Structural insights into Clostridium perfringens delta toxin pore formation

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins

Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation

Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B

B cells and monocytes from patients with active multiple sclerosis exhibit increased surface expression of both HERV-H Env and HERV-W Env, accompanied by increased seroreactivity

<p>Abstract</p> <p>Background</p> <p>The etiology of the neurogenerative disease multiple sclerosis (MS) is unknown. The leading hypotheses suggest that MS is the result of exposure of genetically susceptible individuals to certain environmental factor(s). Herpesviruses and human endogenous retroviruses (HERVs) represent potentially important factors in MS development. Herpesviruses can activate HERVs, and HERVs are activated in MS patients.</p> <p>Results</p> <p>Using flow cytometry, we have analyzed HERV-H Env and HERV-W Env epitope expression on the surface of PBMCs from MS patients with active and stable disease, and from control individuals. We have also analyzed serum antibody levels to the expressed HERV-H and HERV-W Env epitopes. We found a significantly higher expression of HERV-H and HERV-W Env epitopes on B cells and monocytes from patients with active MS compared with patients with stable MS or control individuals. Furthermore, patients with active disease had relatively higher numbers of B cells in the PBMC population, and higher antibody reactivities towards HERV-H Env and HERV-W Env epitopes. The higher antibody reactivities in sera from patients with active MS correlate with the higher levels of HERV-H Env and HERV-W Env expression on B cells and monocytes. We did not find such correlations for stable MS patients or for controls.</p> <p>Conclusion</p> <p>These findings indicate that both HERV-H Env and HERV-W Env are expressed in higher quantities on the surface of B cells and monocytes in patients with active MS, and that the expression of these proteins may be associated with exacerbation of the disease.</p

Comparison of the crystallization and crystal packing of two Fab single-site mutant protein L complexes

Protein L from Peptostreptococcus magnus (PpL) is a multidomain protein composed of four or five immunoglobulin-binding domains that target the light chain of a large repertoire of human and murine antibodies. Thus, a single domain of this protein can be used to aid the crystallization of Fab, free or complexed to their antigen when it is not possible to obtain crystals without it. Each wild-type PpL domain has two light-chain binding sites that target the same region of the light chain and can thus bring together two Fab-antigen complexes within the crystal lattice. In this context the small PpL domain is sandwiched between two Fab and cannot participate in crystal contacts, thus mutants are unlikely to increase the chances of crystallizing a particular complex. However, it is possible to design mutants that can bind at only one site by making use of the crystal structures obtained so far. Such mutants will have a free surface that can participate in crystal contacts and that can be modified to improve its crystal contact-forming properties. Here, a comparison of two single-site mutants that differ at three different positions is reported. In both mutants two different tryptophan residues participate in crystal-packing interactions, suggesting that this residue may be particularly interesting for enhancing crystal-contact formation

Different crystal packing in Fab-protein L semi-disordered peptide complex

Proteins and peptides with variable degrees of disorder are a challenge for protein crystallization. These may be completely disordered or just contain regions with a high degree of mobility that may be represented by a multitude of discretely defined conformations. These difficulties are not insurmountable, but it may be unreasonable to expect a clean result from a structural point of view. The complex between a murine monoclonal antibody (19D9D6) and a synthetic peptide that encompasses the first 45 residues of the core protein of Hepatitis C virus that is poorly structured in solution has been crystallized. In order to make the crystallization possible, use was made of a single immunoglobulin-binding domain of protein L from Peptostreptococcus magnus (PpL), a bacterial protein that can bind the variable region (Fv) of a large population of antibodies through its light chain with no interference with antibody-antigen recognition. Crystals were obtained in different space groups where the size of the cavity that accommodates the peptide is different, although many of the crystal contacts and the overall lattice are preserved. The peptide can be considered to be semi-disordered and the larger cavity accommodates a better ordered peptide than the smaller one. The lattice is of interest for the design of a scaffold system for the crystallization of peptide-tagged proteins since a cavity that accommodates a disordered entity might be able to host ordered proteins of the same size and shape as the cavity. Here, the differences between the lattices formed by this trimolecular complex are described and it is discussed how such a system may be adapted to the crystallization of peptide-tagged proteins

Involvement of the C-terminal end of the prostrate-specific antigen in a conformational epitope: characterization by proteolytic degradation of monoclonal antibody-bound antigen and mass spectrometry.

International audienceProstate-specific antigen (PSA), a 237-amino acid glycoprotein, encoded by the hKLK3 gene, is widely used as a serum marker for the diagnosis and management of prostate cancer. We report here the localization of a conformational epitope recognized by the anti-total PSA monoclonal antibody (mAb) 11E5C6, by proteolytic degradation of mAb-bound antigen followed by mass spectrometric analyses of the peptides generated. These two technologies, combined with molecular display, allowed the identification of amino acid residues contained within three different peptides distant on the PSA sequence, but close in the PSA three-dimensional structure, that may be part of the mAb 11E5C6 epitope. The last four C-terminal amino acid residues are included in this epitope, as well as certain other C-terminal residues between Y225 and T232. The involvement of the PSA C-terminal end in the mAb 11E5C6 epitope was confirmed by western blotting experiments with the recombinant protein proPSA-RP1, resulting from the cloning of an alternative transcript of the hKLK3 gene, in which the PSA C-terminal end was deleted and replaced by another sequence. Although the anti-total PSA mAb 5D5A5 used as a control bound proPSA-RP1, mAb 11E5C6 did not. The requirement of the C-terminal end for the recognition by mAb 11E5C6 may be useful for the discrimination of PSA-related forms

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development

Clostridium perfringens delta toxin Is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences

Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside GM2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da) is a basic protein (pI 9.1) which shows a significant homology with C. perfringens Beta toxin (43% identity), with C. perfringens NetB (40% identity) and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to GM2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader