Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M507690200 on August 30, 2005

J. Biol. Chem., Vol. 280, Issue 44, 36657-36663, November 4, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/44/36657    most recent
M507690200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Valeva, A.
Right arrow Articles by Bhakdi, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Valeva, A.
Right arrow Articles by Bhakdi, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Binding of Escherichia coli Hemolysin and Activation of the Target Cells Is Not Receptor-dependent*

Angela Valeva{ddagger}1, Ivan Walev{ddagger}, Helene Kemmer{ddagger}, Silvia Weis{ddagger}, Isabel Siegel{ddagger}, Fatima Boukhallouk{ddagger}, Trudy M. Wassenaar§, Triantafyllos Chavakis¶2, and Sucharit Bhakdi{ddagger}

From the {ddagger}Institute of Medical Microbiology and Hygiene, University of Mainz, D-55101 Mainz, Germany, §Molecular Microbiology and Genomics Consultants 55576, Zotzenheim, Germany, and Experimental Immunology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, July 15, 2005 , and in revised form, August 30, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Production of a single cysteine substitution mutant, S177C, allowed Escherichia coli hemolysin (HlyA) to be radioactively labeled with tritiated N-ethylmaleimide without affecting biological activity. It thus became possible to study the binding characteristics of HlyA as well as of toxin mutants in which one or both acylation sites were deleted. All toxins bound to erythrocytes and granulocytes in a nonsaturable manner. Only wild-type toxin and the lytic monoacylated mutant stimulated production of superoxide anions in granulocytes. An oxidative burst coincided with elevation of intracellular Ca2+, which was likely because of passive influx of Ca2+ through the toxin pores. Competition experiments showed that binding to the cells was receptor-independent, and preloading of cells with a nonlytic HlyA mutant did not abrogate the respiratory burst provoked by a subsequent application of wild-type HlyA. In contrast to a previous report, expression or activation of the {beta}2 integrin lymphocyte function-associated antigen-1 did not affect binding of HlyA. We conclude that HlyA binds nonspecifically to target cells and a receptor is involved neither in causing hemolysis nor in triggering cellular reactions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hemolysin (HlyA)3 is a major virulence factor of Escherichia coli strains that cause extraintestinal infections. Similar to other members of the RTX family, the toxin binds to target cells and protein-free liposomes and forms transmembrane pores (1, 2). The toxin requires post-translational fatty acylation of two lysine residues (Lys-564 and Lys-690) in order to acquire permeabilizing activity (3). Toxin mutants in which these lysine residues are replaced with arginine are totally nonhemolytic but retain their capacity to bind to erythrocytes (4, 5) and to liposomes (6).

Many attempts have been made to delineate the mode of binding of HlyA to membranes, but the results have proven difficult to accommodate in a single model. Artificial membranes are efficiently permeabilized (7, 8), and initial binding studies indicated that erythrocytes (9) and granulocytes (10) bound the toxin in a nonsaturable manner. In contrast, binding of HlyA to erythrocytes in a saturable manner was reported (11). Subsequently, the lymphocyte function-associated antigen (LFA-1) (CD11a/CD18; {alpha}L{beta}2 integrin), was reported to serve as the receptor for HlyA and Actinobacillus actinomycetemcomitans leukotoxin on polymorphonuclear neutrophils (PMNs) (12). This conclusion was based on the following observations: use of monoclonal antibodies to LFA-1 inhibited binding of HlyA and leukotoxin to cells; immobilized leukotoxin bound LFA-1; expression of LFA-1 in a cell line that normally lacked the integrin and was insensitive to the toxins rendered these cells sensitive to HlyA and leukotoxin. The authors suggested that nonspecific absorption of HlyA to various cell types might additionally occur that could obscure the receptor-mediate interaction (12). Studies with another RTX toxin, the leukotoxin of Mannheimia hemolytica, indicated that binding to integrin on bovine leukocytes results in activation of the tyrosine kinase signaling cascade (13). Binding of collagen to LFA-1 activates this signaling cascade and triggers the respiratory burst in human neutrophils (14). Because HlyA also stimulates the respiratory burst in these cells (15–17), a unifying concept would be that E. coli hemolysin binds to integrin LFA-1, activating the tyrosine kinase signaling cascade and triggering the respiratory burst. Binding to erythrocytes must occur via other mechanisms, however, because these cells do not express LFA-1. According to one report, glycophorin might serve as the receptor for HlyA in these cells (18). Such a concept contradicts the nonsaturable binding previously reported.

All previous investigations on the binding of RTX toxins to cells suffered from the drawback that radioactive toxin tracers were not available for quantitative measurements of binding. Several studies employed crude culture supernatants or cell sonicates rather than purified toxin preparations, and quantification of binding inevitably relied on indirect methods. In this study, we devised a method for radioactive labeling of HlyA and conducted experiments to test the hypothesis that the toxin binds to a receptor. Our results indicate that HlyA does not interact with a receptor on granulocytes. Binding occurs in a nonspecific and nonsaturable manner, and the respiratory burst is triggered directly by pore formation, probably because of flux of extracellular Ca2+ into the cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Expression of hlyA, Mutagenesis, and Toxin Purification—Mutants in which Lys at position 564 and/or 690 was replaced by Arg (3) were kindly provided by Dr. C. Hughes. Mutation of Ser-177 into Cys was described previously (19). The same procedure was followed starting with K564R, K690R and K564R/K690R, to form S177C/K564R, S177C/K690R, and S177C/K564R/K690R, respectively. Protein purification of hemolysin was carried out as described previously (4, 19).

Labeling of Toxin—Mutant toxin containing Cys-177 was labeled with N-ethylmaleimide (NEM) using the following protocol. Alcohol precipitation of toxin was carried out to remove dithiothreitol as described previously (19). The precipitate was dissolved in 8 M guanidine HCl, pH 8.0, and 2 µM toxin was incubated with 50 µM NEM for 1 h at ambient temperature. The reaction was stopped with 5 mM dithiothreitol (10 min), and toxin was alcohol-precipitated. After centrifugation and washing, the labeled toxin was dissolved in 8 M guanidine HCl, 10 mM HEPES, pH 7.5. Before use, toxin was diluted 100-fold in Hanks' balanced salt solution (HBSS) supplemented with 20 mM HEPES, pH 7.5.



View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 1.
Binding of radioactive HlyA to granulocytes. A, increasing numbers of PMN were incubated with 20 pM [3H]HlyA (WT*) for 60 min, and bound radioactivity was determined. The results of triplicate experiments are shown. B, comparison of the binding of 20 pM radioactive HlyA with binding of semi- or nonacylated mutants to 15 x 106 granulocytes. Binding is expressed as a percentage of added labeled toxin. The binding of WT* and monoacylated (K564R*, K690R*) or nonacylated (K564R/K690R*) mutants was comparable.

 
For radioactive labeling with [3H]NEM, 500 nM toxin was incubated with 2 µM [3H]NEM (specific activity, 48.2Ci/mmol, 1 mCi/ml) as described above. After incubation for 1 h, the reaction was completed with excess (50 µM) NEM and toxin was further treated as above.

The homogeneity of the labeled toxins was analyzed by SDS-PAGE. 20 ng of labeled HlyA toxin were mixed with 2 µg of unlabeled toxin, and this mixture was run in two parallel SDS gels. One was stained with Coomassie, and the other cut into thin slices that were homogenized and assayed for radioactivity. Coomassie staining confirmed purity of the toxin bands, and 85% of radioactivity was recovered in the gel slice containing toxin (data not shown).

Cell Assays—Human granulocytes were isolated from heparinized blood of healthy volunteers following conventional procedures. Briefly, 1 volume of 4.5% dextran (Amersham Biosciences) in isotonic salt solution, pH 7.4, was added to 5 volumes of whole blood, and cells were allowed to sediment in tilted plastic centrifugation tubes for 30 min at room temperature. The erythrocyte-depleted supernatants were transferred in 4-ml aliquots to 4 ml of Biocoll Separation Solution gradients (Biochrom AG, Berlin, Germany) and centrifuged for 20 min at 400 x g at 20 °C. Erythrocytes contaminating the cell pellet were lysed in buffer containing 150 mM NH4Cl, 10 mM KHCO3, 10 mM EDTA, pH 7.4. Cells were then resedimented (100 x g, 10 min), washed, suspended in sterile HBSS, and kept on ice. The cell preparations contained ≤3% contaminating lymphocytes as determined with methylene blue staining and ≤4% nonviable cells as determined by staining with trypan blue.



View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 2.
Competition experiments fail to reveal existence of toxin receptors on granulocyte and erythrocyte membranes. [3H]HlyA (20 pM) was incubated with PMN or erythrocyte ghosts at the given temperatures in the presence of unlabeled wild-type toxin (+wt). Bound radiolabeled HlyA was determined after 60 min. A, binding of radiolabeled HlyA to PMN at 4 °C in the presence of unlabeled HlyA. B, binding of radiolabeled HlyA to erythrocyte ghosts in the presence of unlabeled HlyA at 37 °C. C, binding of radiolabeled HlyA to PMN in the presence of unlabeled K690R at 37 °C. Depicted are the results of four experiments ± S.D. in each case.

 
K562 cells, either nontransfected or transfected with LFA-1, were kindly provided by Dr. Y. van Kooyk (Amsterdam, The Netherlands) and cultivated in a mixture of 75% RPMI, 25% Iscov's modified Dulbecco's medium with 10% fetal calf serum. Erythrocyte ghosts were prepared by hypotonic lysis of rabbit erythrocytes in 50 mM phosphate buffer, pH 8, followed by three washes in the same buffer.

Monoclonal antibody KIM-127 (anti-CD18) was a gift from Dr. M. Robinson (Celltech Ltd.). Anti-CD11a antibodies used were TS1/22 (Perbio Science), MCA1848GA (Serotec GmbH, Düsseldorf, Germany), and G43-25B (BD Biosciences). When indicated, cells were preincubated with 1 mM MnCl2 in the presence or absence of 10 µg/ml Kim-127 antibodies prior to HlyA binding.

Toxins used for comparison of cytolytic activity were streptolysin O (20), Staphylococcus aureus {alpha}-toxin (21), and Vibrio cholerae cytolysin (22).

Hemolytic Assays—Serial dilutions of hemolysin were prepared in duplicate with HBBS. To 100 µl of diluted toxin, 100 µl of rabbit erythrocytes were added (2.5 x 108 cells/ml) followed by incubation at 37 °C for 1 h. The absorbance of hemoglobin in supernatants was measured at 412 nm. The HD50 was defined as the concentration of toxin required to lyse 50% of the rabbit erythrocytes. The HD50 of mutant S177C was 30 ng/ml (300 pM), which was comparable with wild-type hemolysin.

Measurement of Intracellular ATP—K562 cells (transfected or nontransfected) were treated with various concentrations of streptolysin O, {alpha}-toxin from S. aureus, V. cholerae cytolysin, or HlyA for 60 min. Cellular ATP concentrations were determined after lysing the cells with 0.5% (v/v) Triton X-100 using a commercial ATP-bioluminescence kit (Roche Applied Science).

Binding Assays—Labeled toxin (20 pM) was added to granulocytes (2 x 107 cells or as indicated), K562 cells (4 x 106 cells), or erythrocyte ghosts (109 cells) in 500 µl of HBSS, and binding was allowed to take place at 37 °C for 1 h. Cells were centrifuged (3000 x g, 10 min) and washed twice, and bound toxin was expressed as the percentage of total toxin in the assay.

Chemiluminescence Measurements—Production of reactive oxygen species was assayed by the luminol-amplified chemiluminescence method as described previously (23). Briefly, 106 granulocytes in HBSS, 20 mM HEPES, pH 7.2, were treated with HlyA or mutant K690R in the presence of 0.2 mM luminol. Chemiluminescence was measured in a luminometer and expressed as relative light units.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 3.
Absence of an effect of LFA-1 in binding of radiolabeled HlyA to K562 cells. A, the fraction of bound toxin to K562 cells. B, cells expressing LFA-1. C, as in B, preincubation with Mn2+. D, as in B, in the presence of the activating antibody Kim-127. E, as in B, in the presence of Mn2+ and Kim-127.

 
Measurement of Intracellular Calcium—Granulocytes were loaded with 5 µM Fura-2 AM (Molecular Probes, Leiden, The Netherlands) in HBSS, 20 mM HEPES, pH 7.2, for 30 min at 37 °C. After granulocytes were washed, toxin was added at the indicated concentrations for 10 min at 37 °C. Fura-2 absorption shift was observed by scanning the excitation spectrum between 300 and 400 nm at an emission wavelength of 510 nm in a Spex-Fluoromax spectrofluorimeter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hemolytic Activities of HlyA and Mutant Toxins and Radioactive Labeling with [3H]NEM—Replacement of Ser-177 with cysteine in HlyA did not result in a loss of hemolytic activity; 300 pM S177C caused hemolysis of ~60% of a rabbit erythrocyte suspension containing 2.5 x 108 cells/ml. Mutant S177C/K564R, lacking the acylation site at position 564, had an ~10-fold reduced activity (HD50 ~ 3 nM). Hemolytically inactive mutants (HD50 > 30 nM) were S177C/K690R, lacking the fatty acid at position 690, and S177C/K564R/K690R, the nonacylated toxin.

Modification of S177C with NEM did not alter the hemolytic activity of the toxin mutants S177C and S177C/K546R. Labeling of S177C (hereafter designated WT*) resulted in a tracer with a specific radioactivity of 40 mCi/mmol, corresponding to a mean incorporation of ~0.8 mol of 3H/mol of HlyA. The other mutants, S177C/K564R, S177C/K690R, and S177C/K564R/K690R, were also labeled and designated K564R*, K690R*, and K564R/K690R*, respectively. Their specific radioactivity was comparable with that of WT*. No incorporation was obtained with cysteine-free toxins.

Binding Characteristics of Radioactive Labeled Toxins—The fraction of bound WT* was determined with increasing numbers of PMNs. Fig. 1A shows that the bindability of labeled toxin was ~40%. Similar results were obtained with erythrocyte ghosts (data not shown). When 20 pM toxin was incubated with 15 x 106 PMNs, about 15–20% of the radioactivity became cell-bound. No decrease in cellular ATP was observed, indicating that this concentration window was sublytic. Similar binding was observed with mutants that were monoacylated (K564R* and K690R*) or nonacylated (K564R/K690R*) (Fig. 1B). Thus, the lack of acylation did not affect the binding of HlyA to granulocytes.

To investigate whether binding was reversible, PMNs were loaded with 20 pM radiolabeled toxin at 37 or 4 °C. After 1 h, unbound toxin was removed by washing. The cells were then incubated with fresh medium for 1 h, and release of toxin was measured in the medium. Radioactive toxin could not be demonstrated in the medium in any case (not shown), indicating that binding was irreversible for all analyzed mutants.

Competition experiments were performed in which radioactively labeled WT* (20 pM) was incubated with granulocytes in the presence of increasing amounts of unlabeled wild-type toxin. These experiments were conducted at 4 °C, because at 37 °C the cells became permeabilized by the unlabeled HlyA and cell swelling caused unspecific increase of bound label. At low temperature, no change in the amount of bound label was found (Fig. 2A). The same findings were obtained at 37 °C using erythrocyte ghosts (Fig. 2B). In another experiment, granulocytes were incubated with 20 pM WT* at 37 °C in the presence of increasing concentrations of the nonlytic mutant K690R (Fig. 2C). Again, no competition of binding was noted despite the high concentrations of unlabeled toxin used.

Role of Integrin in Mediating HlyA Activity—The above results indicated that HlyA bound in a nonspecific manner to cells. However, specific binding of HlyA to LFA-1 integrin has been proposed; therefore PMNs were preincubated with anti-integrin antibodies TS1/22, MCA1848GA, and G43-25B, and binding experiments were repeated. TS1/22 is known to be function-blocking, i.e. to block ligand binding to LFA-1 (24). The antibodies were applied at a final concentration of 10 µg/ml, and under these conditions neither a difference in binding of WT* compared with nontreated cells nor a difference in susceptibility of the cells toward the toxin was observed (data not shown).

Expression of LFA-1 in K562 cells reportedly increases their susceptibility toward HlyA, and this has been taken as another argument for a receptor function (12). Binding experiments were therefore undertaken with K562 cells lacking or expressing LFA-1. Expression of the integrin was confirmed, by flow cytometry and Western blotting, in transfected cells using specific antibodies. Expression of LFA-1 did not significantly increase binding of 20 pM WT* (Fig. 3). Activation of LFA-1 with either manganese ions, stimulating anti-CD18 antibody Kim-127 (25), or a combination of both agonists did not affect HlyA binding to LFA-1-transfected K562 cells, indicating that LFA-1 is not a receptor for HlyA.

The above findings might be reconciled with the report that LFA-1 expression enhances the cytotoxicity of HlyA (12), if LFA-1 expression generally rendered cells more sensitive to pore-forming toxins. Indeed, ATP assays indicated enhanced sensitivity of LFA-1 positive cells toward other toxins. Similar enhancement of susceptibility was noted with streptolysin O, which binds to cholesterol, S. aureus {alpha}-toxin, and V. cholerae cytolysin, for which no evidence exists for an interaction with integrins (TABLE ONE).


View this table:
[in this window]
[in a new window]
  		TABLE ONE
Concentrations of pore-forming toxins eliciting 50% ATP depletion in K562 cells

 
Superoxide Generation by Granulocytes Induced by HlyA—Wild-type HlyA in a concentration range of 0.5 to 200 pM was applied to PMNs and production of reactive oxygen species was determined using the luminol assay. As shown in Fig. 4A, an oxidative burst was induced by HlyA in a narrow concentration range of 5 to 100 pM, with a maximum at 20 pM. At higher toxin concentrations, ATP decrease was apparent, but no oxidative burst could be observed. This accorded with the early report showing that at high concentration cells were killed before a burst could be generated (10).



View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 4.
Production of reactive oxygen species in granulocytes triggered by HlyA. A, cells were incubated with wild-type HlyA at the depicted concentrations and luminol-based chemiluminescence was recorded over time (RLU, relative light units). Maximal production of reactive oxygen species occurred at a concentration of 20 pM HlyA. B, cells were preloaded with nonlytic mutant K690R at 2000 pM, which did not elicit oxidative burst. Subsequent stimulation with 20 pM wild-type HlyA (wt) resulted in an unaltered cellular response. C, measurements of intracellular Ca2+. Cells were loaded with Fura-2 and exposed to wild-type HlyA at the given concentrations. Increases in intracellular Ca2+ were detected by measuring the excitation spectrum at emission of 510 nm. Note the excitation spectrum shifts upon toxin treatment compared with untreated cells (control). D, intracellular Ca2+ did not change when cells were treated with mutant K690R at excess concentration. Measurements of intracellular Ca2+ for untreated cells (control) and HlyA-treated cells are displayed for comparison.

 
When cells were incubated with the nonlytic mutant K690R, no oxidative burst was observed at any toxin concentration from 1 to 2000 pM (Fig. 4B). In a further experiment, cells were preincubated with 2000 pM K690R followed by 20 pM wild-type HlyA. Despite irreversible binding of inactive toxin at this high concentration, the cells responded in a similar manner to stimulation by wild-type toxin (Fig. 4B).

These findings led us to suspect that membrane permeabilization was directly responsible for triggering the oxidative burst, possibly because of Ca2+ influx into the cell. Indeed, Ca2+ influx was found to occur already at a toxin concentration of 10 pM (Fig. 4C). In contrast, no Ca2+ influx was seen with the nonlytic mutant K690R at an excess concentration of 400 pM (Fig. 4D).

In a final experiment, cells were incubated with HlyA on ice in Ca2+-containing buffer for 15 min. This allowed the toxin to bind in the absence of pore formation. Following two washes in ice-cold buffer without Ca2+, cells were resuspended in buffer with or without Ca2+ at 37 °C. As shown in Fig. 5A, superoxide production was observed only in cells that had been resuspended in Ca2+-containing buffer, although pore formation and ATP-depletion occurred to the same extent in cells suspended in Ca2+-free medium (Fig. 5B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The mechanism of binding of RTX toxins to cells has remained an enigmatic and controversial issue to the present day. Because HlyA can efficiently permeabilize artificial protein- and glycolipid-free membranes, it is evident that a receptor need not exist (7, 8). However, low concentrations of HlyA and other RTX toxins trigger cellular events that are classically receptor-mediated, and there have been indications for a dual mechanism of binding of HlyA to nucleated cells; receptor-mediated binding has been proposed to occur at low toxin concentrations and may be responsible for triggering cellular reactions, whereas nonspecific binding of the toxin to membrane lipids may be the primary event leading to pore formation and cell death (13, 15).

A dilemma faced by all workers in the field has been the lack of a sensitive method to quantify toxin binding. It has not been possible to produce functionally active, radioiodinated tracers,4 and all previous investigations therefore had to employ indirect and insensitive assays for binding. These included immunological methods (12), utilization of fluorescence-labeled toxins (26), or just the assessment of cellular cytotoxicity. In general, toxin preparations of undefined purity or even crude culture supernatants or cell lysates were employed. Taken together, these drawbacks have led to some uncertainties regarding the validity of conclusions drawn in previous studies.



View larger version (11K):
[in this window]
[in a new window]
  		FIGURE 5.
Calcium influx is required for production of reactive oxygen species in HlyA-treated cells. A, chemiluminescent assays for reactive oxygen species in cells treated with 20 pM HlyA in the presence or absence of extracellular Ca2+. RLU, relative light units. B, ATP depletion provoked by cell-bound HlyA is not dependent on Ca2+ influx. Cells loaded with HlyA at 0 °C in the presence or absence of extracellular Ca2+ were washed and resuspended in buffer ± Ca2+. Pore formation resulted in both cases leading to ATP depletion.

 

We approached the dilemma by producing the single cysteine substitution mutant S177C, which was found to retain biological activity and to tolerate thiol-specific derivatization with NEM. Using labeled toxin we found that, independently of fatty acylation, HlyA bound to cells with similar efficacy. Our results were in basic accord with earlier data that had shown comparable binding of nonacylated HlyA to liposomes when the toxin was applied at high concentrations (6). This conclusion was now shown to be valid also at low toxin concentrations, and the inability of excess unlabeled toxin to reduce binding of radioactive nonlytic toxin mutants essentially eliminated the possible existence of a receptor.

Expression of LFA-1 had been reported to render cells more sensitive toward the action of HlyA and leukotoxin (12), and we confirmed this finding. However, transfected cells expressing integrin bound HlyA similarly to cells not expressing the integrin. Furthermore, blocking integrins with antibodies did not prevent HlyA binding. Finally, cytotoxicity assays performed with three other pore-forming toxins (unrelated to HlyA and not belonging to the RTX toxin family) indicated that the enhancement in susceptibility of the cells was not specific for HlyA. Together, these findings essentially ruled out a receptor role for LFA-1 in binding of HlyA. The increased sensitivity of LFA-1-expressing cells may be the result of the altered physical properties of the cellular membranes, in particular of the structure of membrane microdomains such as lipid rafts, as LFA-1 may be targeted to such membrane microdomains and affect their structure (27). In fact, after {beta}-methyl-cyclodextrin pretreatment, the susceptibility of K562 cells and the LFA-1-transfected K562 cells to lysis by HlyA was similar (data not shown), indicating that altered membrane structure in the presence of LFA-1 rather than a direct interaction of the toxin with LFA-1 may account for the observed difference in cell lysis by HlyA.

Thus, our data speak against the existence of an HlyA receptor on PMNs and indicate that the toxin binds nonspecifically to lipid bilayers. How irreversible binding of HlyA should occur is unclear, but one possibility is that the toxin domain near the N terminus that harbors anphipathic {alpha}-helices spontaneously inserts into the membranes. This could initially remain without consequence to bilayer integrity, poreformation requiring the additional presence of fatty acids, which might insert or cause insertion of other amino acid sequences into the bilayer. Fatty acylation at position 690 is thereby absolutely essential, whereas lack of fatty acylation at position 564 leads only to reduction of poreforming activity (3). Of distinct interest is the fact that Bordetella pertussis adenylate cyclase, another member of the RTX toxins, is monoacylated at the position corresponding to Lys-690 in HlyA (28).

To summarize, all of our findings indicate that HlyA binds nonspecifically to lipid bilayers and that cellular reactions are triggered by uncontrolled fluxes of ions, particularly Ca2+, through the pores. The reactions are observed only in a narrow range of toxin concentrations, because higher doses will rapidly kill the cells. It is not excluded that other members of the RTX toxin family may bind to receptors; however, this possibility remains to be stringently examined, and the approach described here may prove useful for future studies.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft 490, Project C1/D3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

2 Supported by the Intramural Research Program, NCI, National Institutes of Health. Back

1 To whom correspondence should be addressed: Inst. of Medical Microbiology and Hygiene, University of Mainz, Hochhaus am Augustusplatz, D-55101 Mainz, Germany. Tel.: 49-6131-3936363; Fax: 49-6131-3932359; E-mail: avaleva{at}uni-mainz.de.

3 The abbreviations used are: HlyA, hemolysin; RTX, repeat in toxin; LFA, lymphocyte function-associated antigen; PMN, polymorphonuclear neutrophil; NEM, N-ethylmaleimide; HBSS, Hanks' balanced salt solution; WT*, tritiated wild-type S177C. Back

4 A. Valeva, I. Walev, and S. Bhakdi, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Colin Hughes and Vassilis Koronakis for providing the plasmids carrying HlyA and Lys replacement mutants and Falk Fahrenholz for useful suggestions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bakas, L., Ostolaza, H., Vaz, W. L., and Goni, F. M. (1996) Biophys. J. 71, 1869–1876[Medline] [Order article via Infotrieve]
  2. Welch, R. A. (2001) Curr. Top. Microbiol. Immunol. 257, 85–111[Medline] [Order article via Infotrieve]
  3. Stanley, P., Packman, L. C., Koronakis, V., and Hughes, C. (1994) Science 266, 1992–1996[Abstract/Free Full Text]
  4. Stanley, P., Koronakis, V., and Hughes, C. (1998) Microbiol. Mol. Biol. Rev. 62, 309–333[Abstract/Free Full Text]
  5. Soloaga, A., Ostolaza, H., Goni, F. M., and de la Cruz, F. (1996) Eur. J. Biochem. 238, 418–422[Medline] [Order article via Infotrieve]
  6. Hyland, C., Vuillard, L., Hughes, C., and Koronakis, V. (2001) J. Bacteriol. 183, 5364–5370[Abstract/Free Full Text]
  7. Menestrina, G., Mackman, N., Holland, I. B., and Bhakdi, S. (1987) Biochim. Biophys. Acta 905, 109–117[Medline] [Order article via Infotrieve]
  8. Benz, R., Schmid, A., Wagner, W., and Goebel, W. (1989) Infect. Immun. 57, 887–895[Abstract/Free Full Text]
  9. Eberspacher, B., Hugo, F., and Bhakdi, S. (1989) Infect. Immun. 57, 983–988[Abstract/Free Full Text]
  10. Bhakdi, S., Greulich, S., Muhly, M., Eberspacher, B., Becker, H., Thiele, A., and Hugo, F. (1989) J. Exp. Med. 169, 737–754[Abstract/Free Full Text]
  11. Bauer, M. E., and Welch, R. A. (1996) Infect. Immun. 64, 4665–4672[Abstract]
  12. Lally, E. T., Kieba, I. R., Sato, A., Green, C. L., Rosenbloom, J., Korostoff, J., Wang, J. F., Shenker, B. J., Ortlepp, S., Robinson, M. K., and Billings, P. C. (1997) J. Biol. Chem. 272, 30463–30469[Abstract/Free Full Text]
  13. Jeyaseelan, S., Kannan, M. S., Briggs, R. E., Thumbikat, P., and Maheswaran, S. K. (2001) Infect. Immun. 69, 6131–6139[Abstract/Free Full Text]
  14. Garnotel, R., Monboisse, J. C., Randoux, A., Haye, B., and Borel, J. P. (1995) J. Biol. Chem. 270, 27495–27503[Abstract/Free Full Text]
  15. Bhakdi, S., and Martin, E. (1991) Infect. Immun. 59, 2955–2962[Abstract/Free Full Text]
  16. Grimminger, F., Scholz, C., Bhakdi, S., and Seeger, W. (1991) J. Biol. Chem. 266, 14262–14269[Abstract/Free Full Text]
  17. Grimminger, F., Sibelius, U., Bhakdi, S., Suttorp, N., and Seeger, W. (1991) J. Clin. Investig. 88, 1531–1539
  18. Cortajarena, A. L., Goni, F. M., and Ostolaza, H. (2001) J. Biol. Chem. 276, 12513–12519[Abstract/Free Full Text]
  19. Schindel, C., Zitzer, A., Schulte, B., Gerhards, A., Stanley, P., Hughes, C., Koronakis, V., Bhakdi, S., and Palmer, M. (2001) Eur. J. Biochem. 268, 800–808[Medline] [Order article via Infotrieve]
  20. Weller, U., Muller, L., Messner, M., Palmer, M., Valeva, A., Tranum-Jensen, J., Agrawal, P., Biermann, C., Dobereiner, A., Kehoe, M. A., and Bhakdi, S. (1996) Eur. J. Biochem. 236, 34–39[Medline] [Order article via Infotrieve]
  21. Bhakdi, S., Muhly, M., Korom, S., and Hugo, F. (1989) Infect. Immun. 57, 3512–3519[Abstract/Free Full Text]
  22. Valeva, A., Walev, I., Weis, S., Boukhallouk, F., Wassenaar, T. M., Endres, K., Fahrenholz, F., Bhakdi, S., and Zitzer, A. (2004) J. Biol. Chem. 279, 25143–25148[Abstract/Free Full Text]
  23. Dahlgren, C., and Karlsson A. (1999) J. Immunol. Methods 232, 3–14[CrossRef][Medline] [Order article via Infotrieve]
  24. Van Epps, D. E., Potter, J., Vachula, M., Smith, C. W., and Anderson, D. C. (1989) J. Immunol. 143, 3207–3210[Abstract]
  25. Stephens, P., Romer, J. T., Spitali, M., Shock, A., Ortlepp, S., Figdor, C. G., and Robinson, M. K. (1995) Cell Adhes. Commun. 3, 375–384[Medline] [Order article via Infotrieve]
  26. Herlax, V., and Bakas, L. (2003) Chem. Phys. Lipids 122, 185–190[CrossRef][Medline] [Order article via Infotrieve]
  27. Krauss, K., and Altevogt, P. (1999) J. Biol. Chem. 274, 36921–36927[Abstract/Free Full Text]
  28. Basar, T., Havlicek, V., Bezouskova, S., Hackett, M., and Sebo, P. (2001) J. Biol. Chem. 276, 348–354[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Skals, N. R. Jorgensen, J. Leipziger, and H. A. Praetorius
{alpha}-Hemolysin from Escherichia coli uses endogenous amplification through P2X receptor activation to induce hemolysis
PNAS, March 10, 2009; 106(10): 4030 - 4035.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Kramer, G. Sellge, A. Lorentz, D. Krueger, M. Schemann, K. Feilhauer, F. Gunzer, and S. C. Bischoff
Selective Activation of Human Intestinal Mast Cells by Escherichia coli Hemolysin
J. Immunol., July 15, 2008; 181(2): 1438 - 1445.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
J. Morova, R. Osicka, J. Masin, and P. Sebo
RTX cytotoxins recognize {beta}2 integrin receptors through N-linked oligosaccharides
PNAS, April 8, 2008; 105(14): 5355 - 5360.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
D. N. Atapattu and C. J. Czuprynski
Mannheimia haemolytica Leukotoxin Binds to Lipid Rafts in Bovine Lymphoblastoid Cells and Is Internalized in a Dynamin-2- and Clathrin-Dependent Manner
Infect. Immun., October 1, 2007; 75(10): 4719 - 4727.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Sanchez-Magraner, A. R. Viguera, M. Garcia-Pacios, M. P. Garcillan, J.-L. R. Arrondo, F. de la Cruz, F. M. Goni, and H. Ostolaza
The Calcium-binding C-terminal Domain of Escherichia coli {alpha}-Hemolysin Is a Major Determinant in the Surface-active Properties of the Protein
J. Biol. Chem., April 20, 2007; 282(16): 11827 - 11835.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. Fiser, J. Masin, M. Basler, J. Krusek, V. Spulakova, I. Konopasek, and P. Sebo
Third Activity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin: MEMBRANE TRANSLOCATION OF AC DOMAIN POLYPEPTIDE PROMOTES CALCIUM INFLUX INTO CD11b+ MONOCYTES INDEPENDENTLY OF THE CATALYTIC AND HEMOLYTIC ACTIVITIES
J. Biol. Chem., February 2, 2007; 282(5): 2808 - 2820.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/44/36657    most recent
M507690200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Valeva, A.
Right arrow Articles by Bhakdi, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Valeva, A.
Right arrow Articles by Bhakdi, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement