TY - JOUR A1 - Piteau, Marianne A1 - Papatheodorou, Panagiotis A1 - Schwan, Carsten A1 - Schlosser, Andreas A1 - Aktories, Klaus A1 - Schmidt, Gudula T1 - Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) JF - PLoS Pathogens N2 - The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions. Author Summary We study a crucial virulence factor produced by pathogenic Escherichia coli strains, the Cytotoxic Necrotizing Factor 1 (CNF1). More than 80% of urinary tract infections (UTIs), which are counted among the most common bacterial infections of humans, are caused by Uropathogenic Escherichia coli (UPEC) strains. We and others elucidated the molecular mechanism of the E. coli toxin CNF1. It constitutively activates Rho GTPases by a direct covalent modification. The toxin enters mammalian cells by receptor-mediated endocytosis. Here, we identified the protein receptor for CNF1 by co-precipitation of cell surface molecules with the tagged toxin and subsequent Maldi-TOF analysis. We identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as receptor for CNF1 and located its interaction site to the C-terminal part of the toxin. We performed direct protein-protein interaction analysis and competition studies. Moreover, cells deficient in Lu/BCAM could not bind labeled CNF1. The identification of a toxin's cellular receptor and receptor binding region is an important task for understanding the pathogenic function of the toxin and, moreover, to make the toxin accessible for its use as a cellbiological and pharmacological tool, for example for the generation of immunotoxins. KW - laminin receptor KW - factor-I KW - toxin KW - RHO KW - cells KW - activation KW - protein KW - domain KW - translocation KW - membrane Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117987 SN - 1553-7374 VL - 10 IS - 1 ER - TY - JOUR A1 - Metje-Sprink, Janina A1 - Groffmann, Johannes A1 - Neumann, Piotr A1 - Barg-Kues, Brigitte A1 - Ficner, Ralf A1 - Kühnel, Karin A1 - Schalk, Amanda M. A1 - Binotti, Beyenech T1 - Crystal structure of the Rab33B/Atg16L1 effector complex JF - Scientific Reports N2 - The Atg12-Atg5/Atg16L1 complex is recruited by WIPI2b to the site of autophagosome formation. Atg16L1 is an effector of the Golgi resident GTPase Rab33B. Here we identified a minimal stable complex of murine Rab33B(30-202) Q92L and Atg16L1(153-210). Atg16L1(153-210) comprises the C-terminal part of the Atg16L1 coiled-coil domain. We have determined the crystal structure of the Rab33B Q92L/Atg16L1(153-210) effector complex at 3.47 angstrom resolution. This structure reveals that two Rab33B molecules bind to the diverging alpha -helices of the dimeric Atg16L1 coiled-coil domain. We mutated Atg16L1 and Rab33B interface residues and found that they disrupt complex formation in pull-down assays and cellular co-localization studies. The Rab33B binding site of Atg16L1 comprises 20 residues and immediately precedes the WIPI2b binding site. Rab33B mutations that abolish Atg16L binding also abrogate Rab33B association with the Golgi stacks. Atg16L1 mutants that are defective in Rab33B binding still co-localize with WIPI2b in vivo. The close proximity of the Rab33B and WIPI2b binding sites might facilitate the recruitment of Rab33B containing vesicles to provide a source of lipids during autophagosome biogenesis. KW - autophagosome formation KW - ATG12-ATG5 conjugate KW - LC3 lipidation KW - binding sites KW - ATG proteins KW - RAB GTPases KW - family KW - membrane KW - recognition KW - proppins Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230396 VL - 10 ER - TY - JOUR A1 - Mansour, Ahmad M. A1 - Arevalo, J. Fernando A1 - Al Kahtani, Eman A1 - Zegarra, Hernando A1 - Abboud, Emad A1 - Anand, Rajiv A1 - Ahmadieh, Hamid A1 - Sisk, Robert A. A1 - Mirza, Salman A1 - Tuncer, Samuray A1 - Navea Tejerina, Amparo A1 - Mataix, Jorge A1 - Ascaso, Francisco J. A1 - Pulido, Jose S. A1 - Guthoff, Rainer A1 - Goebel, Winfried A1 - Roh, Young Jung A1 - Banker, Alay S. A1 - Gentile, Ronald C. A1 - Alonso Martinez, Isabel A1 - Morris, Rodney A1 - Panday, Neeraj A1 - Min, Park Jung A1 - Merce, Emilie A1 - Lai, Timothy Y. Y. A1 - Massoud, Vicky A1 - Ghazi, Nicola G. T1 - Role of Intravitreal Antivascular Endothelial Growth Factor Injections for Choroidal Neovascularization due to Choroidal Osteoma JF - Journal of Ophtamology N2 - We treated 26 eyes of 25 young patients having a mean age of 30 years with intravitreal vascular endothelial growth factor (VEGF) inhibitor for choroidal new vessel (CNV) formation overlying choroidal osteoma over a mean follow-up of 26 months. Mean number of injections was 2.4 at 6 months, 3.2 at 12 months, and 5.5 at 24 months. CNV was subfoveal in 14 eyes, juxtafoveal in 5, extrafoveal in 5, and peripapillary in 2. By paired comparison, mean decrease from baseline was 119.7 microns at 6 months (n = 15; P = 0.001), 105.3 microns at 1 year (n = 10; P = 0.03), and 157.6 microns at 2 years (n = 7; P = 0.08). BCVA improved by 3.3 lines at 6 months after therapy (n = 26; P < 0.001), 2.8 lines (n = 20; P = 0.01) at 1 year, and 3.1 lines (n = 13; P = 0.049) at 2 years. We conclude that intravitreal anti-VEGF injections improve vision in majority of eyes with CNV from choroidal osteoma. KW - membrane KW - photodynamic therapy KW - secondary KW - bevacizumab KW - ranibizumab KW - patient Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117923 IS - 210458 ER - TY - JOUR A1 - Hupp, Sabrina A1 - Förtsch, Christina A1 - Wippel, Carolin A1 - Ma, Jiangtao A1 - Mitchell, Timothy J. A1 - Iliev, Asparouh I. T1 - Direct Transmembrane Interaction between Actin and the Pore-Competent, Cholesterol-Dependent Cytolysin Pneumolysin JF - Journal of Molecular Biology N2 - The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170–190 nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought. KW - pore-forming toxin KW - cholesterol-dependent cytolysin KW - actin KW - membrane KW - pneumolysin Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132297 VL - 425 IS - 3 ER - TY - JOUR A1 - Beiss, Veronique A1 - Spiegel, Holger A1 - Boes, Alexander A1 - Scheuermayer, Matthias A1 - Reimann, Andreas A1 - Schillberg, Stefan A1 - Fischer, Rainer T1 - Plant expression and characterization of the transmission-blocking vaccine candidate PfGAP50 JF - BMC Biotechnology N2 - Background: Despite the limited success after decades of intensive research and development efforts, vaccination still represents the most promising strategy to significantly reduce the disease burden in malaria endemic regions. Besides the ultimate goal of inducing sterile protection in vaccinated individuals, the prevention of transmission by so-called transmission blocking vaccines (TBVs) is being regarded as an important feature of an efficient malaria eradication strategy. Recently, Plasmodium falciparum GAP50 (PfGAP50), a 44.6 kDa transmembrane protein that forms an essential part of the invasion machinery (glideosome) multi-protein complex, has been proposed as novel potential transmission-blocking candidate. Plant-based expression systems combine the advantages of eukaryotic expression with a up-scaling potential and a good product safety profile suitable for vaccine production. In this study we investigated the feasibility to use the transient plant expression to produce PfGAP50 suitable for the induction of parasite specific inhibitory antibodies. Results: We performed the transient expression of recombinant PfGAP50 in Nicotiana benthamiana leaves using endoplasmatic reticulum (ER) and plastid targeting. After IMAC-purification the protein yield and integrity was investigated by SDS-PAGE and Western Blot. Rabbit immune IgG derived by the immunization with the plastidtargeted variant of PfGAP50 was analyzed by immune fluorescence assay (IFA) and zygote inhibition assay (ZIA). PfGAP50 could be produced in both subcellular compartments at different yields IMAC (Immobilized Metal Affinity Chromatography) purification from extract yielded up to 4.1 mu g/g recombinant protein per fresh leaf material for ER-retarded and 16.2 mu g/g recombinant protein per fresh leave material for plasmid targeted PfGAP50, respectively. IgG from rabbit sera generated by immunization with the recombinant protein specifically recognized different parasite stages in immunofluorescence assay. Furthermore up to 55 % inhibition in an in vitro zygote inhibition assay could be achieved using PfGAP50-specific rabbit immune IgG. Conclusions: The results of this study demonstrate that the plant-produced PfGAP50 is functional regarding the presentation of inhibitory epitopes and could be considered as component of a transmission-blocking malaria vaccine formulation. KW - PFS25 KW - plastid targeting KW - plant-made vaccines KW - agroinfiltration KW - gametes KW - sexual stage KW - plasmodium falciparum KW - membrane KW - antibodies KW - immunization KW - RTS,S/AS01 malaria vaccine KW - recombinant proteins KW - cost-effectiveness KW - purification Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137327 VL - 15 IS - 108 ER -