Helicobacter pylori-induced homotypic phagosome fusion in human monocytes is independent of the bacterial vacA and cag status.

Following reports that a VacA+cag+ toxigenic but not a VacA-cag- non-toxigenic Helicobacter pylori strain induced homotypic phagosome fusion in murine macrophages, we addressed that phenomenon in human cells. Mononuclear phagocytes and epitheloid cells were challenged with H. pylori strains of different vacA and cag genotypes and with VacA- and Cag- isogenic mutants, and chased in the absence or presence of signal transduction modulators. Electron microscopy revealed that, in monocytes: (i) homotypic phagosome fusion was frequently induced by all live H. pylori strains investigated but not by exogenous VacA; (ii) phagosomes containing bacteria fused, but not those containing latex beads; (iii) fusion resulted in communal compartments resembling giant multivesicular bodies; and (iv) formation of these compartments was blocked by inhibiting the host cell regulators PI 3-kinase, phospholipase C and p42 MAP kinase. Whereas some internalized bacteria remained viable 1 h after uptake, none survived a 24 h period. In contrast to monocytes, infected epitheloid cells rarely developed communal compartments. In combination, these results demonstrate that, in human monocytes, the H. pylori-induced homotypic phagosome fusion depends on neither the vacuolating cytotoxin VacA nor the cag pathogenicity island of H. pylori and does not result in prolonged intracellular survival.

Ribosome-mediated folding of partially unfolded ricin A-chain.

After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.

Proteolytic processing of ricin A chain is not required for cytotoxicity.

[125I]-labeled ricin A chain was endocytosed by macrophages and Vero cells either on its own or as part of the ricin holotoxin. Subsequently [125I]-ricin A chain was recovered from intoxicated cells by immunoprecipitation or acetone precipitation. The recovered protein was shown to have the same electrophoretic mobility as the material supplied to the cells. These data challenge an earlier suggestion that limited intracellular processing of ricin A chain by proteolytic enzymes is required for maximal toxicity.

Introduction of a disulfide bond into ricin A chain decreases the cytotoxicity of the ricin holotoxin.

Wild type ricin A chain (RTA) contains two cysteine residues (Cys171 and Cys259). Cys259 forms the interchain disulfide bond of ricin holotoxin with Cys4 of ricin B chain (RTB). We have used site-directed mutagenesis of RTA cDNA to convert Cys171 to Ser and to introduce a disulfide bond into RTA by converting Ser215 and Met255 to Cys residues. Mutant RTA was expressed in Escherichia coli and directed to the oxidizing environment of the periplasmic space where the Cys215-Cys255 disulfide bond was formed. The disulfide-containing RTA mutant had an in vitro catalytic activity similar to that of an identical form of recombinant RTA that lacked the S215C and M255C mutations. In the presence of glutathione and protein disulfide isomerase, this RTA variant reassociated with RTB to form ricin holotoxin. Incubation of this holotoxin with increasing concentrations of dithiothreitol showed that the interchain disulfide bond joining RTA and RTB was more readily reduced than the intrachain disulfide bond in RTA. Ricin in which the RTA moiety contained the disulfide bond was 15-18-fold less cytotoxic to HeLa or Vero cells than ricin in which the RTA did not contain the stabilizing disulfide cross-link. Since these ricin molecules had identical RTB cell binding and RTA catalytic activities, we suggest that the observed reduction in cytotoxicity caused by the introduced disulfide bond resulted from a constraint on the unfolding of RTA, indicating that such unfolding is necessary for the membrane translocation of RTA during its entry into the cytosol.

Preparation and characterization of recombinant proricin containing an alternative protease-sensitive linker sequence.

The aim of this study was to determine the feasibility of utilizing a factor Xa-specific cleavage site within a recombinant protein containing the ricin A chain (RTA) sequence. Release of RTA is believed to be an essential step during the intracellular phase of ricin intoxication. Failure to incorporate such cleavage sites in fusions containing RTA results in a loss of toxin action (O'Hare, M., et al. (1990) FEBS Lett. 273,200. Kim, J., and Weaver, R.F. (1988) Gene 68,315). In this report we describe the introduction of a factor Xa-specific site in the linker of proricin, which we use here as a model substrate. Upon purification of the recombinant mutant proricin after expression in Xenopus oocytes, we demonstrate that the protease does have access to the engineered recognition sequence (albeit at low efficiency) and that the presence of the latter does not interfere with disulfide bond formation or the lectin activity of the ricin B chain moiety. Upon cleavage and reduction, the RTA polypeptide displays ribosome-inactivating ability, indicating that the presence of the modified linker at its C-terminus does not interfere with its catalytic activity. The general applicability of using such a cleavage site in recombinant fusions with RTA is discussed.