Requirement for prolonged action in the cytosol for optimal protein synthesis inhibition by diphtheria toxin.

Diphtheria toxin A-fragment enters the cytosol of target cells, where it inhibits protein synthesis by catalyzing ADP-ribosylation of elongation factor 2 (EF-2). We have here analyzed toxin-induced protein synthesis inhibition in single cells by autoradiography and compared it with inhibition of protein synthesis in the whole cell culture. The data show that half-maximal protein synthesis inhibition in the whole cell population after a short incubation time is achieved by partially inhibiting protein synthesis in basically all the cells, while half-maximal protein synthesis inhibition after a long incubation time is due to a complete protein synthesis block in about half the cells in the population. We have also compared stable and unstable A-fragment mutants with respect to the kinetics of cell intoxication. While the toxicity of the stable mutants increased with time, the unstable mutants showed a similar toxicity at early and late time points. When studying the kinetics of cell intoxication by toxins with short cytosolic half-life, we could not detect any recovery of protein synthesis at late time points when all the mutant A-fragments should be degraded. This indicates that the ADP-ribosylation of EF-2 cannot be reversed by an endogenous activity in the cells. The data indicate that entry of toxin into a cell is not associated with an immediate block in protein synthesis, and that prolonged action of single A-fragment molecules in the cytosol is sufficient to obtain complete protein synthesis inhibition at low toxin concentrations.

Diagnosis of cytomegalovirus infection in kidney transplant recipients by a quantitative RNA-DNA hybrid capture assay for cytomegalovirus DNA in leukocytes.

The clinical value of a new RNA-DNA hybridization assay for quantification of cytomegalovirus (CMV) DNA in leukocytes [Hybrid Capture CMV DNA Assay (HCA); Murex Biotech, UK] was evaluated. The HCA was compared with an assay for CMV pp65 antigen in leukocytes and an in-house CMV polymerase chain reaction PCR (CMV-PCR) on parallel blood samples. The HCA and the CMV-PCR were less sensitive than the CMV pp65 assay, but the positive predictive value of all three methods for CMV disease was 50% or less. However, when quantitation of viral load by HCA and CMV pp65 assay was taken into consideration, both assays were superior to CMV-PCR in predicting CMV disease.

Colony variation of Helicobacter pylori: pathogenic potential is correlated to cell wall lipid composition.

BACKGROUND: Differences in expression of disease after infection with Helicobacter pylori have so far been connected with host factors and bacterial interstrain variation. In this study, spontaneous and ecology-mediated intrastrain variation was examined. METHODS: Four clinical isolates of H. pylori were shown to give rise to two colony forms. Bacterial morphology was examined by electron microscopy. Bacterial fractions were examined for proteins using ion exchange chromatography and SDS-PAGE; for lipids using thin-layer chromatography, lipid anion-exchange chromatography, column chromatography on silica gel, 31P-NMR, gas chromatography and mass spectrometry. Bacterial in vitro invasiveness and adhesiveness were examined in two different systems, and urease and VacA toxin were assayed by Western blot analysis. RESULTS: H. pylori was shown to give rise to two colony forms: at normal pH the population was dominated by L colonies. One strain was chosen for further studies. Bacteria from L colonies retained VacA toxin and urease, did not invade or adhere to epithelial cells, and contained normal quantities of phosphatidylethanolamine. In a small frequency, spontaneous S colonies were formed. Bacteria from these colonies released VacA and urease, adhered to and invaded epithelial cells and contained increased amounts of lysophosphatidyl ethanolamine and phosphatidyl serine. After addition of HCl to the culture medium (pH6), almost only S colonies were formed. The results demonstrate that environmental factors, such as HCl, can change the bacterial cell wall, and thereby enhance expression of virulence factors of H. pylori in vitro. A similar in vivo variation would have implications for our understanding of the interaction between HCl secretion in the gastric mucosa and H. pylori in the development of peptic ulcer disease.

Membrane translocation of diphtheria toxin A-fragment: role of carboxy-terminal region.

The C-terminal end of diphtheria toxin A-fragment was altered and the consequences for toxicity and translocation of the A-fragment to the cytosol were studied. Mutations and deletions in the protease-sensitive, disulfide-bridged region linking the two functional parts of the toxin, the A- and B-fragments, reduced the toxicity of the protein as such, but when the mutant toxins were cleaved ("nicked") by trypsin before being added to cells, the toxicity was restored. Prevention of disulfide formation by removal of Cys186 resulted in complete loss of toxicity. To circumvent the nicking step, toxin was formed by reconstitution from separate A- and B-fragments where the A-fragments varied in the C-terminal sequences. The amino acids C-terminal to Cys186 were found not to be required for translocation. Furthermore, both charged and uncharged residues near the C-terminal end were compatible with translocation. The data indicate that the C-terminal amino acid sequence is not decisive for translocation of diphtheria toxin A-fragment to the cytosol.

Interactions of diphtheria toxin B-fragment with cells. Role of amino- and carboxyl-terminal regions.

The B-fragment of diphtheria toxin binds to cell surface receptors and facilitates entry of the enzymatically active A-fragment into the cytosol. The roles of the amino- and carboxyl-terminal regions of the B-fragment in interactions with the cell membrane were studied by measuring specific binding, insertion into membranes at low pH, and formation of cation-selective channels, as well as by toxicity measurements after association with active A-fragment. Deletion of the amino-terminal 12 amino acids of the B-fragment did not affect its ability to bind to receptors and to form ion channels at low pH, whereas both abilities were strongly impaired when one more amino acid (Trp206) was removed. Replacement of the amino-terminal 31 residues with an amphipathic sequence from human apolipoprotein A1 restored receptor binding but not ion channel formation. The binding to cells was virtually abolished when 9 residues were deleted from the carboxyl terminus. Deletion of only 4 residues or extension by 12 residues did not prevent specific binding, but reduced insertion, channel formation, and toxicity. Those deletions that reduced receptor binding ability increased the trypsin sensitivity of the B-fragment. The results indicate that the amino- and carboxyl-terminal regions of diphtheria toxin B-fragment are important for receptor binding, possibly because they contribute to keep the B-fragment in a binding-competent conformation. Small alterations in the carboxyl-terminal end reduced insertion, channel formation, and toxicity more than the ability of the B-fragment to bind to cells.

Association between diphtheria toxin A- and B-fragment and their fusion proteins.

Natural diphtheria toxin is synthesized as a single polypeptide chain that is activated by cleavage into an A- and a B-fragment, which are linked by a disulphide bond. In the present work the ability of independently translated A- and B-fragments to associate was investigated. Low amounts of A- and B-fragments synthesized in vitro were mixed under conditions that allowed formation of a disulphide bridge between the fragments. Under these conditions toxin was reconstituted in close to 100% yield and found to be as toxic to Vero cells as natural diphtheria toxin. Efficient association between the A- and B-fragment was dependent on the formation of a disulphide bridge. Reconstituted toxin obtained from one [35S]methionine-labelled fragment and one unlabelled fragment proved useful in translocation studies. Addition of a number of different polypeptides to the N- and C-termini of either fragment did not, in most cases, prevent reconstitution. The ready reconstitution allows easy manipulations with the toxin to form targeted molecules and to develop diphtheria toxin as a vector for translocation of peptides to the cytosol. The fact that the reconstituted toxin does not need to be nicked with proteinases to be active allows experimentation with proteinase-sensitive constructs.