Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo.

The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a CTL response against the epitope in BALB/c mice. As little as 300 fmol of fusion could stimulate a response. The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn. Upon challenge with L. monocytogenes, mice previously injected with LFn-LLO91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice. These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications.

Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen.

The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets. Genetically fusing the amino-terminal domain of LF (LFN; residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol. We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery. Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis. The efficiency of translocation was dependent on tract length: (LFN > Lys8 > Lys6 > Lys3). Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive. Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA. The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs. Excess free LFN blocked the action of LFNDTA, but not of Lys6DTA. This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site. Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells.

Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors.

We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly). The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly. Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase. We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor. We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment. To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification.

Differential effects of staphylococcal toxic shock syndrome toxin-1 on B cell apoptosis.

Superantigens, such as toxic shock syndrome toxin 1 (TSST-1), have been implicated in the pathogenesis of several autoimmune and allergic diseases associated with polyclonal B cell activation. In this report, we studied the in vitro effects of TSST-1 on B cell activation. We show herein that TSST-1 produced antagonistic effects on Ig synthesis by peripheral blood mononuclear cells (PBMC) from normal subjects, depending on the concentration used; Ig production was inhibited at 1000 pg/ml (P < 0.01) and enhanced at 1 and 0.01 pg/ml (P < 0.01) of toxin. Cultures of PBMC were then examined for morphologic features and DNA fragmentation characteristic for apoptosis. B cells exhibited a significantly higher (P < 0.01) incidence of apoptosis after stimulation with 1000 pg/ml of TSST-1 compared with 1 or 0.01 pg/ml of toxin or medium alone. Abundant expression of Fas, a cell surface protein that mediates apoptosis, was detected on B cells after stimulation with 1000 pg/ml of TSST-1 and was significantly higher on B cells undergoing apoptosis than on live cells (P = 0.01). Additionally, increased Fas expression and B cell death occurred at concentrations of TSST-1 inducing the production of high amounts of gamma interferon (IFN-gamma), and both events could be blocked by neutralizing anti-IFN-gamma antibody. These findings suggest that high concentrations of TSST-1 can induce IFN-gamma-dependent B cell apoptosis, whereas at low concentrations it stimulates Ig synthesis by PBMC from normal subjects. These findings support the concept that staphylococcal toxins have a role in B cell hyperactivity in autoimmunity and allergy.

Transgenic mice carrying the diphtheria toxin A chain gene under the control of the granzyme A promoter: expected depletion of cytotoxic cells and unexpected depletion of CD8 T cells.

We have generated transgenic mice bearing the diphtheria toxin A chain (DTA) gene under the control of granzyme A (GrA) promoter sequences (GrA-DTA). GrA is expressed in activated cytotoxic cells but not in their immediate progenitors. These GrA-DTA mice are deficient in cytotoxic functions, indicating that most cytotoxic cells express GrA in vivo. Surprisingly, one founder strain containing a multicopy GrA-DTA insert show a marked and selective deficiency in CD8+ cells in peripheral lymphoid organs. This depletion was not observed in thymus, where the distribution of CD4+ and CD8+ cells is normal. Moreover, the emigration of T cells from thymus is normal, indicating that the depletion occurs in the periphery. GrA-DTA mice should be useful as models to dissect the role of cytotoxic cells in immune responses and as recipients of normal and neoplastic hematopoietic cells. The selective depletion of CD8+ cells in one founder strain could have implications for postthymic T-cell development.

A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein.

A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels.