Channel Formation by the Glycosylphosphatidylinositol-anchored Protein Binding Toxin Aerolysin Is Not Promoted by Lipid Rafts

Nelson, Kim L.; Buckley, J. Thomas

doi:10.1074/jbc.m002785200

Cited by 34 publications

(22 citation statements)

References 35 publications

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“…We believe that the cyclic AMP is specifically released outside of the cell. The reports from Buckley's group support our hypothesis about the specific release of cyclic AMP (10,11,12,13). These authors showed that channels that are relatively nonselective are formed in the cell membrane after the incubation with aerolysin.…”

Section: Fig 2 Effects Of Ada Xacsupporting

confidence: 75%

Studies of the Mechanism of Action of the Aerolysin-Like Hemolysin of Aeromonas sobria in Stimulating T84 Cells To Produce Cyclic AMP

et al. 2003

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We previously reported that the aerolysin-like hemolysin of Aeromonas sobria stimulates T84 cells to produce cyclic AMP, which then emerges in the culture medium. In order to clarify the mechanism of action of the hemolysin, we examined the involvement of adenosine nucleotide. The results show that the hemolysin stimulates T84 cells to release ATP, which is then converted to adenosine by ectonucleotidase. The adenosine generated might stimulate the P1 adenosine receptors of T84 cells to produce cyclic AMP.We purified the enterotoxin from the culture supernatant of Aeromonas sobria and found that it possessed hemolytic activity in addition to enterotoxic activity (5). The analysis of the DNA sequence (GenBank accession number AY157998) showed that the hemolysin is homologous with aerolysin produced by Aeromonas hydrophila. Homology between the two toxins is 68.5% at the amino acid level (5). The aerolysin-like hemolysin and aerolysin bound to cells act to form small pores in the membrane and induce cell damage (5, 11). These results suggest that the mode of action of the hemolysin is identical to that of aerolysin. Since cultured intestinal cells are destroyed by incubation with the hemolysin, we had predicted that the diarrhea caused by the hemolysin was a result of such cytotoxic action of the hemolysin. However, micropathological examination showed that the diarrheic syndrome appeared before the cell damage occurred (5). So, we speculated that the diarrhea was not due to the cytotoxic action of the hemolysin but rather that the hemolysin stimulated the intestinal cells to send some signal(s) to induce diarrhea. Thus, we examined the levels of cyclic nucleotides in T84 cells, i.e., human colonic epithelial cells, after incubation with the hemolysin to obtain a clue as to the signal and found that the level of cyclic AMP in the culture medium increased after the incubation (6). However, it has remained unsolved how the hemolysin induces the elevation of the level in the extracellular cyclic AMP.In the meantime, the action of adenosine existing outside of the cell has been studied extensively and its receptor, P1, has been found (4). At present, P1 receptors are divided into three groups, A 1 to A 3 . A 2 is divided into two subtypes: A 2A and A 2B . These P1 receptors couple to adenylate cyclase, whose activity is regulated by G proteins. A 1 and A 3 function to inhibit the cyclase and A 2 , both A 2A and A 2B , functions to activate the cyclase (3, 4). It was also shown that T84 cells, which we used to examine the activity of the hemolysin, possessed the A 2B receptor (14). Thus, we suspected that the adenosine was involved in the action of the hemolysin.Addition of papaverine to the medium. We reported that the level of cyclic AMP in the medium increased on incubation with the hemolysin (6). The excess cyclic AMP produced by the cells is decomposed by phosphodiesterase. Therefore, the amount of cyclic AMP reported in a previous study should be the residual amount after the attack by phosphodiesterase. To exami...

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Section: Fig 2 Effects Of Ada Xacsupporting

confidence: 75%

Studies of the Mechanism of Action of the Aerolysin-Like Hemolysin of Aeromonas sobria in Stimulating T84 Cells To Produce Cyclic AMP

et al. 2003

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“…It has been further reinforced by the finding that aerolysin heptamers formed in DRMs move to a non-DRM fraction upon treatment with saponin, a cholesterol-binding agent (9). The cytotoxicity and heptamerization of aerolysin in T lymphocytes, however, are insensitive to MbCD treatment, thus arguing against the dependence of aerolysin heptamerization on DRMs (33). A plausible explanation for these apparently contradictory observations is that cholesterol depletion affects heptamerization in most cell types but not in T lymphocytes expressing unusually high levels of aerolysin receptors (10).…”

Section: Discussionmentioning

confidence: 89%

Clostridium perfringens ε-Toxin Forms a Heptameric Pore within the Detergent-insoluble Microdomains of Madin-Darby Canine Kidney Cells and Rat Synaptosomes

Miyata

Minami

Tamai

et al. 2002

Journal of Biological Chemistry

130

138

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Clostridium perfringens ⑀-toxin, which is responsible for enterotoxaemia in ungulates, forms a heptamer in rat synaptosomal and Madin-Darby canine kidney (MDCK) cell membranes, leading to membrane permealization. Thus, the toxin may target the detergent-resistant membrane domains (DRMs) of these membranes, in analogy to aerolysin, a heptameric pore-forming toxin that associates with DRMs. To test this idea, we examined the distribution of radiolabeled ⑀-toxin in DRM and detergent-soluble membrane fractions of MDCK cells and rat synaptosomal membranes. When MDCK cells and synaptosomal membranes were incubated with the toxin and then fractionated by cold Triton X-100 extraction and flotation on sucrose gradients, the heptameric toxin was detected almost exclusively in DRMs. The results of a toxin overlay assay revealed that the toxin preferentially bound to and heptamerized in the isolated DRMs. Furthermore, cholesterol depletion by methyl-␤-cyclodextrin abrogated their association and lowered the cytotoxicity of the toxin toward MDCK cells. When ⑀-protoxin, an inactive precursor able to bind to but unable to heptamerize in the membrane, was incubated with MDCK cell membranes, it was detected mainly in their DRMs. These results suggest that the toxin is concentrated and induced to heptamerize on binding to a putative receptor located preferentially in DRMs, with all steps from initial binding through pore formation completed within the same DRMs.

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“…Approximately 90% of the total hRBC cholesterol was removed primarily because erythrocyte cholesterol is almost entirely associated with the plasma membrane of erythrocytes (20).…”

Section: Methodsmentioning

confidence: 99%

“…The effect of cholesterol depletion on the sensitivity of hRBCs to lysis by PFO, SLO, or ILY was initially examined to confirm that the cytolytic mechanism of all three toxins was indeed sensitive to the loss of membrane cholesterol. The hemolytic dose for 50% lysis (HD 50 , see Materials and Methods) was determined for each toxin by using native hRBCs and hRBCs depleted of Ϸ90% of their membrane cholesterol (Table 1) with the cholesterol-sequestering agent M␤CD (20,26).…”

Section: Depletion Of Membrane Cholesterol Abolishes Cdc-dependent Ermentioning

confidence: 99%

Redefining cholesterol's role in the mechanism of the cholesterol-dependent cytolysins

Giddings

Johnson

Tweten

2003

Proc. Natl. Acad. Sci. U.S.A.

176

190

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The cholesterol-dependent cytolysins (CDCs) constitute a large family of pore-forming toxins that function exclusively on cholesterol-containing membranes. A detailed analysis of the various stages in the cytolytic mechanism of three members of the CDC family revealed that significant depletion of cholesterol from the erythrocyte membrane stalls these toxins in the prepore complex. Therefore, the depletion of membrane cholesterol prevents the insertion of the transmembrane ␤-barrel and pore formation. These unprecedented findings provide a paradigm for the involvement of cholesterol in the CDC cytolytic mechanism and that of other pore-forming toxins whose activity is enhanced by the presence of membrane cholesterol.

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Channel Formation by the Glycosylphosphatidylinositol-anchored Protein Binding Toxin Aerolysin Is Not Promoted by Lipid Rafts

Cited by 34 publications

References 35 publications

Studies of the Mechanism of Action of the Aerolysin-Like Hemolysin of Aeromonas sobria in Stimulating T84 Cells To Produce Cyclic AMP

Studies of the Mechanism of Action of the Aerolysin-Like Hemolysin of Aeromonas sobria in Stimulating T84 Cells To Produce Cyclic AMP

Clostridium perfringens ε-Toxin Forms a Heptameric Pore within the Detergent-insoluble Microdomains of Madin-Darby Canine Kidney Cells and Rat Synaptosomes

Redefining cholesterol's role in the mechanism of the cholesterol-dependent cytolysins

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