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Background/Aims: Genetic cell ablation using the human diphtheria toxin receptor (hDTR) is a new strategy used for analysing cellular function. Diphtheria toxin (DT) is a cytotoxic protein that leaves mouse cells relatively unaffected, but upon binding to hDTR it ultimately leads to cell death. We used a podocyte-specific hDTR expressing (Pod-DTR) mouse to assess the anti-permeability and cyto-protective effects of the splice isoform vascular endothelial growth factor (VEGF-A165b). Methods: The Pod-DTR mouse was crossed with a mouse that over-expressed VEGF-A165b specifically in the podocytes (Neph-VEGF-A165b). Wild type (WT), Pod-DTR, Neph-VEGF-A165b and Pod-DTR X Neph-VEGF-A165b mice were treated with several doses of DT (1, 5, 100, and 1,000 ng/g bodyweight). Urine was collected and the glomerular water permeability (LpA/Vi) was measured ex vivo after 14 days. Structural analysis and podocyte marker expression were also assessed. Results: Pod-DTR mice developed an increased glomerular LpA/Vi 14 days after administration of DT (all doses), which was prevented when the mice over-expressed VEGF-A165b. No major structural abnormalities, podocyte ablation or albuminuria was observed in Pod-DTR mice, indicating this to be a mild model of podocyte disease. However, a change in expression and localisation of nephrin within the podocytes was observed, indicating disruption of the slit diaphragm in the Pod-DTR mice. This was prevented in the Pod-DTR X Neph-VEGF-A165b mice. Conclusion: Although only a mild model of podocyte injury, over-expression of the anti-permeability VEGF-A165b isoform in the podocytes of Pod-DTR mice had a protective effect. Therefore, this study further highlights the therapeutic potential of VEGF-A165b in glomerular disease.

Frontiers in immunology 8, 140 (2017). doi:10.3389/fimmu.2017.00140, Published by Frontiers Media, Lausanne

The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin, Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin bat Plot by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.

In this paper it is demonstrated that reconstituted influenza virus envelopes (virosomes) fuse efficiently with membranes of the endosomal cell compartment of cultured cells, after internalization through receptor-mediated endocytosis. As a consequence, molecules, encapsulated in the virosomal interior, are transferred to the cell cytoplasm. This process was monitored on the basis of delivery of subunit A of diphtheria toxin (DTA), initially encapsulated in the virosomal lumen. Virosome-mediated cytoplasmic delivery of DTA resulted in a virtually complete inhibition of cellular protein synthesis. DTA delivery was blocked by factors inhibiting the pH-dependent fusion activity of viral hemagglutinin, i.e., 20 mM NH4Cl, preincubation of the virosomes at low pH, and anti-hemagglutinin antibodies. Quantitation of the extent of virosome-mediated delivery of biologically active DTA demonstrated that a lower bound of approximately 10% of the virosomes entering the cells deposited their aqueous contents into the cytosol. This is in good agreement with the final extent of virosome fusion of 40%, as determined by a fluorescence lipid mixing assay based on the dilution of pyrene-labeled phosphatidylcholine from the virosomal into the endosomal membrane. At an added concentration of 1 mu M virosomal lipid per 50 000 cells, depending on the condition of the experiment, a minimum of 4000 virosomes per cell were found to be internalized. Virosomes could also be induced to fuse with the cell plasma membrane by a transient lowering of the extracellular pH, as detected by appearance of virosome-encapsulated DTA in the cytoplasm. The results demonstrate that virosome-mediated delivery of polypeptide toxin molecules can be used as a sensitive means to study the interactions of enveloped viruses with host cells. In addition, virosomes may be used as versatile vehicles for the delivery of water-soluble molecules to the cytoplasm of cultured cells.

Biological toxicity of E. coli heat-labile enterotoxin and the closely related cholera toxin requires that the assembled toxin be activated by proteolytic cleavage of the A subunit and reduction of a disulfide bond internal to the A subunit. The structural role served by this reduction and cleavage is not known, however. We have crystallographically determined the structure of the E. coli heat-labile enterotoxin AB, hexamer in which the A subunit has been cleaved by trypsin between residues 192 and 195. The toxin is thus partially activated, in that it has been cleaved but the disulfide bond has not been reduced. The structure of the A subunit in the cleaved toxin is substantially the same as that previously observed for the uncleaved AB, structure, suggesting that although such cleavage is required for biological activity of the toxin it does not by itself cause a conformational change.

Background Human central nervous system-stem cells grown as neurospheres (hCNS-SCns) self-renew, are multipotent, and have potential therapeutic applications following trauma to the spinal cord. We have previously shown locomotor recovery in immunodeficient mice that received a moderate contusion spinal cord injury (SCI) and hCNS-SCns transplantation 9 days post-injury (dpi). Engrafted hCNS-SCns exhibited terminal differentiation to myelinating oligodendrocytes and synapse-forming neurons. Further, selective ablation of human cells using Diphtheria toxin (DT) abolished locomotor recovery in this paradigm, suggesting integration of human cells within the mouse host as a possible mechanism for the locomotor improvement. However, the hypothesis that hCNS-SCns could alter the host microenvironment as an additional or alternative mechanism of recovery remained unexplored; we tested that hypothesis in the present study. Methods and Findings Stereological quantification of human cells using a human-specific cytoplasmic marker demonstrated successful cell engraftment, survival, migration and limited proliferation in all hCNS-SCns transplanted animals. DT administration at 16 weeks post-transplant ablated 80.5% of hCNS-SCns. Stereological quantification for lesion volume, tissue sparing, descending serotonergic host fiber sprouting, chondroitin sulfate proteoglycan deposition, glial scarring, and angiogenesis demonstrated no evidence of host modification within the mouse spinal cord as a result of hCNS-SCns transplantation. Biochemical analyses supplemented stereological data supporting the absence of neural stem-cell mediated host repair. However, linear regression analysis of the number of engrafted hCNS-SCns vs. the number of errors on a horizontal ladder beam task revealed a strong correlation between these variables (r = −0.78, p

Examination of the structure of Escherichia coli heat-labile enterotoxin in the AB5 complex at a resolution of 2.3 angstrom reveals that the doughnut-shaped B pentamer binds the enzymatic A subunit using a hairpin of the A2 fragment, through a highly charged central pore. Putative ganglioside G(M1-) binding sites on the B subunits are more than 20 angstrom removed from the membrane-crossing A1 subunit. This ADP-ribosylating (A1) fragment of the toxin has structural homology with the catalytic region of exotoxin A and hence also to diphtheria toxin.

International audience; The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.

International audience; In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M. fortuitum beta-lactamase promoter, pBlaF*. Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol. Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile. Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response. Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin. Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin. Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases.

The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin, Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin bat Plot by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.

The insertion of soluble proteins into membranes has been a topic of considerable interest. We have studied the insertion of bovine alpha-lactalbumin into single-bilayer vesicles prepared from egg phosphatidylcholine (PC). Fluorescence studies indicated rapid and tight binding of apo-alpha-lactalbumin (apo-alpha-LA) to PC vesicles as a function of pH. The binding was maximal at pH values which favor the formation of the molten globule state. As an increase of hydrophobic surface is observed in the molten globule state, this conformational state can provide a molecular basis for insertion of soluble proteins into membranes. The membrane-bound complex formed at low pH (3.0) could be isolated and was found to be stable at neutral pH. The structural characterization of the apo-alpha-LA-PC complex was studied by fluorescence quenching using iodide, acrylamide, and 9,10-dibromostearic acid. The results obtained indicated that some of the tryptophans of apo-alpha-LA were buried in the membrane interior and some were exposed on the outer side. Fluorescence quenching and CD studies indicated the membrane-bound conformation of apo-alpha-LA was some conformational state that is between the soluble, fully folded conformation and the molten globule state.

In this paper it is demonstrated that reconstituted influenza virus envelopes (virosomes) fuse efficiently with membranes of the endosomal cell compartment of cultured cells, after internalization through receptor-mediated endocytosis. As a consequence, molecules, encapsulated in the virosomal interior, are transferred to the cell cytoplasm. This process was monitored on the basis of delivery of subunit A of diphtheria toxin (DTA), initially encapsulated in the virosomal lumen. Virosome-mediated cytoplasmic delivery of DTA resulted in a virtually complete inhibition of cellular protein synthesis. DTA delivery was blocked by factors inhibiting the pH-dependent fusion activity of viral hemagglutinin, i.e., 20 mM NH4Cl, preincubation of the virosomes at low pH, and anti-hemagglutinin antibodies. Quantitation of the extent of virosome-mediated delivery of biologically active DTA demonstrated that a lower bound of approximately 10% of the virosomes entering the cells deposited their aqueous contents into the cytosol. This is in good agreement with the final extent of virosome fusion of 40%, as determined by a fluorescence lipid mixing assay based on the dilution of pyrene-labeled phosphatidylcholine from the virosomal into the endosomal membrane. At an added concentration of 1 mu M virosomal lipid per 50 000 cells, depending on the condition of the experiment, a minimum of 4000 virosomes per cell were found to be internalized. Virosomes could also be induced to fuse with the cell plasma membrane by a transient lowering of the extracellular pH, as detected by appearance of virosome-encapsulated DTA in the cytoplasm. The results demonstrate that virosome-mediated delivery of polypeptide toxin molecules can be used as a sensitive means to study the interactions of enveloped viruses with host cells. In addition, virosomes may be used as versatile vehicles for the delivery of water-soluble molecules to the cytoplasm of cultured cells.

Biological toxicity of E. coli heat-labile enterotoxin and the closely related cholera toxin requires that the assembled toxin be activated by proteolytic cleavage of the A subunit and reduction of a disulfide bond internal to the A subunit. The structural role served by this reduction and cleavage is not known, however. We have crystallographically determined the structure of the E. coli heat-labile enterotoxin AB, hexamer in which the A subunit has been cleaved by trypsin between residues 192 and 195. The toxin is thus partially activated, in that it has been cleaved but the disulfide bond has not been reduced. The structure of the A subunit in the cleaved toxin is substantially the same as that previously observed for the uncleaved AB, structure, suggesting that although such cleavage is required for biological activity of the toxin it does not by itself cause a conformational change.

The anthrax toxin is a tripartite toxin, where the two enzymatic subunits require the third subunit, the protective antigen (PA), to interact with cells and be escorted to their cytoplasmic targets. PA binds to cells via one of two receptors, TEM8 and CMG2. Interestingly, the toxin times and triggers its own endocytosis, in particular through the heptamerization of PA. Here we show that PA triggers the ubiquitination of its receptors in a β-arrestin-dependent manner and that this step is required for clathrin-mediated endocytosis. In addition, we find that endocytosis is dependent on the heterotetrameric adaptor AP-1 but not the more conventional AP-2. Finally, we show that endocytosis of PA is strongly dependent on actin. Unexpectedly, actin was also found to be essential for efficient heptamerization of PA, but only when bound to one of its 2 receptors, TEM8, due to the active organization of TEM8 into actin-dependent domains. Endocytic pathways are highly modular systems. Here we identify some of the key players that allow efficient heptamerization of PA and subsequent ubiquitin-dependent, clathrin-mediated endocytosis of the anthrax toxin., Author Summary Bacillus anthracis is the bacterium responsible for the anthrax disease. Its virulence is mainly due to 2 factors, the anthrax toxin and the anti-phagocytic capsule. This toxin is composed of three independent polypeptide chains. Two of these have enzymatic activity and are responsible for the effects of the toxin. The third has no activity but is absolutely required to bring the 2 enzymatic subunits into the cell where they act. If one blocks entry into the cells, one blocks the effects of these toxins, which is why it is important to understand how the toxin enters into the cell at the molecular level. Here we identified various molecules that are involved in efficiently bringing the toxin into the cell. First, we found that the actin cytoskeleton plays an important role in organizing one of the two anthrax toxin receptors at the cell surface. Second, we found a cytosolic protein, β-arrestin, that is required to modify the intracellular part of the toxin receptor, to allow uptake. Finally, we directly show, for the first time, that anthrax toxin uptake is mediated by the so-called clathrin-dependent pathway, a very modular entry pathway, but that the toxin utilizes this pathway in an unconventional way.

Examination of the structure of Escherichia coli heat-labile enterotoxin in the AB5 complex at a resolution of 2.3 angstrom reveals that the doughnut-shaped B pentamer binds the enzymatic A subunit using a hairpin of the A2 fragment, through a highly charged central pore. Putative ganglioside G(M1-) binding sites on the B subunits are more than 20 angstrom removed from the membrane-crossing A1 subunit. This ADP-ribosylating (A1) fragment of the toxin has structural homology with the catalytic region of exotoxin A and hence also to diphtheria toxin.