Transcutaneous immunization with bacterial ADP-ribosylating exotoxins as antigens and adjuvants.
Transcutaneous immunization (TCI) is a new technique that uses the application of vaccine antigens in a solution on the skin to induce potent antibody responses without systemic or local toxicity. We have previously shown that cholera toxin (CT), a potent adjuvant for oral and nasal immunization, can induce both serum and mucosal immunoglobulin G (IgG) and IgA and protect against toxin-mediated mucosal disease when administered by the transcutaneous route. Additionally, CT acts as an adjuvant for coadministered antigens such as tetanus and diphtheria toxoids when applied to the skin. CT, a member of the bacterial ADP-ribosylating exotoxin (bARE) family, is most potent as an adjuvant when the A-B subunits are present and functional. We now show that TCI induces secondary antibody responses to coadministered antigens as well as to CT in response to boosting immunizations. IgG antibodies to coadministered antigens were also found in the stools and lung washes of immunized mice, suggesting that TCI may target mucosal pathogens. Mice immunized by the transcutaneous route with tetanus fragment C and CT developed anti-tetanus toxoid antibodies and were protected against systemic tetanus toxin challenge. We also show that bAREs, similarly organized as A-B subunits, as well as the B subunit of CT alone, induced antibody responses to themselves when given via TCI. Thus, TCI appears to induce potent, protective immune responses to both systemic and mucosal challenge and offers significant potential practical advantages for vaccine delivery. (+info)
Pseudomonas aeruginosa exoenzyme S is a biglutamic acid ADP-ribosyltransferase.
Kinetic analysis of two mutations within Pseudomonas aeruginosa exoenzyme S (ExoS) showed that a E379D mutation inhibited expression of ADP-ribosyltransferase activity but had little effect on the expression of NAD glycohydrolase activity while a E381D mutation inhibited expression of both activities. These data identify ExoS as a biglutamic acid ADP-ribosyltransferase, where E381 is the catalytic residue and E379 contributes to the transfer of ADP-ribose to the target protein. (+info)
Correlation of activity regulation and substrate recognition of the ADP-ribosyltransferase that regulates nitrogenase activity in Rhodospirillum rubrum.
In Rhodospirillum rubrum, nitrogenase activity is regulated posttranslationally through the ADP-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered both in the posttranslational regulation of DRAT activity and in the ability to recognize variants of dinitrogenase reductase have been found. This correlation suggests that these two properties are biochemically connected. (+info)
Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway.
Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho proteins. (+info)
Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras at multiple sites. One site appeared to be Arg41, but the second site could not be localized. In this study, the sites of ADP-ribosylation of c-Ha-Ras by ExoS were directly determined. Under saturating conditions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose incorporated per mol of Ras. Nucleotide occupancy did not influence the stoichiometry or velocity of ADP-ribosylation of Ras by ExoS. Edman degradation and mass spectrometry of V8 protease generated peptides of ADP-ribosylated Ras identified the sites of ADP-ribosylation to be Arg41 and Arg128. ExoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Ras, which indicated that Ras possessed an alternative site of ADP-ribosylation. The alternative site of ADP-ribosylation on Ras was identified as Arg135, which was on the same alpha-helix as Arg128. Arg41 and Arg128 are located within two different secondary structure motifs, beta-sheet and alpha-helix, respectively, and are spatially separated within the three-dimensional structure of Ras. The fact that ExoS could ADP-ribosylate a target protein at multiple sites, along with earlier observations that ExoS could ADP-ribosylate numerous target proteins, were properties that have been attributed to several vertebrate ADP-ribosyltransferases. This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology with the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases. These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ribosyltransferases. (+info)
Chemotaxin-dependent translocation of immunoreactive ADP-ribosyltransferase-1 to the surface of human neutrophil polymorphs.
mRNA from human polymorphonuclear neutrophil leucocytes (PMNs) was probed with cDNA encoding human skeletal muscle arginine-specific ADP-ribosyltransferase (ART1). A single 2.6-kb transcript was identified, which was similar in size to that observed in human skeletal muscle RNA. An 872-bp cDNA fragment, corresponding to the amino acid sequence of the processed human skeletal muscle enzyme, was generated by reverse transcription-PCR amplification of RNA from human PMNs, and was found to be identical to the ART1 cDNA derived from human skeletal muscle. ART1 was expressed as a fusion protein with glutathione S-transferase (GST) in insect cells, and antibodies were raised against the fusion protein in a rabbit. Following removal of GST immunoreactivity by immunoprecipitation, these antibodies were used to measure the abundance of immunoreactive ART1 on the surface of PMNs. Exposure of PMNs to formyl-Met-Leu-Phe (FMLP) was followed by a rapid increase in the abundance of cell surface ART1 (T1/2 = 1.9 min), and the concentration of FMLP for half-maximum response was 28.6 nM. Similar responses were observed after exposure of the cells to platelet-activating factor or interleukin-8, and we conclude that some of the effects of these chemotaxins are mediated by translocation of an intracellular pool of ART1 to its site of catalytic activity on the outer aspect of the plasma membrane. (+info)
Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome.
The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins. (+info)
Heparin-binding EGF-like growth factor interacts with mouse blastocysts independently of ErbB1: a possible role for heparan sulfate proteoglycans and ErbB4 in blastocyst implantation.
Blastocyst implantation requires molecular and cellular interactions between the uterine luminal epithelium and blastocyst trophectoderm. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is induced in the mouse luminal epithelium solely at the site of blastocyst apposition at 16:00 hours on day 4 of pregnancy prior to the attachment reaction (22:00-23:00 hours), and that HB-EGF promotes blastocyst growth, zona-hatching and trophoblast outgrowth. To delineate which EGF receptors participate in blastocyst activation, the toxicity of chimeric toxins composed of HB-EGF or TGF-(&agr;) coupled to Pseudomonas exotoxin (PE) were used as measures of receptor expression. TGF-(&agr;) or HB-EGF binds to EGF-receptor (ErbB1), while HB-EGF, in addition, binds to ErbB4. The results indicate that ErbB1 is inefficient in mediating TGF-(&agr;)-PE or HB-EGF-PE toxicity as follows: (i) TGF-(&agr;)-PE was relatively inferior in killing blastocysts, 100-fold less than HB-EGF-PE, (ii) analysis of blastocysts isolated from cross-bred egfr+/- mice demonstrated that HB-EGF-PE, but not TGF-(&agr;)-PE, killed egfr-/- blastocysts, and (iii) blastocysts that survived TGF-(&agr;)-PE were nevertheless killed by HB-EGF-PE. HB-EGF-PE toxicity was partially mediated by cell surface heparan sulfate proteoglycans (HSPG), since a peptide corresponding to the heparin-binding domain of HB-EGF as well as heparitinase treatment protected the blastocysts from the toxic effects of HB-EGF-PE by about 40%. ErbB4 is a candidate for being an HB-EGF-responsive receptor since RT-PCR analysis demonstrated that day 4 mouse blastocysts express two different erbB4 isoforms and immunostaining with anti-ErbB4 antibodies confirmed that ErbB4 protein is expressed at the apical surface of the trophectoderm cells. It is concluded that (i) HB-EGF interacts with the blastocyst cell surface via high-affinity receptors other than ErbB1, (ii) the HB-EGF interaction with high-affinity blastocysts receptors is regulated by heparan sulfate, and (iii) ErbB4 is a candidate for being a high-affinity receptor for HB-EGF on the surface of implantation-competent blastocysts. (+info)