digitonin and sodium-chlorate

Group B Streptococcus (GBS) colonizes mucosal surfaces of the human gastrointestinal and gynecological tracts and causes disease in a wide range of patients. Invasive illness occurs after organisms traverse an epithelial boundary and enter deeper tissues. Previously we have reported that the alpha C protein (ACP) on the surface of GBS mediates GBS entry into ME180 cervical epithelial cells and GBS translocation across layers of these cells. We now demonstrate that ACP interacts with host cell glycosaminoglycan (GAG); the interaction of ACP with ME180 cells is inhibited if cells are pretreated with sodium chlorate, an inhibitor of sulfate incorporation, or with heparitinases. The interaction is also inhibited in the presence of soluble heparin or heparan sulfate or host cell-derived GAG. In addition, ACP binds soluble heparin specifically in inhibition and dot blot assays. After interaction with host GAG, soluble ACP enters ME180 cells and fractionates to the eukaryotic cell cytosol. These events are inhibited in cells pretreated with cytochalasin D or with Clostridium difficile toxin B. These data indicate that full-length ACP interacts with ME180 cell GAG and enters the eukaryotic cell cytosol by a mechanism that involves Rho GTPase-dependent actin rearrangements. We suggest that these molecular interactions drive ACP-mediated translocation of GBS across epithelial barriers, thereby facilitating invasive GBS infection.

Topics: Actins; Antigens, Surface; Bacterial Proteins; Bacterial Toxins; Cell Line, Tumor; Cell Membrane; Cervix Uteri; Chlorates; Cytochalasin D; Cytoplasm; Cytosol; Cytotoxins; Digitonin; Dose-Response Relationship, Drug; Epithelial Cells; Female; Flow Cytometry; Glycosaminoglycans; Heparin; Humans; Immunoassay; Kinetics; Microscopy, Confocal; Polysaccharide-Lyases; Protein Binding; Protein Structure, Tertiary; Protein Transport; Streptococcus agalactiae; Subcellular Fractions; Time Factors

The intact Photosystem I core protein, containing the psaA and psaB polypeptides, and electron transfer components P-700 through FX, was isolated from cyanobacterial and higher plant Photosystem I complexes with chaotropic agents followed by sucrose density ultracentrifugation. The concentrations of NaClO4, NaSCN, NaI, NaBr or urea required for the functional removal of the 8.9 kDa, FA/FB polypeptide was shown to be inversely related to the strength of the chaotrope. The Photosystem I core protein, which was purified to homogeniety, contains 4 mol of acid-labile sulfide and has the following properties: (i) the FX-containing core consists of the 82 and 83 kDa reaction center polypeptides but is totally devoid of the low-molecular-mass polypeptides; (ii) methyl viologen and other bipyridilium dyes have the ability to accept electrons directly from FX; (iii) the difference spectrum of FX from 400 to 900 nm is characteristic of an iron-sulfur cluster; (iv) the midpoint potential of FX, determined optically at room temperature, is 60 mV more positive than in the control; (v) there is indication by ESR spectroscopy of low-temperature heterogeneity within FX; and (vi) the heterogeneity is seen by optical spectroscopy as inefficiency in low-temperature electron flow to FX. The constraints imposed by the amount of non-heme iron and labile sulfide in the Photosystem I core protein, the cysteine content of the psaA and psaB polypeptides, and the stoichiometry of high-molecular-mass polypeptides, cause us to re-examine the possibility that FX is a [4Fe-4S] rather than a [2Fe-2S] cluster ligated by homologous cysteine residues on the psaA and psaB heterodimer.

Topics: Bromides; Centrifugation, Density Gradient; Chlorates; Chlorophyll; Cold Temperature; Cross-Linking Reagents; Cyanobacteria; Digitonin; Electron Spin Resonance Spectroscopy; Electron Transport; Glutaral; Kinetics; Light-Harvesting Protein Complexes; Molecular Weight; Octoxynol; Oxidation-Reduction; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Photosystem I Protein Complex; Plant Proteins; Polyethylene Glycols; Sodium; Sodium Compounds; Sodium Iodide; Spectrophotometry; Thiocyanates; Urea