The recent emergence of a highly related virulent Clostridium difficile clade with unique characteristics.

OBJECTIVES: Clostridium difficile is a major global human pathogen divided into five clades, of which clade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this clade. METHODS: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized. RESULTS: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among clade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype. CONCLUSIONS: Given their recent emergence, virulence and genomic characteristics, the surveillance of clade 3 strains should be more highly prioritized.

The crystal structure of tetanus toxin Hc fragment complexed with a synthetic GT1b analogue suggests cross-linking between ganglioside receptors and the toxin.

Tetanus toxin, a member of the family of Clostridial neurotoxins, is one of the most potent toxins known. The crystal structure of the complex of the COOH-terminal fragment of the heavy chain with an analogue of its ganglioside receptor, GT1b, provides the first direct identification and characterization of the ganglioside-binding sites. The ganglioside induces cross-linking by binding to two distinct sites on the Hc molecule. The structure sheds new light on the binding of Clostridial neurotoxins to receptors on neuronal cells and provides important information relevant to the design of anti-tetanus and anti-botulism therapeutic agents.

Interaction of tetanus toxin derived hybrid proteins with neuronal cells.

The non-toxic ganglioside binding domain of tetanus toxin (Hc fragment C or TTC) has been studied as a vector for delivering therapeutic proteins to neurons. There is little information on the cellular processing of proteins delivered by linkage to TTC. We have evaluated the cellular handling of a multi-domain hybrid protein containing TTC and both the human enzyme superoxide dismutase and the maltose binding protein from E. coli. Binding, internalization, and cleavage of this protein during prolonged incubation with fetal cortical neurons or cells of the N18-RE-105 line was evaluated by immunoblot analysis, ELISA, and immunocytochemistry. Hybrid proteins were bound and internalized in a manner very similar to TTC. Internalized proteins showed long-term stability within cells, and were degraded into predictable large protein fragments in both cell types. Fragments that were cleaved away from the TTC domain were released into extracellular fluid after internalization. Proteins coupled to TTC share its long-term stability after cellular internalization. After internalization, dissociation of proteins linked to TTC facilitates their release from the cell, but not into other cellular compartments such as the cytosol. TTC linked proteins are probably enclosed within a stable endosomal compartment throughout their cellular lifetime.

Retargeting of adenoviral vectors to neurons using the Hc fragment of tetanus toxin.

The Hc fragment of tetanus toxin (Hc) retains the specific nerve cell binding and transport properties of the holotoxin, but lacks any toxicity. We are investigating the potential for utilising its neurotropism for targeted gene delivery to the central nervous system. Previously we reported the use of Hc-polylysine conjugates for selective gene transfer into neuronal cells in vitro. However, as attempts to apply these constructs in vivo were not successful, we have extended these studies to modification of the tropism of adenoviral vectors. Either Hc-polylysine conjugates or the Fab fragment of a neutralising anti-knob antibody covalently bound to Hc were attached to the virus. Infection of neuronal and non-neuronal cell lines with retargeted virus showed highly increased neuronal cell selectivity, but no significant enhancement of gene delivery into these cells. High concentrations of free Hc blocked the infectivity of the retargeted vector efficiently. Intramuscular injection of retargeted virus into mouse tongues resulted in selective gene transfer to the neurons of the hypoglossal nucleus, where no pathological changes were observed. As differentiated neurons do not undergo cell division, appropriate vectors carrying a thymidine kinase gene, which allows selective elimination of dividing cells, may be exploitable for the treatment of tumours of the central nervous system. The demonstrated suitability of the Hc fragment of tetanus toxin as targeting moiety for viral vectors also indicates a potential for gene therapy of inherited neurodegenerative diseases such as spinal muscular atrophy.

Protective effect of supplemental superoxide dismutase on survival of neuronal cells during starvation. Requirement for cytosolic distribution.

There is evidence that raising cellular levels of Cu2+/Zn2+ superoxide dismutase (SOD1) can protect neurons from oxidative injury. We compared a novel method of elevating neuronal SOD activity using a recombinant hybrid protein composed of the atoxic neuronal binding domain of tetanus toxin (C fragment or TTC) and human SOD1 (hSOD1) with increasing cellular SOD levels through overexpression. Fetal murine cortical neurons or N18-RE-105 cells were incubated with the TTC-hSOD1 hybrid protein and compared to cells constitutively expressing hSOD1 for level of SOD activity, cellular localization of hSOD1, and capacity to survive glucose and pyruvate starvation. Cells incubated with TTC-hSOD1 showed a threefold increase in cellular SOD activity over control cells. This level of increase was comparable to fetal cortical neurons from transgenic mice constitutively expressing hSOD1 and transfected N18-RE-105 cells expressing a green fluorescent protein-hSOD1 fusion protein (GFP-hSOD1). Human SOD1 was distributed diffusely throughout the cytoplasm of the transgenic murine neurons and transfected N18-RE-105 cells. In contrast, cells incubated with TTC-hSOD1 showed hSOD1 localized to the cell surface and intra-cytoplasmic vesicles. The cells expressing hSOD1 showed enhanced survival in glucose- and pyruvate-free medium. Neither cortical neurons nor N18-RE-105 cells incubated in TTC-hSOD1 showed increased survival during starvation. Access to the site where toxic superoxides are generated or their targets may be necessary for the protective function of SOD1.

The structures of the H(C) fragment of tetanus toxin with carbohydrate subunit complexes provide insight into ganglioside binding.

The entry of tetanus neurotoxin into neuronal cells proceeds through the initial binding of the toxin to gangliosides on the cell surface. The carboxyl-terminal fragment of the heavy chain of tetanus neurotoxin contains the ganglioside-binding site, which has not yet been fully characterized. The crystal structures of native H(C) and of H(C) soaked with carbohydrates reveal a number of binding sites and provide insight into the possible mode of ganglioside binding.

Modulation by epitope-specific antibodies of class II MHC-restricted presentation of the tetanus toxin antigen.

Above a certain affinity the dissociation rate of monovalent antigen from antibody becomes slower than the time taken for antigen capture, endocytosis and processing by professional antigen presenting cells. Thus, when high affinity antibodies drive antigen uptake, either directly via B-cell membrane immunoglobulin or indirectly via Fc receptors, the substrate for processing may frequently be an antigen/antibody complex. Here we review studies using the tetanus toxin antigen which show that bound antibodies can dramatically affect proteolytic processing, dependent on the epitope specificity and multiplicity of antibodies bound. Certain antibodies protect or 'footprint' specific domains of the antigen during processing in B-cell clones resulting in modulation of loading of class II MHC-restricted T-cell epitopes. Processing and class II MHC loading of some T-cell epitopes within the footprinted region was hindered, as might be expected, but, surprisingly, presentation of other T-cell epitopes was boosted considerably. These studies show that protein/protein complexes can be processed in an unpredictable fashion by antigen presenting cells and indicate a possible mechanism whereby cryptic T-cell epitopes might be revealed in autoimmune disease.

Delivery of recombinant tetanus-superoxide dismutase proteins to central nervous system neurons by retrograde axonal transport.

The nontoxic C fragment of tetanus toxin (TC) can transport other proteins from the circulation to central nervous system (CNS) motor neurons. Increased levels of CuZn superoxide dismutase (SOD) are protective in experimental models of stroke and Parkinson's disease, whereas mutations in SOD can cause motor neuron disease. We have linked TC to SOD and purified the active recombinant proteins in both the TC-SOD and SOD-TC orientations. Light microscopic immunohistochemistry and quantitative enzyme-linked immunosorbant assays (ELISA) of mouse brainstem, after intramuscular injection, demonstrate that the fusion proteins undergo retrograde axonal transport and transsynaptic transfer as efficiently as TC alone.

Structure of Bordetella pertussis virulence factor P.69 pertactin.

A new generation of whooping-cough vaccines contain P.69 pertactin, a surface-exposed domain of an outer membrane protein expressed by the virulent bacterium Bordetella pertussis. This protein is a virulence factor that mediates adhesion to target mammalian cells, a reaction that is in part mediated by an RGD sequence. The X-ray crystal structure of P.69 pertactin has been determined to 2.5 A. The protein fold consists of a 16-stranded parallel beta-helix with a V-shaped cross-section, and is the largest beta-helix known to date. Several between-strand weakly conserved amino-acid repeats form internal and external ladders. The structure appears as a helix from which several loops protrude, which contain sequence motifs associated with the biological activity of the protein. One particular (GGXXP)5 sequence is located directly after the RGD motif, and may mediate interaction with epithelial cells. The carboxy-terminal region of P.69 pertactin incorporates a (PQP)5 motif loop containing the major immunoprotective epitope.

A single mutation in the recombinant light chain of tetanus toxin abolishes its proteolytic activity and removes the toxicity seen after reconstitution with native heavy chain.

Specific proteolysis by the tetanus toxin light chain of a vesicle-associated membrane protein (VAMP) involved in exocytosis is thought to underlie its intracellular blockade of neurotransmitter release. To substantiate this mechanism, recombinant light chain was expressed as a maltose binding protein-light chain fusion product in Escherichia coli. After purification of affinity chromatography and cleavage with factor Xa, the resultant light chain was isolated and its identity confirmed by Western blotting and N-terminal sequencing. It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-residue synthetic polypeptide spanning the cleavage site of the substrate. The importance of Glu234 in the catalytic activity of the light chain, possibly analogous to Glu143 of thermolysin, was examined using site-directed mutagenesis. Changing Glu234 to Ala abolished the protease activity of the light chain, but its ability to bind the polypeptide substrate was retained. Each recombinant light chain could be reconstituted with the heavy chain of tetanus toxin, yielding the same level of disulfide-linked species as the two native chains. Whereas the toxin formed with wild-type light chain exhibited appreciable neuromuscular paralysis activity and mouse lethality, the equivalent dichain material containing the Ala234 mutant lacked neurotoxicity in both the in vitro and in vivo assays. Thus, these results demonstrate directly, for the first time, that the lethality of tetanus toxin and its inhibition of exocytosis in intact neurons are attributable largely, if not exclusively, to endoprotease activity.

Crystallographic characterization of pertactin, a membrane-associated protein from Bordetella pertussis.

Pertactin, a membrane-associated protein of Bordetella pertussis, has been crystallized in the presence of 28% ammonium sulphate. The space group is P6(3)22 with cell dimensions a = b = 178.2 A and c = 106.8 A. The crystals diffract to 3.3 A using a rotating anode source and are suitable for an X-ray structure determination. Assuming one molecule in the asymmetric unit, 70% of the cell is occupied by solvent.

Production of biologically active light chain of tetanus toxin in Escherichia coli. Evidence for the importance of the C-terminal 16 amino acids for full biological activity.

The activity of the light (L) chain of tetanus toxin, and of mutants constructed by site-directed mutagenesis, was studied by expression and purification of the proteins from E. coli. Wild-type recombinant L chain (pTet87) was active in the inhibition of exocytosis from cultured bovine adrenal chromaffin cells, although at a level 5-15% of that of L chain purified from tetanus toxin. L chain mutants which terminated at Leu-438 (pTet89), or which contained a Cys-to-Ser mutation at residue 439 (pTet88) were equally as active as the full-length recombinant protein. The reduced activity of pTet87 L chain correlated with C-terminal proteolysis of the protein upon purification. A tryptic fragment derived from native light chain and which terminated at Leu-434 also showed reduced activity in the exocytosis assay, consistent with a requirement of the C-terminal region of the L chain for maximal activity. pTet87 L chain, but neither of the mutants, could be associated with purified H (heavy) chain to form a covalent dimer which induced the symptoms of tetanus in mice. The ability to form biologically active toxin using recombinant L chain will be of great value in structure-function studies of tetanus toxin.

Cloning, nucleotide sequence and heterologous expression of the protective outer-membrane protein P.68 pertactin from Bordetella bronchiseptica.

The prn gene encoding the 68 kDa protective outer-membrane protein of Bordetella bronchiseptica (P.68 pertactin) was cloned, sequenced and expressed in Escherichia coli. The gene was isolated by DNA:DNA hybridization experiments using a radioactively-labelled fragment of the homologous prn gene from Bordetella parapertussis. DNA sequence analysis reveals that the gene is capable of encoding a protein with a molecular mass of 93996 Da (P.94); this precursor molecule is processed to form the P.68 antigen on the surface of B. bronchiseptica. Heterologous expression of the full-length gene encoding P.94 in Escherichia coli results in similar processing, with the P.68 antigen targeted to the bacterial outer membrane. Comparison of P.94 with the P.93 and P.95 precursors, encoding homologous proteins from Bordetella pertussis and B. parapertussis, shows a high degree (greater than 90%) of homology. The major differences between all three proteins occur in the number of repeats of the two families (Gly-Gly-Xaa-Xaa-Pro)n and (Pro-Gln-Pro)n of reiterated sequence motifs.

Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus vaccine.

Plasmid pTETnir15, which directs the expression of the non-toxic immunogenic fragment C of tetanus toxin from the anaerobically inducible nirB promoter, was introduced into the Salmonella typhimurium aroA aroD live oral vaccine strain BRD509. The resulting strain, designated BRD847, was used to vaccinate orally BALB/c mice and was tested for plasmid stability and its ability to protect against a lethal tetanus toxin challenge. pTETnir15 was stably inherited by bacteria growing or persisting in the tissues of immunized mice whereas another BRD509 derivative, designated BRD753, harboring plasmid pTET85 which directs fragment C expression from the tac promoter, was highly unstable. Mice immunized with a single oral dose of BRD847 developed high levels of circulating anti-fragment C antibodies and were solidly protected against tetanus toxin challenge. Mice immunized with a single oral dose of BRD753 developed no detectable anti-fragment C antibodies. After boosting, antibodies were detected, but the mice were only partially protected against tetanus toxin challenge. Thus the use of an in vivo inducible promoter such as nirB may be a generally applicable approach to obtaining the stable in vivo expression of heterologous antigens in Salmonella vaccine strains.

Recombinant P.69/pertactin: immunogenicity and protection of mice against Bordetella pertussis infection.

The immunogenicity of recombinant (r-) pertactin was examined. Parenteral immunization of mice with natural or r-pertactin produced a similar increase in serum anti-pertactin antibodies and a decrease in Bordetella pertussis lung counts following aerosol challenge. Study of the kinetics of B. pertussis growth in the respiratory tract of immunized and control mice revealed that immunization with r-pertactin halted the multiplication of B. pertussis in the lungs and facilitated the early onset of bacterial clearance. In the trachea, bacterial numbers declined sharply in immunized animals during the first 3 days after challenge but thereafter B. pertussis numbers remained fairly constant throughout the rest of the experiment. Very low doses (0.1 micrograms) of r-pertactin were immunogenic and protective but only if the antigen was absorbed to alhydrogel. In vitro proliferation assays with lymphocytes from mice primed with either natural or r-pertactin indicated that the major T-cell epitopes of pertactin are conserved in the recombinant protein.

Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: high-level production and immunological properties.

Acellular whooping cough vaccines are based on pertussis toxoid but their effectiveness may be increased by the addition of other Bordetella pertussis antigens. We expressed the immunogenic outer membrane protein pertactin (P69) from B. pertussis to high levels in multi-copy transformants of the industrial yeast Pichia pastoris. In high-density fermentations, engineered P. pastoris yielded greater than 3 g of the protein per litre of culture. Purified recombinant pertactin was able to stimulate the incomplete protection afforded by toxoid to the level of the whole-cell vaccine, as shown by the Kendrick test, supporting its inclusion in future acellular vaccines.

Construction and characterization of Bordetella pertussis mutants lacking the vir-regulated P.69 outer membrane protein.

The Bordetella pertussis P.69 protein is an immunogen with vaccine potential. The role of this protein in pathogenesis is unclear; it has been associated with the toxic adenylate cyclase and adhesion to eukaryotic cells. For further analysis of the role of P.69 in the biology of B. pertussis, we have constructed strains which specifically lack P.69. The cloned P.69 (prn) gene of B. pertussis was insertionally inactivated with a kanamycin-resistance cassette. This inactivated gene was used to construct P.69- mutants of B. pertussis by allelic exchange using plasmid pRTP1. B. pertussis P.69- strains produced normal levels of other vir-regulated factors, including adenylate cyclase. The serotype of B. pertussis, determined by Eldering and Preston typing sera and monoclonal antibodies, was also unaffected by the presence or absence of P.69. The ability of a prn mutant to adhere to and invade HEp2 cells was not significantly different from that of its parent strain. A strain containing a mutation in fhaB was significantly less adhesive and invasive than its parent, and a prn fhaB double mutant exhibited an even greater reduction in adhesiveness and invasiveness down to levels comparable with a Vir- strain. However, strains harbouring mutations in FHA and/or P.69 were able to colonize or multiply in the murine respiratory tract, although a Vir- strain was unable to survive and proliferate in the same infection model.

Intranasal immunization using the B subunit of the Escherichia coli heat-labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen.

The plasmid pBRD026, which directs expression of the B subunit of the Escherichia coli heat-labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3' end of the gene. An oligonucleotide linker containing restriction sites for BglII and SpeI was inserted at the SpeI site at the 3' end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly-Pro-Gly-Pro which we propose acts as a 'hinge' between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetella pertussis P.69 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB-specific and P.69-specific antibody-secreting cells in the lungs of immunized mice.

Synthesis of tetanus toxin fragment C in insect cells by use of a baculovirus expression system.

The baculovirus expression vector p36C was used to express in cells of the insect Spodoptera frugiperda fragment C of tetanus toxin under the control of the strong polyhedrin promoter. Fragment C was expressed intracellularly at a high level and was soluble, allowing it to be purified by affinity chromatography with monoclonal antibody TT08. Purified fragment C from baculovirus was used to immunize mice and was shown to successfully prevent the symptoms of tetanus following a toxin challenge. The ganglioside-binding properties of baculovirus-derived fragment C were compared with those of intact tetanus toxin and native fragment C and were found to be dissimilar.