The aim of this work was isolation and purification of the major immunodominant protein, Outer surface protein C (OspC) of three members of the species group Borrelia burgdorferi, the causative agent of Lyme disease. Our aim was to obtain this protein in a quantity and purity sufficient for immunization of experimental animals. For optimalization of protein purification's yield we used immobilized metal ion affinity chromatography (IMAC) under different conditions. The greatest efficiency was achieved by using of HiTrap Chelating Column under native conditions.

• MeSH
• antigeny bakteriální biosyntéza   izolace a purifikace   MeSH
• DNA vakcíny biosyntéza   izolace a purifikace   MeSH
• Escherichia coli MeSH
• genetické vektory MeSH
• proteiny vnější bakteriální membrány biosyntéza   izolace a purifikace   MeSH
• vakcína proti lymeské nemoci biosyntéza   izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• DNA vakcíny MeSH
• OspC protein MeSH Prohlížeč
• proteiny vnější bakteriální membrány MeSH
• vakcína proti lymeské nemoci MeSH

We investigated the presence of glycoproteins in Borrelia burgdorferi. We did not find any evidence for glycosylation of the major outer membrane proteins OspA and OspB or the structural flagellar proteins FlaB and FlaA. We suggest that glycoproteins present on the surface of B. burgdorferi may be tightly bound culture medium glycoproteins.

• MeSH
• antigeny bakteriální genetika   metabolismus   MeSH
• antigeny povrchové genetika   metabolismus   MeSH
• bakteriální vakcíny genetika   metabolismus   MeSH
• Borrelia burgdorferi genetika   metabolismus   ultrastruktura   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• elektronová kryomikroskopie MeSH
• flagelin genetika   metabolismus   MeSH
• glykosylace MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• lipoproteiny genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• proteiny vnější bakteriální membrány genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• antigeny povrchové MeSH
• bakteriální vakcíny MeSH
• flaA protein, bacteria MeSH Prohlížeč
• flaB flagellin MeSH Prohlížeč
• flagelin MeSH
• lipoproteiny MeSH
• OspA protein MeSH Prohlížeč
• OspB protein, Borrelia burgdorferi MeSH Prohlížeč
• proteiny vnější bakteriální membrány MeSH

In current work, we used recombinant OspC protein derived from B. afzelii strain BRZ31 in the native homodimeric fold for mice immunization and following selection process to produce three mouse monoclonal antibodies able to bind to variable parts of up to five different OspC proteins. Applying the combination of mass spectrometry assisted epitope mapping and affinity based theoretical prediction we have localized regions responsible for antigen-antibody interactions and approximate epitopes' amino acid composition. Two mAbs (3F4 and 2A9) binds to linear epitopes located in previously described immunogenic regions in the exposed part of OspC protein. The third mAb (2D1) recognises highly conserved discontinuous epitope close to the ligand binding domain 1.

• Klíčová slova
• Borrelia burgdorferi, OspC, epitope mapping, immunoprecipitation, monoclonal antibody, outer surface protein C, protein alignment,
• MeSH
• antigeny bakteriální chemie   genetika   imunologie   MeSH
• Borrelia burgdorferi chemie   genetika   imunologie   MeSH
• mapování epitopu * MeSH
• multimerizace proteinu * MeSH
• myši inbrední BALB C MeSH
• myší monoklonální protilátky chemie   imunologie   MeSH
• myši MeSH
• proteiny vnější bakteriální membrány chemie   genetika   imunologie   MeSH
• sbalování proteinů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• myší monoklonální protilátky MeSH
• OspC protein MeSH Prohlížeč
• proteiny vnější bakteriální membrány MeSH

The formation of biomolecular coronas around nanoparticles as soon as they come in contact with biological media is nowadays well accepted. The self-developed biological outer surfaces can affect the targeting capability of the colloidal carriers as well as their cytotoxicity and cellular uptake behavior. In this framework, we explored the structural features and biological consequences of protein coronas around block copolymer assemblies consisting of a common pH-responsive core made by poly[2-(diisopropylamino) ethyl methacrylate] (PDPA) and hydrophilic shells of different chemical natures: zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) or highly hydrophilic poly(ethylene oxide) (PEO) and poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA). We demonstrated the presence of ∼50 nm protein coronas around the nanoparticles regardless of the chemical nature of the polymeric shells. The thickness is understood as the sum of the soft and hard layers and it is the actual interface seen by the cells. Although the soft corona composition is difficult to determine because the proteins are loosely bound to the outer surface of the assemblies, the tightly bound proteins (hard corona) could be identified and quantified. The compositional analysis of the hard corona demonstrated that human serum albumin (HSA), immunoglobulin G (IgG) and fibrinogen are the main components of the protein coronas, and serotransferrin is present particularly in the protein corona of the zwitterionic-stabilized assemblies. The protein coronas substantially reduce the cellular uptake of the colloidal particles due to their increased size and the presence of HSA which is known to reduce nanoparticle-cell adhesion. On the other hand, their existence also reduces the levels of cytotoxicity of the polymeric assemblies, highlighting that protein coronas should not be always understood as artifacts that need to be eliminated due to their positive outputs.

• MeSH
• buněčná adheze MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• mechanické jevy * MeSH
• nanočástice chemie   MeSH
• polymery chemie   MeSH
• povrchové vlastnosti MeSH
• proteinová korona chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• polymery MeSH
• proteinová korona MeSH

The influences of the hydrophilic chain length, morphology and chemical nature have been probed with regard to the adsorption of model proteins onto the surface of soft nanoparticles (crew-cut micelles and polymersomes). The investigations were based on assemblies manufactured from PEOm-b-PLAn (poly(ethylene oxide)-b-poly(lactic acid)), which is a well-established block copolymer platform towards the manufacturing of drug delivery vehicles, and PHPMAm-b-PDPAn (poly([N-(2-hydroxypropyl)]methacrylamide)-b-poly[2-(diisopropylamino)ethyl methacrylate]), which is pH-responsive and therefore potentially able to target damaged cells in slightly acid microenvironments. Besides, protein adsorption onto PHPMA-stabilized nanoparticles has been seldom explored up-to-date. The morphologies were produced using two different approaches (nanoprecipitation and thin-film hydration) and afterwards, the protein-repelling property of the assemblies in model protein environments (BSA - bovine serum albumin, lysozyme and IgG - immunoglobulin G) was evaluated. We report that, regardless the morphology, PHPMA35-b-PDPA42 block copolymer assemblies are highly stable with negligible protein binding. On the other hand, PEOm-b-PLAn nanostructures are susceptible to protein adsorption and the phenomenon is protein-dependent. The nanoparticles are more susceptible to adsorption of the model positively charged biomacromolecule (lysozyme). The adsorption phenomenon is thermodynamically complex with simultaneous endothermic and exothermic processes involved. Although the experimental data highlight that qualitatively the morphology plays negligible effects on the event, fluorescence spectroscopy measurements evidenced that the binding is stronger onto the surface of nanoparticles stabilized by shorter hydrophilic shells. Nevertheless, the adsorption does not affect the secondary structure of the model proteins as confirmed by circular dichroism spectroscopy. Overall, by comparing soft nanoparticles stabilized by PEO and PHPMA, the latter is herein proved to be a better choice towards the manufacturing of non-fouling structures (either core-shell or hollow spheres) where even a reasonably short hydrophilic chain confers outstanding protein-repelling feature.

• MeSH
• adsorpce MeSH
• akrylamidy chemie   MeSH
• nanočástice chemie   MeSH
• polymery chemie   MeSH
• povrchové vlastnosti MeSH
• proteiny chemie   MeSH
• termodynamika MeSH
• velikost částic MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• akrylamidy MeSH
• N-(2-hydroxypropyl)methacrylamide MeSH Prohlížeč
• polymery MeSH
• proteiny MeSH

In recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) of Treponema pallidum subspecies pallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions of tprC, tprD, and bamA in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we used T. pallidum 0548 (tp0548) genotyping and tp0558 sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants in T. pallidum subsp. pallidum reference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands of T. pallidum repeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures drive T. pallidum subsp. pallidum OMP diversity and that genetic exchange contributes to the evolutionary biology of T. pallidum subsp. pallidum They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development.IMPORTANCE Despite recent progress characterizing outer membrane proteins (OMPs) of Treponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci, tprC, tprD, and bamA, in T. pallidum subsp. pallidum isolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations drive T. pallidum subsp. pallidum OMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance of T. pallidum subsp. pallidum and frame strategies for vaccine development based upon conserved OMP extracellular loops.

• Klíčová slova
• Treponema pallidum, molecular subtyping, outer membrane proteins, spirochetes, syphilis,
• MeSH
• fylogeneze MeSH
• genetická variace MeSH
• lidé MeSH
• molekulární evoluce * MeSH
• molekulární sekvence - údaje MeSH
• proteinové domény MeSH
• proteiny vnější bakteriální membrány chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• Spirochaetales klasifikace   genetika   růst a vývoj   izolace a purifikace   MeSH
• syfilis mikrobiologie   MeSH
• Treponema pallidum klasifikace   genetika   růst a vývoj   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• proteiny vnější bakteriální membrány MeSH

The iron-regulated FrpD protein is a unique lipoprotein embedded into the outer membrane of the Gram-negative bacterium Neisseria meningitidis. The biological function of FrpD remains unknown but might consist in anchoring to the bacterial cell surface the Type I-secreted FrpC protein, which belongs to a Repeat in ToXins (RTX) protein family and binds FrpD with very high affinity (K(d) = 0.2 nM). Here, we report the backbone (1)H, (13)C, and (15)N chemical shift assignments for the FrpD(43-271) protein that allow us to characterize the intimate interaction between FrpD and the N-terminal domain of FrpC.

• MeSH
• lipoproteiny chemie   MeSH
• Neisseria meningitidis metabolismus   MeSH
• nukleární magnetická rezonance biomolekulární * MeSH
• proteiny vnější bakteriální membrány chemie   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipoproteiny MeSH
• proteiny vnější bakteriální membrány MeSH

Leptospirosis is an underestimated tropical disease caused by the pathogenic Leptospira species and responsible for several serious health problems. Here, we aimed to develop an ultrasensitive DNA biosensor for the rapid and on-site detection of the Loa22 gene of Leptospira interrogans using a gold nanoparticle-carbon nanofiber composite (AuN/CNF)-based screen-printed electrode. Cyclic voltammetry and electrochemical impedance were performed for electrochemical analysis. The sensitivity of the sensor was 5431.74 μA/cm2/ng with a LOD (detection limit) of 0.0077 ng/μL using cyclic voltammetry. The developed DNA biosensor was found highly specific to the Loa22 gene of L. interrogans, with a storage stability at 4 °C for 180 days and a 6% loss of the initial response. This DNA-based sensor only takes 30 min for rapid detection of the pathogen, with a higher specificity and sensitivity. The promising results obtained suggest the application of the developed sensor as a point of care device for the diagnosis of leptospirosis.

• Klíčová slova
• AuN/CNF, DNA sensor, Leptospira interrogans, Loa22 gene,
• MeSH
• kovové nanočástice * MeSH
• Leptospira interrogans * genetika   MeSH
• leptospiróza * diagnóza   MeSH
• lidé MeSH
• membránové proteiny MeSH
• zlato MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• membránové proteiny MeSH
• zlato MeSH

Extensive exploitation of titanium dioxide nanoparticles (TiO2NPs) augments rapid release into the marine environment. When in contact with the body fluids of marine invertebrates, TiO2NPs undergo a transformation and adhere various organic molecules that shape a complex protein corona prior to contacting cells and tissues. To elucidate the potential extracellular signals that may be involved in the particle recognition by immune cells of the sea urchin Paracentrotus lividus, we investigated the behavior of TiO2NPs in contact with extracellular proteins in vitro. Our findings indicate that TiO2NPs are able to interact with sea urchin proteins in both cell-free and cell-conditioned media. The two-dimensional proteome analysis of the protein corona bound to TiO2NP revealed that negatively charged proteins bound preferentially to the particles. The main constituents shaping the sea urchin cell-conditioned TiO2NP protein corona were proteins involved in cellular adhesion (Pl-toposome, Pl-galectin-8, Pl-nectin) and cytoskeletal organization (actin and tubulin). Immune cells (phagocytes) aggregated TiO2NPs on the outer cell surface and within well-organized vesicles without eliciting harmful effects on the biological activities of the cells. Cells showed an active metabolism, no oxidative stress or caspase activation. These results provide a new level of understanding of the extracellular proteins involved in the immune-TiO2NP recognition and interaction in vitro, confirming that primary immune cell cultures from P. lividus can be an optional model for swift and efficient immune-toxicological investigations.

• Klíčová slova
• biocorona, echinoderm, extracellular signaling, immune-adhesome, in vitro-ex vivo model, proxy to human,
• MeSH
• buněčná adheze imunologie   MeSH
• fagocyty imunologie   MeSH
• galektiny imunologie   MeSH
• glykoproteiny imunologie   MeSH
• ježovky imunologie   MeSH
• nanočástice aplikace a dávkování   MeSH
• nektiny imunologie   MeSH
• Paracentrotus imunologie   MeSH
• proteinová korona imunologie   MeSH
• proteom imunologie   MeSH
• titan imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• galektiny MeSH
• glykoproteiny MeSH
• nektiny MeSH
• proteinová korona MeSH
• proteom MeSH
• titan MeSH
• titanium dioxide MeSH Prohlížeč
• toposome glycoprotein complex MeSH Prohlížeč

Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc2Man5 glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

• Klíčová slova
• C-type lectin-like ligand, LLT1,
• MeSH
• glykosylace MeSH
• kvarterní struktura proteinů MeSH
• lektinové receptory NK-buněk - podrodina B chemie   genetika   metabolismus   MeSH
• lektiny typu C chemie   genetika   metabolismus   MeSH
• lidé MeSH
• multimerizace proteinu * MeSH
• receptory buněčného povrchu chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CLEC2D protein, human MeSH Prohlížeč
• KLRB1 protein, human MeSH Prohlížeč
• lektinové receptory NK-buněk - podrodina B MeSH
• lektiny typu C MeSH
• receptory buněčného povrchu MeSH