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Keywords:

  • clathrin;
  • endocytosis;
  • Shiga toxin

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Shiga toxin can be internalized by clathrin-dependent endocytosis in different cell lines, although it binds specifically to the glycosphingolipid Gb3. It has been demonstrated previously that the toxin can induce recruitment of the toxin–receptor complex to clathrin-coated pits, but whether this process is concentration-dependent or which part of the toxin molecule is involved in this process, have so far been unresolved issues. In this article, we show that the rate of Shiga toxin uptake is dependent on the toxin concentration in several cell lines [HEp-2, HeLa, Vero and baby hamster kidney (BHK)], and that the increased rate observed at higher concentrations is strictly dependent on the presence of the A-subunit of cell surface-bound toxin. Surface-bound B-subunit has no stimulatory effect. Furthermore, this increase in toxin endocytosis is dependent on functional clathrin, as it did not occur in BHK cells after induction of antisense to clathrin heavy chain, thereby blocking clathrin-dependent endocytosis. By immunofluorescence, we show that there is an increased colocalization between Alexa-labeled Shiga toxin and Cy5-labeled transferrin in HeLa cells upon addition of unlabeled toxin. In conclusion, the data indicate that the Shiga toxin A-subunit of cell surface-bound toxin stimulates clathrin-dependent uptake of the toxin. Possible explanations for this phenomenon are discussed.

Abbreviations
BHK

baby hamster kidney

CHC

clathrin heavy chain

CT

cholera toxin

MESNa

mercaptoethanesulfonic acid

STEC

Shiga toxin producing Escherichia coli

Stx

Shiga toxin

Stx1

Shiga toxin 1

Stx2

Shiga toxin 2

StxB

Shiga toxin B subunit

The Shiga toxins consist of one enzymatically active A-subunit noncovalently linked to a stable pentamer of binding subunits (StxB), which in most cases bind specifically to the glycosphingolipid Gb3 in the plasma membrane [1]. To exert its toxic effect, the holotoxin must be endocytosed and retrogradely transported via the Golgi apparatus to the endoplasmic reticulum, where the A-subunit is translocated to the cytosol and inhibits protein synthesis [2,3]. The Shiga family of toxins is divided into two main groups, based on antigenic differences. Shiga toxin (Stx) produced by Shigella dysenteriae and Shiga toxin 1 (Stx1) secreted by certain strains of Eshcerichia coli (Shiga toxin producing E. coli; STEC) are virtually identical and differ only in one amino acid. The toxins in the Shiga toxin 2 family (Stx2, Stx2c, Stx2d and Stx2e) are also secreted by STEC and have a similar structure to the toxins in the first group, but differ both functionally and immunologically [1,4,5]. To understand the differences between the effects of the various Stx, it is important to clarify the uptake mechanisms for these toxins.

It has been shown that Stx is internalized via clathrin-dependent endocytosis in several cell types, although there is evidence that also clathrin-independent mechanisms are partly involved [2,6–8]. Importantly, several studies have been concerned with the uptake and intracellular transport of the Stx B-subunit [8–21] but so far, investigations of a possible role of the A-subunit for uptake and intracellular routing of the toxin have been few. By electron microscopy studies, intact Stx has been shown to preferentially localize to clathrin-coated pits in HeLa cells [22,23], and both acidification and potassium depletion of cytosol, which inhibit clathrin-dependent endocytosis, have been shown to protect the cells against Stx [22–24]. Furthermore, induced expression of antisense to clathrin heavy chain (CHC) in a baby hamster kidney (BHK) cell line [25–27], thereby blocking the clathrin-dependent endocytosis, also protects against Stx [2] and reduces the endocytic uptake of the toxin [8]. By using this BHK cell line, we could quantify the initial Stx internalization, both when clathrin-dependent endocytosis was operating and when it was blocked by antisense expression. Toxin uptake was decreased by about 50% upon inhibition of clathrin-dependent endocytosis, but it should be noted that a block in clathrin-dependent endocytosis might lead to increased toxin uptake via other mechanisms.

Importantly, Stx seems to be able to induce its own entry from clathrin-coated pits. Electron microscopy studies revealed that bound Stx is evenly distributed on the cell surface at low temperature, while after shifting to 37 °C, the toxin is aggregated in clathrin-coated pits [22,23]. There is no obvious explanation as to how the Stx–Gb3 complex is recruited to clathrin-coated pits, given that the receptor is a glycosphingolipid and not a protein with specific sequences required for interaction with the sorting machinery. One possible explanation is that the Stx–Gb3 complex interacts with another protein that is internalized via clathrin-dependent endocytosis. In fact, it has been shown by crosslinking experiments that members of the Shiga family of toxins can interact with 27 and 40 kDa molecules at the cell surface of Vero and CaCo2 cells [28], suggesting that interaction with accessory proteins might facilitate clathrin-mediated uptake. However, other possible explanations do exist. The toxin could induce toxin-specific signaling leading to recruitment of clathrin. Furthermore, crosslinking of Gb3 and perhaps of lipid rafts might be important for this process. Interesting in this connection, is the finding that the epidermal growth factor receptor present in lipid rafts recruits clathrin to the membrane upon binding of epidermal growth factor [29].

We here demonstrate that the rate of Stx uptake is up-regulated by increasing toxin concentrations by a mechanism in which the A-subunit of the surface-bound toxin is required. Importantly, this increased endocytosis is clathrin-dependent, and it seems to be caused by an increased recruitment of the toxin–receptor complex to clathrin-coated pits.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Stx stimulates its own rapid entry in several cell lines

We have shown previously that Stx, by a so far unknown mechanism, is able to mediate redistribution of the Stx–Gb3 complex to clathrin-coated pits, before being internalized [22]. In order to investigate whether Stx internalization is dependent on the amount of toxin that is bound to the membrane, different cell types were incubated with increasing concentrations of unlabeled Stx or Stx1 (0–20 nm) in addition to a constant concentration of TAG- and biotin-labeled Stx (0.33 nm), before the amount of internalized Stx was measured and compared to untreated control (no unlabeled Stx added). As shown in Fig. 1A, treating cells with increasing concentrations of unlabeled Stx1 increased the fraction of internalized TAG-labeled Stx in nearly all the cell lines tested (BHK, Vero, HEp-2 and HeLa) by a factor of ≈ 2 during a 10 min incubation at 37 °C. Thus, Stx seemed to have a stimulatory effect on its own uptake in these cells. In one cell line tested, A431, only a slight stimulatory effect was observed.

image

Figure 1. Endocytosis of Stx at increasing toxin concentrations. (A) Stimulation of Stx uptake in different cell lines. The cells were preincubated with increasing concentrations of unlabeled Stx1 (0–20 nm) in addition to a constant concentration of TAG- and biotin-labeled Stx (0.33 nm) in Hepes buffered MEM at 0 °C for 30 min. Then the cells were washed, and the incubation continued for 10 min at 37 °C, except for BHK cells, which were incubated for 20 min. Bound and endocytosed toxin were quantified as described in Experimental procedures. The endocytic values obtained upon Stx stimulation were compared to control values (no unlabeled Stx1 added) in one representative experiment from each cell line (mean values ± deviations between duplicates). The experiment was repeated separately 3–6 times for every cell line. (B) Uptake of Stx with or without unbound Stx1 in solution. HEp-2 cells were preincubated with TAG- and biotin-labeled Stx (0.33 nm) in addition to increasing concentrations of Stx1 (0–20 nm) for 30 min at 0 °C. Then the medium was collected and warmed, and the cells were washed, before endocytosis was measured at 37 °C for 10 min either in the presence of the warmed preincubation medium (i.e., with unbound toxin present) or with warmed toxin-free medium. The endocytic values are compared to untreated control (no unlabeled Stx1 added at 0 °C) (mean values ± deviations between two independent experiments, each done in duplicate).

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To investigate whether this stimulatory effect was affected by unbound toxin in the medium, increasing concentrations of Stx1 (0–20 nm) and a constant concentration of TAG- and biotin-labeled Stx (0.33 nm) were prebound to HEp-2 cells for 30 min on ice. Then the medium was collected and warmed to 37 °C, while the cells were washed twice. Endocytic uptake at 37 °C was measured for 10 min in the presence or absence of the warmed preincubation medium (i.e., with or without unbound toxin in the solution), and the results are compared in Fig. 1B. Clearly, the presence of unbound toxin in the medium does not affect the stimulation of toxin uptake.

To study the actual rate of Stx uptake, and more specifically to compare the rate of control and stimulated Stx uptake, TAG- and biotin-labeled Stx (0.33 nm) was added to HEp-2 cells, and internalized toxin was measured after different time periods in the presence or absence of a stimulatory dose of unlabeled Stx1 (13 nm). As shown in Fig. 2A, the internalization of Stx is quite rapid both at high and low toxin concentrations, but less toxin is internalized at low toxin concentrations even during the first 10 min of incubation. The fact that the toxin accumulation leveled off at later time points may be due to recycling. For easy comparison, the endocytic control values obtained in the absence of unlabeled Stx1 were normalized to 100% (Fig. 2B). The data show that whereas no significant difference was seen after 2.5 min, there was a clear difference after 5 min and no further increase after 7.5 min. Thus, the stimulation of Stx uptake is a rapid process.

image

Figure 2. Uptake of Stx at high or low toxin concentration with time. (A) HEp-2 cells were incubated with (○) or without (•) unlabeled Stx1 (13 nm) in addition to TAG- and biotin-labeled Stx (0.33 nm) in Hepes buffered MEM at 37 °C for 2.5–60 min, before bound and endocytosed Stx were quantified as described in Experimental procedures (mean values ± deviations between two independent experiments, each done in duplicate). (B) Control values (filled bars) were normalized to 100% for comparison to the endocytic values obtained upon Stx1 stimulation (open bars).

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The stimulation of Stx uptake is specific for Stx and requires surface-bound toxin A-subunit

In order to investigate whether the stimulation of toxin uptake at high toxin concentrations was an effect specific for Stx, experiments using cholera toxin (CT) were also conducted. A possible cross-stimulation between CT and Stx was investigated because both toxins bind to glycosphingolipids in the plasma membrane (Gb3 and GM1, respectively), and might be localized in the same microdomain. Increasing the toxin concentrations would presumably give increased crosslinking and aggregation of toxin–receptor complexes, and the possibility existed that the two toxins would be endocytosed together in these aggregates. Importantly, Stx uptake was unchanged upon addition of increasing concentrations of CT (0–20 nm) as shown in Fig. 3A. Also, endocytosis of TAG- and biotin-labeled CT (0.12 nm) was measured in HEp-2 cells incubated with increasing concentrations of unlabeled Stx1 (0–20 nm). There was no increase in CT uptake under these conditions (data not shown). Furthermore, endocytosis of TAG- and biotin-labeled CT (0.12 nm) was measured at increasing CT concentrations (0–20 nm) in A431 cells. There was no stimulation of CT endocytosis with increasing toxin concentrations (data not shown), which is in accordance with data obtained previously for CT uptake in BHK cells [30]. Thus, stimulation of toxin uptake at high toxin concentrations seems to be an effect specific for Stx. We next investigated whether Stx2, which also binds to Gb3, could stimulate Stx uptake. Stx1 and Stx2 show 55% and 57% amino acid similarity in the A- and B-chain, respectively [31], but despite this similarity they are immunologically distinct. Endocytosis of Stx was measured upon addition of increasing amounts of unlabeled Stx2 (0–20 nm), and as shown in Fig. 3A, only at very high concentrations of Stx2 was a slight stimulation of Stx uptake observed.

image

Figure 3. Endocytosis of Stx in cells incubated with increasing concentrations of Stx, Stx1, Stx2, CT or Stx B-subunit. (A) Effect of surface-bound Stx1, Stx2 or CT. HEp-2 cells were preincubated with TAG- and biotin-labeled Stx (0.33 nm) in addition to increasing concentrations of Stx1, Stx2 or CT (0–20 nm) in Hepes buffered MEM for 30 min at 0 °C. Then the cells were washed, and the incubation continued for 10 min at 37 °C, before bound and endocytosed toxin were quantified as described in Experimental procedures. The endocytic values obtained were compared to untreated control values (no unlabeled toxin added) in a representative experiment (mean values ± deviations between duplicates). Each experiment was repeated separately 3–4 times. (B) Effect of surface-bound Stx or StxB. Increasing concentrations of Shiga holotoxin or Stx B-subunit, both at 0–20 nm, were added to HEp-2 or HeLa cells in Hepes buffered MEM in addition to a constant concentration of TAG- and biotin-labeled Stx (0.33 nm). The cells were incubated for 10 min at 37 °C, before bound and endocytosed toxin were quantified as described in Experimental procedures. The amount of internalized toxin is presented as percent of total cell-associated toxin (mean values ± deviations between duplicates in a representative experiment.) Stimulation with the holotoxin or the B-subunit was assayed in parallel in four independent experiments in each cell type.

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To explore the mechanism of this specific stimulation of Stx uptake, we wanted to investigate whether an aggregation of Gb3 in the membrane, mediated by the pentameric B-subunit, would be sufficient to increase the toxin uptake. To this end, endocytosis of TAG- and biotin-labeled Stx (0.33 nm) was compared in cells treated with either increasing concentrations of Shiga holotoxin or Stx B-subunit (0–20 nm) in parallel experiments, in both HEp-2 and HeLa cells. As shown in Fig. 3B, there was no stimulatory effect on Stx endocytosis upon addition of Stx B-subunit alone in either cell type; thus, the stimulation seems to require the presence of the Stx A-subunit.

The self-stimulated Stx uptake is clathrin-dependent

In the internalization of Stx, both clathrin-dependent and clathrin-independent mechanisms seem to be involved in different cell types [2,6–8]. To further elucidate the role of clathrin in Stx uptake, and more specifically, to study the involvement of clathrin in the self-stimulated uptake of Stx, a BHK cell line with inducible expression of antisense to CHC was used. Upon induction, the clathrin-dependent endocytosis is completely inhibited in these cells. After two days of expression of antisense to CHC, uptake of TAG- and biotin-labeled Stx (0.33 nm) was measured, and as shown in Fig. 4, when clathrin-dependent endocytosis was inhibited, Stx endocytosis was reduced by 50% when no unlabeled toxin was added; confirming previously published data on clathrin-dependent uptake of Stx in these cells [8]. Interestingly, as shown in Fig. 4, the presence of increasing concentrations of unlabeled Stx1 (0–20 nm) lead to a marked increase in Stx uptake only in cells where the clathrin-dependent uptake was functional. In contrast, toxin endocytosis was not increased at high toxin concentrations after induction of antisense CHC. This suggests that high toxin concentrations preferentially stimulate the clathrin-dependent fraction of Stx uptake. To rule out the possibility that high Stx concentrations lead to a stimulation of clathrin-dependent uptake in general, the endocytosis of transferrin was measured in the presence of increasing Stx1 concentrations (0–20 nm). Under these conditions, the transferrin uptake remained unchanged (data not shown). In addition, uptake of the plant toxin ricin, which binds to both glycoproteins and glycolipids with terminal galactose, and which presumably is internalized by different endocytic mechanisms, was not affected by high concentrations of Stx1 (data not shown).

image

Figure 4. Endocytosis of Stx in noninduced and induced BHK cells with inducible expression of antisense CHC, which inhibits clathrin-dependent endocytosis. After two days of induction, the cells were washed and preincubated with increasing concentrations of unlabeled Stx1 (0–20 nm) in addition to TAG- and biotin-labeled Stx (0.33 nm) in Hepes buffered MEM at 0 °C for 30 min. Then the cells were washed, and the incubation continued for 20 min at 37 °C, before bound and endocytosed toxin were quantified as described in Experimental procedures. The amount of internalized toxin is presented as percent of total cell-associated toxin in noninduced cells (filled bars) and induced cells (open bars) (mean values ± deviations between duplicates) in a representative experiment. The experiment was repeated separately four times.

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Alexa-Stx is redistributed to transferrin-containing domains upon addition of unlabeled Stx1

Increasing amounts of Stx1 might lead to aggregation of Stx–Gb3 complexes into large clusters that subsequently could increase the uptake efficiency of the toxin in a manner where apparently also the A-subunit plays a role. Because stimulating the cells with increasing concentrations of Stx1 does not increase the clathrin-dependent endocytosis in general, the increase in Stx uptake that we observe might be explained by an increased recruitment of Stx–Gb3 complexes to clathrin-coated pits. To explore this, HeLa cells grown on coverslips were treated with Alexa-labeled Stx (1.3 nm) and Cy5-labeled transferrin (130 nm) as a marker of clathrin-coated pits, and incubated with or without unlabeled Stx1 (20 nm). Then single plane sections were made by confocal microscopy and the fluorescence intensities were quantified. Earlier results from electron microscopy indicate that Stx is evenly distributed at the cell surface of HeLa cells at 0 °C, and there is a redistribution of the toxin to clathrin-coated pits only at higher temperatures [22]. In agreement with these data there was a diffuse labeling of HeLa cells incubated with Alexa-labeled Stx at 0 °C (data not shown). To allow for slow redistribution of toxin, the temperature was raised to 10 °C, and colocalization of Alexa-Stx and Cy5-transferrin was quantified and compared in cells treated with or without unlabeled Stx1. As shown in Fig. 5, incubation of the cells with unlabeled Stx1 (in addition to Alexa-Stx) lead to a marked increase in colocalization of Alexa-Stx and Cy5-transferrin, indicating an increased redistribution of Stx to transferrin-containing microdomains/clathrin-coated pits at the plasma membrane.

image

Figure 5. Localization of Alexa-labeled Stx and Cy5-labeled transferrin in cells treated with or without unlabeled Stx1. HeLa cells were incubated with Alexa-labeled Stx (1.3 nm) and Cy-5 labeled transferrin (130 nm), and one half of the cells was in addition incubated with 20 nm of Stx1 (high unlabeled Stx1). All the cells were incubated for 40 min at 10 °C before fixation. Quantification of the extent of colocalization between Stx and transferrin (as percentage of total amount of Stx) when the cells were incubated without unlabeled Stx1 (black bars, n = 29) or with high unlabeled Stx1 (grey bars, n = 43) is shown in the lower panel. Data were pooled from four independent experiments.

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Recent data indicate a correlation between the level of the proteoglycan heparan sulfate on the cell surface and the rate of prion protein endocytosis [32]. In order to investigate whether proteoglycans are involved in Stx endocytosis, HEp-2 cells were treated with chlorate, which inhibits the sulfation of proteoglycans [33], or treated with heparinase I, which cleaves off surface-bound heparan sulfate [34]. Neither treatment reduced the endocytic uptake of Stx (data not shown). Thus, there is no evidence for involvement of proteoglycans in Stx internalization. Furthermore, to investigate whether the charge of Stx is of importance for the toxin uptake, internalization of Stx was measured at different pH values, ranging from 5.5 to 8.5, after different time points. However, under these conditions the rate of Stx uptake was unchanged (data not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

In the present article, we show that Stx is able to stimulate its own entry by a process dependent on the toxin concentration, the surface-binding of the complete AB5 toxin and clathrin-dependent endocytosis. This self-stimulatory effect of Stx uptake seems to be mediated by an increased recruitment of the Stx–Gb3 complex to clathrin-coated pits.

Stx can be internalized by clathrin-dependent endocytosis, despite the lipid nature of its receptor Gb3 [2,4,7,22,35]. To study this process we decided to investigate whether Stx uptake was dependent on the toxin concentration added to the cells, and indeed this seemed to be the case. When increasing concentrations of unlabeled Stx were added to different cell lines (HEp-2, BHK, Vero, HeLa and A431 cells), uptake of labeled Stx was increased approximately twofold in all the cell lines except A431 cells. This self-stimulated Stx uptake was found to be mediated by clathrin-dependent endocytosis, as suggested from experiments using BHK cells with inducible expression of antisense to CHC. Based on this result, one might speculate whether the difference in stimulation of Stx uptake between cell lines is due to differential involvement of clathrin-dependent endocytosis of Stx in the cells. Interestingly, there were large differences in the actual amount of Stx internalized in each cell line after 10 min at low toxin concentrations, ranging from 13% in A431 cells to 33% in BHK cells (internalized toxin as percent of total cell-associated toxin; data not shown). In general, clathrin-dependent endocytosis is much faster than clathrin-independent mechanisms studied so far, due to the ≈ 1 min half-life of clathrin-coated pits at the cell surface [36]. Thus, a high rate of toxin uptake might reflect that a larger fraction of toxin endocytosis is clathrin-dependent, even at low toxin concentrations.

Upon stimulation of Stx endocytosis with increasing concentrations of toxin, clathrin-dependent and -independent endocytosis were not increased in general. There was no increase in the uptake of transferrin or ricin, rather the Stx uptake seemed to be increased specifically. This could be explained by an increased localization of Stx–Gb3 complexes in clathrin-coated pits, and was investigated by immunofluorescence using Alexa-labeled Stx (in the absence and presence of unlabeled toxin) and Cy5-labeled transferrin as a marker of clathrin-coated pits. Although Alexa-Stx gave a heterogeneous labeling of the cells, as reported previously by others [9], the results clearly showed an increased colocalization of Stx and transferrin in cells incubated with unlabeled Stx1 compared to control cells incubated with Alexa-Stx only. The increased colocalization of Stx and transferrin did not result from an increased amount of transferrin receptor on the plasma membrane, because neither the binding nor the endocytosis of transferrin was increased upon incubation with high concentrations of Stx1 (data not shown). Thus, it seems likely that Stx–Gb3 localization in transferrin-containing clathrin-coated pits is increased upon stimulation with high concentrations of Stx1. These new results can explain our previously published electron microscopy data regarding Stx redistribution to clathrin-coated pits upon shifting the temperature from 0 °C to 37 °C [22]. In those experiments, Stx at a concentration as high as 133 nm was added to HeLa cells, and in agreement with the data in the present article, a high degree of Stx redistribution to clathrin-coated pits was observed by electron microscopy upon shifting the temperature.

It seems that only cell surface-bound toxin molecules are responsible for the self-stimulation of Stx uptake, as was shown by comparing the stimulation of toxin uptake in the presence or absence of free toxin molecules in the medium. This could be due to the much higher local toxin concentration at the cell surface compared to that in the medium. Calculations show that there is ≈ 10 000 times higher concentration of labeled Stx at the cell periphery than in the rest of the medium. (For the assumptions involved, see Experimental procedures.)

Importantly, binding of the complete AB5 Stx structure seems to be specifically required for the ability of high toxin concentrations to induce an increased rate of toxin endocytosis. Control experiments using CT showed that the observed stimulation of Stx endocytosis was not due to an unspecific aggregation of glycosphingolipids/lipid rafts in the plasma membrane, nor was a mere aggregation of Gb3 by Stx B-subunits sufficient to stimulate Stx internalization. Thus, the presence of the A-subunit of the Shiga holotoxin seems to be crucial for the self-stimulated uptake to occur. In order to explain this, one might envision that the A-subunits are directly involved in important interactions either to other A-subunits, which could possibly cluster the toxins, or to other plasma membrane proteins, which might facilitate toxin internalization. Alternatively, the A-subunit might influence the toxin internalization indirectly by affecting the exposure of its associated B-subunits. This could change the surface location of the toxin or facilitate interactions with other membrane proteins that might induce toxin internalization. Also, as mentioned above, both toxin subunits (A and/or B) might induce signaling that could mediate toxin internalization, and this toxin-induced signaling might differ, depending on whether the intact toxin or the B-subunit bind to Gb3. However, these signaling pathways are largely unknown.

Interestingly, increasing concentrations of Stx2, which also binds to Gb3 and has an A-subunit with 55% amino acid similarity to Stx1, gave only a slight stimulation of Stx uptake in HEp-2 cells, a further demonstration of the specificity of this process. Important in this connection is that although Stx1 and Stx2 are structurally very similar, the A-subunit of Stx2 has a different orientation with respect to the B-subunits than in Stx1 [37]. This might influence the ability of Stx2 to stimulate Stx1 uptake. The finding that Stx2 behaves differently from Stx1 is in agreement with their different effects on cells and in disease (for review see [4,5]).

The data shown in this article clearly reveal that the Stx A-subunit bound to the cells via the B-pentamer is responsible for the stimulation of endocytosis. However, the requirement for the A-subunit could be mediated via an effect of the B-moiety. This illustrates the importance of detailed studies of the role of the different subunits/toxin domains for toxin uptake and intracellular transport. In order to understand the action of Stx and the Shiga-like toxins on the cellular level as well as in disease, each step along the retrograde pathway from the cell surface to the cytosol should be characterized.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Materials

Hepes, BSA, mercaptoethanesulphonic acid (MESNa), n-octyl β-d-glucopyranoside, tetracycline and transferrin were purchased from Sigma Chemical Co., St. Louis, MO, USA. CT was obtained from Calbiochem, San Diego, CA, USA. Stx was provided by JV Kozlov (Academy of Sciences of Russia, Moscow, Russia), and by JE Brown (USAMRIID, Fort Detrick, MD, USA), who also provided us with Shiga toxin B-subunit. The plasmid encoding StxB-Sulf2 was a kind gift from B Goud (The Curie Institute, Paris, France). Stx1 and Stx2 were purchased from Toxin Technology, Sarasota, FL, USA. Stx was labeled with Alexa from Molecular Probes (Leiden, the Netherlands) according to the procedure given by the company, and Cy5-labeled transferrin was purchased from the same company.

Cells

HEp-2, HeLa, Vero and A431 cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 units·mL−1 penicillin and 100 µg·mL−1 streptomycin. HEp-2 cells were seeded out in 24-well plates at a density of 5 × 104 cells per well one day prior to experiments, while HeLa and Vero cells were seeded at 2 × 104 cells per well two days prior to experiments. A431 cells were seeded at 5 × 104 cells per well in the presence of 1.5 mm butyric acid two days prior to experiments. BHK21-tTA cells transfected with antisense CHC were grown in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 units·mL−1 penicillin, 100 µg·mL−1 streptomycin, 0.5 µg·mL−1 puromycin, 0.2 mg·mL−1 geneticin and 2 µg·mL−1 tetracycline. Two days before experiments the cells were seeded with and without tetracycline in 24-well plates at a density of 2 × 104 cells per well.

Endocytosis of TAG- and biotin-labeled Stx, CT and transferrin

Endocytosis of Stx, CT and transferrin was performed essentially as described previously [8,30]. Briefly, this special method is based on double-labeling of the proteins of interest with both a capture-label (biotin) and a detection-label (electrochemiluminescent label), which are selectively captured and quantitated in a highly specialized electrochemiluminescent detection instrument produced by BioVeris Corporation (Gaithersburgh, MD, USA). Stx, CT or transferrin were labeled with the detection label BV-TAG® (BioVeris Corporation), which was stably bound to the proteins via an amide bond. The central atom of this specific label is a tris (bipyridine)-chelated ruthenium (II), which emits light when electrochemically stimulated, and the photons are measured in a photomultiplier tube in less than a second. Capture of the protein is mediated via streptavidin-coated magnetic beads, and the protein must therefore be simultaneously labeled with biotin. Once bound to the cell surface, neither Stx nor CT is easily removed, even with proteases. Therefore, to distinguish internalized toxin from total cell-associated toxin (bound + internalized), the toxins were labeled with the reducible EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA). Then, upon internalization of labeled toxin, the biotin linked to surface-bound toxin was cleaved off in some of the wells by the cell-impermeable reducing agent MESNa, thereby preventing detection of the still surface-bound toxins [38].

In the experiments, the cells were washed once with Hepes buffered MEM and then incubated with TAG- and biotin-labeled Stx (0.33 nm), CT (0.12 nm) or transferrin (0.64 nm) in Hepes buffered MEM in the presence of BSA (0.2%) for different time periods at 37 °C. In some experiments the cells were preincubated with the labeled toxins for 30 min at 0 °C, followed by a brief wash, and endocytosis was measured after incubation at 37 °C for the indicated time periods. After internalization of toxin, the cells were washed with cold buffer (0.14 m NaCl, 2 mm CaCl2, 20 mm Hepes; pH 8.6), and one half of the wells were treated with 0.1 m MESNa in the same buffer for 20 min at 0 °C to reduce the SS-linked biotin in cell surface-bound toxin, while the other half were mock treated. The cells were washed in cold buffer (0.14 m NaCl, 2 mm CaCl2, 20 mm Hepes; pH 7.0) and lysed (1% Triton, 60 mm n-octyl β-d-glucopyranoside, 100 mm NaCl, 5 mm MgCl2, 50 mm Hepes). Streptavidin-coated magnetic beads (Dynal, Oslo, Norway) were added to the lysate, and the samples were shaken for 20 min. The amount of TAG-labeled toxin captured by the beads was detected in a BioVeris Detection System instrument (BioVeris Corporation). Counts from cells treated with MESNa represent the amount of internalized toxin, while counts from untreated cells represent the total amount of toxin associated with the cells (bound + internalized). Endocytosis of Stx was reported as internalized toxin in percent of total cell-associated toxin. The background in this system, as measured by treating the cells with unlabeled toxin alone, was low (≈ 1200 counts) and equal to counting lysis buffer alone. The background value was subtracted from all the values in each experiment. The endocytic values, with background subtracted, ranged from 5000 to 10 000, depending on the cell line, and the values for total cell-associated toxin ranged from 20 000 to 40 000. It should be noted that even for the lowest experimental values used, the signal : background ratio was not lower than ≈ 3. Furthermore, upon binding of labeled toxin to cells on ice, treatment with MESNa reduced the detected amount of surface-bound toxin to 98%. Thus, there was a 2% constant background value after MESNa treatment, which was subtracted from all the experimental values.

Stx produced by S. dysenteriae and Stx1 produced by E. coli are virtually identical and differ only in one amino acid. Replacing Stx with Stx1 in the endocytosis experiment shown in Fig. 1, gave nearly identical results (data not shown). Unless otherwise stated, Stx was substituted with the commercially available Stx1 in the remaining experiments. Similarly, identical results were obtained by StxB-Sulf2and StxB from S. dysenteriae (data not shown), and both proteins have been used.

Preparation of StxB

The StxB containing a tandem of sulfation sites in the C-terminus (StxB-Sulf2) was produced in E. coli BL21 (DE3) cells essentially as described previously [39]. Briefly, a 10 mL overnight bacterial culture grown at 37 °C was inoculated in 500 mL of LB medium and further grown to an attenuance at 600 nm of 0.6. The culture was heat-induced for 4 h at 42 °C, and the cells were harvested by centrifugation. The pellet was washed twice with 10 mm Tris/HCl (pH 8.0), resuspended in 25% (w/v) sucrose, 1 mm Na2EDTA, and 10 mm Tris/HCl (pH 8.0), and gently shaken at 30 °C for 10 min. Cells were harvested by centrifugation and resuspended in ice-cold distilled water. After centrifugation the supernatant was dialyzed overnight against 20 mm Tris/HCl (pH 7.5), loaded on a Resource Q column (Amersham Biosciences, Uppsala, Sweden), and eluted with a 0–600 mm NaCl gradient in 20 mm Tris/HCl (pH 7.5). At physiological pH StxB is in a stable pentameric form, and the dialysis was actually performed in tubing with cut-off 12–14 000 Da. Purified StxB is therefore structurally equivalent to the B-moiety of the holotoxin [40–42].

Immunofluorescence

HeLa cells grown on coverslips were washed with cold Hepes medium and cooled to 10 °C before incubation with Alexa-labeled Stx (1.3 nm) and Cy-5 labeled transferrin (130 nm) for 40 min at 10 °C. The cells were washed twice with NaCl/Pi and fixed with 3% (v/v) paraformaldehyde in NaCl/Pi, before analysis in a LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany). Pictures were taken of thin single plane sections. The extent of colocalization between Stx (green channel) and transferrin (blue channel, changed to red for quantification) was quantified as reported previously [8] by calculating the ratio between the number of yellow (= colocalization) pixels (fluorescence level between 150 and 255) and the number of green pixels (fluorescence level between 150 and 255) using the Adobe photoshop 7.0 software.

Estimation of cell surface-bound toxin concentration

To be able to compare the local concentration of Stx on the cell surface and in the medium, the labeled toxin bound to the cells were assumed to be distributed in a restricted volume surrounding the cell from the outside of the plasma membrane and 10 nm outwards in the medium. Counts per µL in this volume were compared to the counts of free toxin molecules per µL of medium. The volume of a HEp-2 cell has been estimated to be 4.5 × 10−12µL, and the number of cells after one day of growth was approximated to be 7.5 × 104 per well.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References

We thank Dr Tore-Geir Iversen for critically reading the manuscript. This work was supported by The Norwegian Cancer Society, The Norwegian Research Council for Science and the Humanities, the Novo-Nordisk Foundation, the Jahre Foundation and Jeanette and Søren Bothners legacy.

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Experimental procedures
  6. Acknowledgements
  7. References
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