In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951–962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (K d ≈ 5 nM), only CT elicited a cAMP-dependent Cl− secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.
The phagocyte chemotactic receptors, including the formyl peptide receptor (FPR), 1 the lipoxin A 4 receptor, the C5a receptor, the platelet-activating factor receptor, and the interleukin-8 receptor are involved in inflammation and are all members of the G protein-coupled receptor (GPCR) superfamily. Among the most studied in this inflammatory receptor family is neutrophil FPR (1). FPR binds N-formyl peptides, such as formyl-Met-Leu-Phe (fMLF), with nanomolar affinity (2). Such N-formyl peptides are indicators of the presence of bacteria (3) or damage to host cell mitochondria (4,5). Binding of N-formyl peptides to FPR thus provides phagocytes with signals for infection or injury and results in activation of chemotaxis and other host defensive processes including lysosomal enzyme secretion, stimulation of production of inflammatory mediators, and generation of superoxide.The effects of amino acid substitutions and modifications of fMLF peptides on binding to FPR and activation have been studied extensively (6 -8). The formyl group, the methionine at position 1, and phenylalanine at position 3 have been shown to be necessary for high affinity binding. Decarboxylation of the C-terminal phenylalanine markedly reduces activity, but esters or amides of this residue or peptides with C-terminal amino acid additions exhibit similar activity to the tripeptide with the free acid. None of the fMLF functional groups have been shown to be absolutely essential for activity but rather they appear to individually contribute to the overall free energy of binding.Chemical and photoaffinity cross-linking of fMLF analogs to FPR has been achieved by a number of groups (9 -12). However, none of these studies have identified the site of labeling. The residue in the second position of N-formylated peptides appears to be the most tolerant of modification (6, 13) and both of the flanking residues are critical for high affinity binding (14); so the second residue was chosen to accommodate the photoreactive amino acid benzoylphenylalanine (Bpa). Bpa is chemically stable in the absence of photoexcitation, can be directly introduced into peptide ligands by solid phase peptide synthesis, and has been photocross-linked into several peptide receptors (15). Here we report that a fluorescent photoaffinity analog of fMLF, formyl-Met-p-benzoyl-L-phenylalanine-PheTyr-Lys-⑀-N-fluorescein (fMBpaFYK-fl), efficiently photocrosslinks to FPR residues 83-85. Derivatization of these residues in FPR by Bpa supports recent site-directed mutagenesis stud-* This work was supported in part by National Science Foundation EPSCoR Grant RII-891879 (to E. A. D.), a Grant from the Pittsburgh Supercomputing Centers through the National Institutes of Health resource Grant 2p41RR06009, a grant from the Rocky Mountain Chapter of the Arthritis Foundation and the Harmon Foundation (to J. S. M.), a grant from the Rocky Mountain Chapter of the Arthritis Foundation (to H. M. M.), and Public Health Service Grants 1RO1A40108-01 and RO122735 (to A. J. J.). The costs of publicati...
Culture supernatants prepared from reactogenic strains of Vibrio cholerae cause a decrease in the transcellular epithelial resistance of T84 intestinal cells. This decrease correlates with the presence of hemagglutinin/ protease but not with the presence of other potential accessory toxins or proteases. These data suggest a possible role for hemagglutinin/protease in reactogenicity, although other factors may also contribute.The potentially life-threatening disease cholera is caused by toxigenic strains of the gram-negative organism Vibrio cholerae. The hallmark symptom of cholera, profuse, watery diarrhea, is caused primarily by cholera toxin (CT). The genes encoding CT, ctxAB, are carried on a transducing phage, CTX⌽, that integrates into the V. cholerae genome (29). The "core" element of the phage genome carries four genes in addition to ctxAB : cep, orfU, ace, and zot (29). Both zot and orfU are known to be essential for phage morphogenesis (29). In addition, the recombinant products of the zot and ace genes have also been associated with changes in intestinal tissue conductance, suggesting that these genes encode accessory toxins of V. cholerae (6,27,28).The development of a safe and effective vaccine to protect against cholera is a multifaceted problem. An ideal cholera vaccine must confer long-lasting protective immunity, be inexpensive, and be easy to use. A vaccine strategy employing live attenuated V. cholerae strains should meet each of these requirements (15, 17). However, production of a safe, attenuated strain has been problematic. Shortly after the discovery of the genes zot and ace, one would have predicted that a core deletion, resulting in mutants unable to produce CT, zonula occludens toxin (Zot), and accessory cholera enterotoxin (Ace), would produce ideal vaccine candidate strains. However, even with the core element deleted, strains CVD110, CVD111, and CVD112 were still "reactogenic," causing residual side effects in volunteer recipients, including mild diarrhea, nausea, vomiting, abdominal cramps, and fever (21, 23). Similarly, vaccine strains with deletions of the entire integrated CTX⌽ element (attRS deletions) are also mildly reactogenic (5, 24). These data indicate that these strains encode additional reactogenic factors encoded at loci other that the integrated CTX⌽ prophage. Curiously, the reactogenic effect of these undefined factors is absent from vaccine candidate strains that have additional defects in motility (5,11,24). This paper describes experiments using transcellular epithelial resistance (TER) across polarized T84 epithelial cells to monitor potential reactogenic factors in various vaccine strains. We show that an activity associated with the zot gene is not detected in this system. However, we find that a decrease in TER correlates with the presence of the genes for production of hemagglutinin/protease (HA/protease), indicating that HA/protease may be a significant contributor to the reactogenicity of some V. cholerae vaccine strains.Addition of supernatant fluids to pol...
We examined the entry of anthrax edema toxin (EdTx) into polarized human T84 epithelial cells using cyclic AMP-regulated Cl−secretion as an index of toxin entry. EdTx is a binary A/B toxin which self assembles at the cell surface from anthrax edema factor and protective antigen (PA). PA binds to cell surface receptors and delivers EF, an adenylate cyclase, to the cytosol. EdTx elicited a strong Cl− secretory response when it was applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface. PA alone had no effect when it was applied to either surface. T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane of these cells. The PA receptor did not fractionate with detergent-insoluble caveola-like membranes as cholera toxin receptors do. These findings have implications regarding the nature of the PA receptor and confirm the view that EdTx and CT coopt fundamentally different subcellular systems to enter the cell and cause disease.
Culture supernatants prepared from reactogenic strains of Vibrio cholerae cause a decrease in the transcellular epithelial resistance of T84 intestinal cells. This decrease correlates with the presence of hemagglutinin/ protease but not with the presence of other potential accessory toxins or proteases. These data suggest a possible role for hemagglutinin/protease in reactogenicity, although other factors may also contribute.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.