The Ebola virus ribonucleoprotein complex: a novel VP30-L interaction identified.

The ribonucleoprotein (RNP) complex of Ebola virus (EBOV) is known to be a multiprotein/RNA structure, however, knowledge is rather limited regarding the actual protein-protein interactions involved in its formation. Here we show that singularly expressed VP35 and VP30 are present throughout the cytoplasm, while NP forms prominent cytoplasmic inclusions and L forms smaller perinuclear inclusions. We could demonstrate the existence of NP-VP35, NP-VP30 and VP35-L interactions, similar to those described for Marburg virus (MARV) based on the redistribution of protein partners into NP and L inclusion bodies. Significantly, a novel VP30-L interaction was also identified and found to form as part of an NP-VP30-L bridge structure, similar to that formed by VP35. The identification of these interactions allows a preliminary model of the EBOV RNP complex structure to be proposed, and may provide insight into filovirus transcriptional regulation.

Electrically neutral microheterogeneity of human plasma transthyretin (prealbumin) detected by isoelectric focusing in urea gradients.

Mutants of the human plasma transthyretin (TTR, prealbumin) have attracted interest due to their rather frequent association with the autosomal dominant disease familial amyloidotic polyneuropathy (FAP). Some three quarters of known TTR mutations produce electrically neutral amino acid substitutions undetectable via separation by charge. We have developed an electrophoretic procedure sensitive to differences in the stability of tetramers and monomers under partially denaturing conditions. The differential folding states were found to be fully reversible. Applying the procedure we found 14 electrically silent mutants of TTR among 2000 plasma samples from German donors. We demonstrate that the normal TTR monomer exists in different forms of variable stability and/or charge due to binding of sulfhydryls from plasma to the unique cysteine at position 10 of the primary structure as well as due to modification by treatment with an oxidant. We found that reduction of Cys10 increases the stability of the folded monomeric and tetrameric conformations. The conformational changes of TTR induced by isoelectric focusing in a urea gradient were found to be associated by a gain of three positive charge units. Using published crystallographic data we present structural sites in the TTR molecule which could explain the observed effects.

Insights into carbohydrate recognition by Narcissus pseudonarcissus lectin: the crystal structure at 2 A resolution in complex with alpha1-3 mannobiose.

Carbohydrate recognition by monocot mannose-binding lectins was studied via the crystal structure determination of daffodil (Narcissus pseudonarcissus) lectin. The lectin was extracted from daffodil bulbs, and crystallised in the presence of alpha-1,3 mannobiose. Molecular replacement methods were used to solve the structure using the partially refined model of Hippeastrum hybrid agglutinin as a search model. The structure was refined at 2.0 A resolution to a final R -factor of 18.7 %, and Rfreeof 26.7 %. The main feature of the daffodil lectin structure is the presence of three fully occupied binding pockets per monomer, arranged around the faces of a triangular beta-prism motif. The pockets have identical topology, and can bind mono-, di- or oligosaccharides. Strand exchange forms tightly bound dimers, and higher aggregation states are achieved through hydrophobic patches on the surface, completing a tetramer with internal 222-symmetry. There are therefore 12 fully occupied binding pockets per tetrameric cluster. The tetramer persists in solution, as shown with small-angle X-ray solution scattering. Extensive sideways and out-of-plane interactions between tetramers, some mediated via the ligand, make up the bulk of the lattice contacts.A fourth binding site was also observed. This is unique and has not been observed in similar structures. The site is only partially occupied by a ligand molecule due to the much lower binding affinity. A comparison with the Galanthus nivalis agglutinin/mannopentaose complex suggests an involvement of this site in the recognition mechanism for naturally occurring glycans.

The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality.

We have investigated the ability of a recombinant protein (REP231), derived from Clostridium difficile toxin A C-terminal domain, to protect against toxin A (TcdA) intoxication in vitro and in vivo. REP231 was cloned, expressed and purified by thyroglobulin affinity chromatography, and demonstrated identical binding properties to TcdA. Immunofluorescence experiments and in vitro cytotoxicity assays using mouse teratocarcinoma cells F9 showed that specific binding of TcdA to F9 cells through its C-terminal domain is essential for producing cytotoxic effects. TcdA binding and cytotoxicity was inhibited by REP231 and a monoclonal antibody directed against the C-terminal domain. Toxin B did not bind to F9 cells and was consequently inactive in cytotoxicity assays. Inhibition studies with lectins and a Le(x)-specific antibody supported earlier findings that a terminal galactose is part of the bound saccharide but excluded Le(x) as a receptor for TcdA. Mice immunised with REP231 were protected against a threefold lethal dose of TcdA. Thus, REP231 appeared to be a suitable candidate to develop an alternative therapeutic agent, which is able to neutralise carbohydrate-mediated TcdA binding and might act as a vaccine.

Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile.

To analyse the transcription pattern of the five tcdA-E genes of the pathogenicity locus (PaLoc) of Clostridium difficile a protocol was established to purify RNA from strain VPI10463. Transcription analysis of the five tcdA-E genes showed that they were all transcribed. In the early exponential phase, a high level of tcdC and low levels of tcdA,B,D,E transcripts were detectable; this was inverted in the stationary phase, suggesting that TcdC might have a negative influence on transcription of the other genes. Three transcription initiation sites, one for tcdA and two for tcdB were determined by primer extension analysis. Readthrough transcripts from outside the locus were not obtainable, so that parts of the transcription of tcdD, tcdB, tcdA and tcdC must occur by monocistronic transcription. Within the locus all possible intergenic readthrough transcripts were detectable except that between tcdC and tcdA, a stretch of DNA interrupted by a functional transcription terminator. Thus we found mono- and polycistronic transcription of tcdA and tcdB to occur which should lead to production of a surplus of tcdA over tcdB transcripts. This would explain the surplus of TcdA over TcdB expression observed in vitro. Due to its basic nature and similarity to BcnA of Clostridium perfringens and to Orf-22 of Clostridium botulinum, TcdD is most probably a regulatory protein with DNA-binding properties. On the basis of the presented study we discuss a model for the growth-phase-related, coordinate regulation of toxin expression wherein tcdC has a negative and tcdD a positive regulatory function on transcription of the tcdD,B,E and tcdA genes.

Definition of the single integration site of the pathogenicity locus in Clostridium difficile.

We determined the nucleotide sequence 3.8 kb upstream and 5.2 kb downstream of the toxin genes A and B of Clostridium difficile. Nine ORFs were discovered. Based on PCR-directed approaches, two were attributed to the pathogenicity locus (PaLoc). The other seven were found in every C. difficile isolate obtained from the human gastrointestinal tract, respectless of their toxinogenicity. The ORFs cdu1 and cdu2/2' upstream of the PaLoc displayed similarity to repressors of Gram-positive bacteria (cdu1), and to an Na+/H+ antiporter described for Enterococcus hirae (cdu2/2'). Downstream of the locus a putative ABC transporter (cdd2-4) was identified. With a set of three paired primers used in polymerase chain reactions we succeeded in delineating the PaLoc. Sequencing of the appropriate stretch of DNA in C. difficile VPI10463 and four additional toxinogenic strains proved a high conservation of the borders of the PaLoc in all these strains. Our data define the locus as a distinct genetic element. Comparing the sequences of five toxinogenic and five non-toxinogenic strains the integration site of the PaLoc was defined. This showed that a stretch of 115 bp found in non-toxinogenic strains is replaced by the 19-kb locus in toxinogenic strains. Analysis of the boundary sequences showed that the locus is obviously not a mobile genetic element by itself. Instead we propose that it is the independent pathogenic part of a more extended genetic element associated with virulence. The 115 bp of non-toxinogenic strains replaced by the locus in toxinogenic strains carry the putative transcription terminator of the cdu1, a predicted repressor protein. A possible polar effect of the loss of this terminator on transcription of the TcdABCDE genes is discussed. Such an effect would explain the unidirectional insertion of the PaLoc at a single site of the C. difficile genome and might give a rationale for the development of the disease which is induced after antibiotical treatment.

Large clostridial cytotoxins--a family of glycosyltransferases modifying small GTP-binding proteins.

Some Clostridium species produce ABX-type protein cytotoxins of high molecular weight. These toxins constitute the group of large clostridial cytotoxins (LCTs), which have homologous protein sequences, exert glycosyltransferase activity and modify GTP-binding proteins of the Ras-superfamily. These characteristics render the LCTs valuable tools for developmental and cell biologists.

Evidence for a modular structure of the homologous repetitive C-terminal carbohydrate-binding sites of Clostridium difficile toxins and Streptococcus mutans glucosyltransferases.

The homologous C-terminal repeats of Clostridium difficile toxins (ToxA and ToxB) and streptococcal glucosyltransferases appear to mediate protein-carbohydrate interactions at cellular binding sites with sugar moieties as substrates. A consensus sequence of 134 repeating units from gram-positive bacteria indicates that these repeats have a modular design with (i) a stretch of aromatic amino acids proposed to be involved in the primary carbohydrate-protein interaction, (ii) an amplification of this interaction by repetition of the respective sequences, and (iii) a second domain, not characterized, that is responsible for carbohydrate specificity.

Comparative sequence analysis of the Clostridium difficile toxins A and B.

The six clones pTB112, pTB324, pTBs12, pCd122, pCd14 and pCd13 cover the tox locus of Clostridium difficile VPI 10463. This region of 19 kb of chromosomal DNA contains four open reading frames including the complete toxB and toxA genes. The two toxins show 63% amino acid (aa) homology, a relatedness that had been predicted by the cross-reactivity of some monoclonal antibodies (mAb) but that is in contrast to the toxin specificity of polyclonal antisera. A special feature of ToxA and ToxB is their repetitive C-termini. We define herein 19 individual CROPs (combined repetitive oligopeptides of 20-50 aa length) in the ToxB C-terminus, which are separable into five homologous groups. Comparison of the aa sequences of the N-terminal two-thirds of ToxA and ToxB revealed three marked structures, a cluster of 172 hydrophobic, highly conserved aa in the centre of both toxins, a sequence of 120 residues with an accumulation of highly conserved arginine, cysteine, histidine, methionine, and tryptophan residues, and a stretch of 248 less conserved aa. The probable function of these domains is discussed. Structural and functional homologies of ToxA and ToxB indicate that both genes have a common ancestor and may have evolved by gene duplication, with subsequent recombination and mutation, as has been reported for streptococcal glucosyltransferases (Gtf).

Clostridium difficile toxin A carries a C-terminal repetitive structure homologous to the carbohydrate binding region of streptococcal glycosyltransferases.

A detailed analysis of the 8130-bp open reading frame (ORF) of gene toxA and of an upstream ORF designated utxA, indicates the presence of a transcription terminator stem-loop for toxA, promoter sequences, and Shine-Dalgarno boxes for toxA and utxA. No transcription terminator between toxA and utxA is suggested by the sequence. ToxA contains two domains, one-third (C-terminal) with a repetitive structure and the residual two-thirds with no repetitions. The 2499-bp sequence encoding the repetitive structure is composed of nine groups of different short repetitive oligodeoxyribonucleotides (SRONs). A combination of these SRONs codes for five groups of combined repetitive oligopeptides (CROPs). Seven 50-amino acid (aa) CROPs and 23 CROPs of 21 aa in length are noticed. The CROPs are generally highly conserved, but four exhibit variability and possibly represent 'hot spots' of the repetitive structure. The reactivity of the C-terminal repeat with monoclonal antibody 1337C8 indicates that this part contains the carbohydrate-binding domain of ToxA. In this region homology exists between the ToxA repeats and the glucosyltransferases of Streptococci. We propose that binding of ToxA to cells occurs via the C-terminal repeat domain, with the N-terminal domain being responsible for toxic function.

Cloning of Clostridium difficile toxin B gene and demonstration of high N-terminal homology between toxin A and B.

High titered Clostridium sordellii lethal toxin antiserum, cross-reactive with C. difficile cytotoxin B (ToxB), was used to isolate toxB fragments from a C. difficile expression library. Recombinant clones containing toxB fragments of the 5' and 3' end were isolate. A 2.5-kb HincII fragment of chromosomal DNA overlaps both groups of clones. A partial restriction map of the total toxB gene is presented. The gene is positioned upstream of utxA and toxA, toxB has a size of 6.9 kb, corresponding to a 250-kDa polypeptide. A partial sequence of the 5' end of toxB was determined. The sequence contains 398 bp upstream of toxB with a putative Shine-Dalgarno box (AGGAGA) and 609 bp of the toxB open reading frame. The N-terminal 203 amino acids of ToxB were compared with the N-terminal amino acids of the enterotoxin A (ToxA). A homology of 64% of the residues was detected, which proves the relatedness of ToxA and ToxB of C. difficile.