3389/fcimb.2017.00464). Frontiers in Cellular and Infection Microbiology. 10. doi: 10.3389/fcimb.2020.00220. Full text in Research Archive
• Linke, Dirk (2020). Biofilm-forming adhesins of the fish pathogen Yersinia ruckeri. Full text in Research Archive
• Tietgen, M; Weidensdorfer, M; Leukert, L; Djahanshiri, Bardya; Iruegas, R & Ebersberger, Ingo [Show all 9 contributors for this article] (2019). Virulence of Acinetobacter baumannii depends on the Acinetobacter trimeric autotransporter adhesin (Ata). Full text in Research Archive
• Kandanur, Sai Priya Sarma; Linke, Dirk & Leo, Jack Christopher (2019). Hairpin Formation and Passenger Secretion in the Inverse Autotransporter Intimin. Full text in Research Archive
• Chauhan, Nandini; Hatlem, Daniel; Orwick-Rydmark, Marcella; Schneider, Kenneth; Linke, Dirk & Leo, Jack Christopher (2019). Using the SpyCatcher-SpyTag technology for topology mapping of outer membrane proteins in Gram-negative bacteria. Full text in Research Archive
• Linke, Dirk (2019). Insights into the autotransport process of a trimeric autotransporter, YadA. Full text in Research Archive
• Linke, Dirk (2019). Type V secretion: membrane transport and anchoring of bacterial adhesins. Full text in Research Archive
• Røsjø, Bjarne; Orwick-Rydmark, Marcella; Linke, Dirk; Leo, Jack Christopher & Hatlem, Daniel (2019). Ny antibiotika-idé: Vil hindre bakteriene i å feste seg. [Internet]. TITAN.NO. Full text in Research Archive
• Tietgen, M; Weidensdorfer, M; Djahanshiri, Bardya; Linke, Dirk; Kempf, Volkhard A.J. & Gottig, Stephan (2019). Virulence of Acinetobacter baumannii depends on the Acinetobacter trimeric autotransporter adhesin Ata. Full text in Research Archive
• Meuskens, Ina; Higson, Seamus & Linke, Dirk (2019). Innovative training networks: a new way of collaboration-propped PhD training. Medical Microbiology and Immmunology. ISSN 0300-8584. 209, p. 215–216. doi: 10.1007/s00430-019-00647-0. Full text in Research Archive
• Leva, Juan; Meuskens, Ina; Gibson, Timothy M.; Linke, Dirk & Millner, Paul (2019). Biosensors for pathogen detection. Full text in Research Archive
• Saragliadis, Athanasios & Linke, Dirk (2019). Assay development for the discovery of small-molecule inhibitors of YadA adhesion to collagen. Full text in Research Archive
• Meuskens, Ina; Leva, Juan; Millner, Paul & Linke, Dirk (2019). Differential Adhesion of Trimeric Autotransporter Adhesins to Extracellular Matrix Proteins. Full text in Research Archive
• Chowdhury, Sounak; Happonen, Lotta Johanna; Linke, Dirk & Malmström, Johan (2019). QUANTITATIVE AFFINITY-PURIFICATION AND SURFACE-ADSORPTION MASS SPECTROMETRY TO MAP STREPTOCOCCUS PYOGENES AND HUMAN PROTEIN INTERACTION. Full text in Research Archive
• Linke, Dirk (2019). Autotransporter Adhesins in the fish pathogen Yersinia ruckeri. Full text in Research Archive
• Thibau, Arno; Vaca, Diana; Linke, Dirk; Ballhorn, Wibke & Kempf, Volkhard A.J. (2019). Analysing the Fibronectin Binding Domain of the Bartonella henselae Adhesin A. Full text in Research Archive
• Leukert, L; Weidensdorfer, M; Djahanshiri, Bardya; Linke, Dirk; Kempf, Volkhard A.J. & Gottig, Stephan (2019). The Acinetobacter Trimeric Autotransporter Adhesin Ata Controls Key Virulence Traits of Acinetobacter baumannii. Full text in Research Archive
• Linke, Dirk (2019). The Gram-Negative Bacterial Cell Surface: How to Study Its Protein Components and How to Remove Endotoxin. Full text in Research Archive
• Linke, Dirk & Saragliadis, Athanasios (2019). Bacterial adhesion to living and non-living surfaces. Full text in Research Archive
• Joudeh, Nadeem; Røyne, Anja; Mikheenko, Pavlo & Linke, Dirk (2019). Bio-Engineered Palladium Nanoparticles (BEDPAN). Full text in Research Archive
• Joudeh, Nadeem & Linke, Dirk (2019). Bio-engineered Palladium Nanoparticles. Full text in Research Archive
• 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. Full text in Research Archive Show summary

  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.

• Hatlem, Daniel; Linke, Dirk & Krengel, Ute (2022). Probing the role of lipopolysaccharides in Gram-negative protein secretion and biogenesis. Universitetet i Oslo. Full text in Research Archive
• Bergvoll, Helga; Linke, Dirk & Saragliadis, Athanasios (2022). Secreted toxin components in the fish pathogen Tenacibaculum maritimum. Universitetet i Oslo. Full text in Research Archive
• Joudeh, Nadeem; Linke, Dirk & Mikheenko, Pavlo (2022). Escherichia coli-mediated palladium nanoparticle synthesis. Universitetet i Oslo. Full text in Research Archive Show summary

  Palladium nanoparticles (Pd NPs) offer a wide range of novel and exciting applications in catalysis, medicine, engineering, and electronics. Currently, these NPs are synthesized using conventional physical and chemical methods. These methods have several disadvantages as the physical methods usually require energy-intensive processes, and the chemical methods often involve harmful solvents or produce toxic by-products. Lately, more environmentally friendly approaches for the synthesis of NPs have emerged using microorganisms. Several bacteria were reported for their ability to biosynthesize Pd NPs. In this process, Pd ions are biologically transported into the bacterial cells and subsequently reduced to metallic Pd, forming Pd NPs. The current knowledge on the proteins, enzymes, or pathways involved in the transport and reduction of Pd is very limited, which hinders the ability to genetically engineer bacterial strains capable of producing Pd NPs with fine-tuned properties. This thesis provides new insights on the genes that might be responsible for the transport of Pd ions in E. coli, the toxicological effects of Pd, and the potential detoxification strategies that E. coli employ to deal with Pd stress. In addition, this thesis describes an E. coli-mediated approach for the synthesis of 1-2 nm Pd and Pd-Fe NPs displaying novel ferromagnetic properties. At the same time, this thesis shows different methods for the characterization of the magnetic properties of these NPs in biological samples.

• Hovland, Hege; Linke, Dirk & Saragliadis, Athanasios (2022). Palladium nanoparticles in copper transport mutants of Escherichia coli. Universitetet i Oslo. Full text in Research Archive
• Meuskens, Ina; Linke, Dirk & Bardiaux, Benjamin (2022). Insights into the Binding Mechanism and Function of Bacterial Adhesins - Interactions between YadA and Host Extracellular Matrix Components. Universitetet i Oslo. Full text in Research Archive
• 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. Full text in Research Archive
• Hermansen, Simen; Linke, Dirk & Orwick-Rydmark, Marcella (2020). Investigating the effects of loop modifications on the folding of outer membrane protein X. Universitetet i Oslo. Full text in Research Archive Show summary

  The surface of Gram-negative bacteria is perforated by β-barrel proteins called outer membrane proteins (OMPs). These β-barrels are integral to the outer membrane (OM) and are essential for the viability of the bacterial cell. OMPs often have large loops that protrude into the extracellular environment. These loops show promise for biotechnological applications and as therapeutic targets. The loops can be utilized to attach heterologous proteins at the surface of bacteria. Understanding how modifications to these loops affect the stability and folding of outer membrane proteins is essential for their efficient utilization for biotechnological purposes. In this work, the small outer membrane protein OmpX was used a model system to examine the effects of loop insertions on folding and stability. The insertions were varied according to hydrophobicity and size. The effects of the loop inserts were determined by assaying folding into detergent micelles in vitro by SDS-PAGE. The folding capacity of the constructs were also examined in vivo, by isolating the OM of cells expressing the constructs. The results indicate that folding of OMPs is effected by the hydrophobic character of the extracellular loops. Small insertions of five residues were found to improve the folding efficiency of OmpX, while large hydrophilic inserts reduced folding efficiency. 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 OM in vivo, but remained unfolded. The results have important biotechnological implications as they could improve the design and efficiency of recombinant OMPs used for surface display.

• Trunk, Thomas; Leo, Jack Christopher & Linke, Dirk (2020). Characterization and comparative analyses of type Vd-secreted phospholipases expressed in pathogenic bacteria. Universitetet i Oslo. Full text in Research Archive Show summary

  Autotransporters, or type 5 secretion systems (T5SS), are widespread surface proteins of Gram-negative bacteria often associated with virulence. Three novel T5dSSs expressed in the fish pathogen Aeromonas hydrophila, the phytopathogen Ralstonia solanacearum and the human pathogen Burkholderia pseudomallei are presented in this thesis alongside the complementation of the enzymatic characterization of the T5dSS phospholipases from Pseudomonas aeruginosa and Fusobacterium nucleatum, already known to literature. Detailed information on the phospholipases’ relative enzyme activities, substrate specificities as well as the role of quaternary structure for enzyme activity and stability are shown. Furthermore, a low resolution (6 Å) structure of the T5dSS phospholipase from the fish pathogen Aeromonas hydrophila is presented, showing high structural similarity with the T5dSS present in Pseudomonas aeruginosa. Additionally, the results in the thesis show the role of the periplasmic chaperone SurA on integration of the T5dSS of Pseudomonas aeruginosa into the outer membrane as well as the chaperones effect on membrane integrity and outer membrane composition. With an increased accessibility as well as sensitivity to antibiotics resulting from a conditional deletion of SurA, the chaperone might qualify as potential novel antimicrobial target.

• Wrobel, Agnieszka & Linke, Dirk (2019). Structural and functional studies of the Yersinia ruckeri inverse autotransporters. Universitetet i Oslo. Full text in Research Archive

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