cytochalasin-d and sodium-chlorate

Generally, biomacromolecules, such as DNA, RNA, and proteins, cannot freely permeate into cells from outside the membrane. Protein transduction domains (PTDs) are peptides containing a large number of basic amino acids that can deliver macromolecules into living cells. Arginine-rich intracellular delivery (AID) peptides are more effective than other PTD peptides at carrying large molecules across cellular membranes. In the present study, we demonstrated that AID peptides are able to deliver cargo proteins into living cells in both covalent and noncovalent protein transductions (CNPT) synchronously. Human A549 cells were treated with a fluorescent protein (FP) that was noncovalently premixed with another AID-conjugated FP, which emitted a different color. After the delivery of carrier AID-FP and cargo FP into cells, the emission and merge of fluorescence were observed and recorded with a confocal microscope, while the internalization efficiency was quantitatively analyzed with a flow cytometer. The optimal molecular ratio between carrier AID-FP and cargo FP for CNPT is about 1:1/3. Fluorescence resonance energy transfer (FRET) assay further confirmed AID-conjugates can physically interact with its cargo FPs in CNPT in cells. Potential uptake mechanisms of CNPT may involve a combination of multiple internalization pathways. After delivery, intracellular distributions of AID-conjugates and FPs may possibly colocalize with lysosomes. These results will facilitate the understanding of multiple mechanisms of PTDs, and provide a powerful tool for simultaneously delivering several proteins or compounds in protein internalization.

Topics: Amiloride; Arginine; beta-Cyclodextrins; Cell Line, Tumor; Cell Membrane; Cell Survival; Chlorates; Cytochalasin D; Drug Carriers; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; Humans; Luminescent Proteins; Lysosomes; Mitochondria; Nocodazole; Oligopeptides; Peptides; Pinocytosis; Plasmids; Protein Transport; Recombinant Fusion Proteins; Red Fluorescent Protein; Temperature

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