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Keywords:

  • alpha-toxin;
  • Clostridium septicum;
  • cytotoxicity;
  • receptor

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Clostridium septicum alpha-toxin has a unique tryptophan-rich region (302NGYSEWDWKWV312) that consists of 11 amino acid residues near the C-terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan-rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)-anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI-anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan-rich region plays an important role in the cytotoxic mechanism of alpha-toxin, especially in the binding to GPI-anchored proteins as cell receptors.

Abbreviations
CDC

cholesterol-dependent cytolysin

C. septicum

Clostridium septicum

D

domain

DRM

detergent-resistant membrane

EC50

half maximal effective concentration

E. coli

Escherichia coli

GPI

glycosylphosphatidylinositol

PI-PLC

phosphatidylinositol-specific phospholipase C

PVDF

polyvinylidene difluoride

Clostridium septicum is a gram-positive, spore-forming anaerobe that causes a variety of disease syndromes in humans and animals [1, 2]. This organism produces several extracellular factors, including deoxyribonuclease, hyaluronidase, neuraminidase and alpha-toxin [3]. Alpha-toxin, the major virulent factor, has hemolytic, lethal and necrotizing activities [4, 5]. Alpha-toxin is a pore-forming toxin that belongs to the same family as aerolysin from Aeromonas hydrophila [6] and epsilon-toxin from Clostridium perfringens [7]. Alpha-toxin is secreted by the organism as an inactivated 46 kDa protoxin [8, 9]. The protoxin binds to GPI-anchored proteins on cell surfaces with high affinity [10, 11]. The protoxin is then cleaved to its 43 kDa active form by host cell proteases, such as furin [5, 8]. Activated toxin monomers interact with each other on DRMs to form oligomeric prepore complexes [12, 13]. The prepore complexes ultimately insert into the plasma membrane, generating pores that are approximately 1.3–1.6 nm in diameter [4, 8].

Alpha-toxin and aerolysin show structural and functional similarities, at the level of 72%, with 27% identity [6, 9]. Although GPI-anchored proteins also act as receptors for aerolysin, each toxin binds to different subsets of GPI-anchored proteins [10]. Furthermore, the most striking difference between alpha-toxin and aerolysin is that D1 of aerolysin is missing from the amino terminus in alpha-toxin, implying that alpha-toxin is a single-lobed structure consisting of three domains, D1, D2, and D3, which are homologous to D2, D3, and D4 of aerolysin, respectively (Fig. 1) [6, 14, 15]. The functional domains and amino acids of alpha-toxin involved in receptor binding, oligomerization and pore formation have been identified by Melton et al. [14, 16]. Binding of alpha-toxin to GPI-anchored proteins is restricted to D1 [16].

image

Figure 1. Crystal structure of aerolysin as molecular model of alpha-toxin. Areas enclosed by circles show tryptophan-rich regions in each toxin. The area enclosed by a dotted circle shows D1 of aerolysin. D1 to D4 indicate domains 1 to 4.

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Because cholesterol is essential to binding and stability on the cellular membrane for many kinds of pore-forming toxins from gram-positive bacteria, these toxins have been named CDCs. CDCs that bind specifically to membrane cholesterol, such as perfringolysin O [17], streptolysin O [18], pneumolysin [19] and listeriolysin O [20], have a region of 11 highly conserved amino acid residues, ECTGLAWEWWR (tryptophan-rich motif) that is located in the C-terminal region of each toxin. In perfringolysin O, three tryptophan residues in the tryptophan-rich motif have an important role in binding to the cell membrane [21]. Although C. septicum alpha-toxin does not bind to cholesterol but to GPI-anchored proteins on the cell surface, alpha-toxin also has a tryptophan-rich region lying within an 11 amino acid sequence in D1 near the C terminus (NGYSEWDWKWV; residues 302–312; Fig. 1) [6]. In a previous study, Melton-Witt et al. found that cytotoxic activity on cells and binding of alpha-toxin to GPI-anchored proteins are restricted by replacing some amino acids in D1 by alanine [16]. This suggests that the receptor-binding region is present in D1. Three tryptophans in the tryptophan-rich region have been found to be associated with the loss of >90% of the lethal activity of wild-type alpha-toxin [16].

In this study, we examined the contribution of individual amino acids in the tryptophan-rich region to the activity of alpha-toxin by preparing mutant toxins with amino acid residues with different side chains and electric charges.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Construction of recombinant alpha-toxin and mutagenesis

The protoxin gene was cloned in pET 30(a) (Novagene, Madison, WI, USA) by PCR amplification of the gene from C. septicum NCTC 547 chromosomal DNA with the following pair of synthetic primers: 5′-CGGGATCCCGACTTACAAATCTTGAAGA-3′ and 5′-CCCAAGCTTGGGTTATATATTATTAATTAATATCA-3′. These primers add BamHI and HindIII sites to the 5′ and 3′ ends, respectively, of the protoxin gene. The BamHI–HindIII fragment containing protoxin gene was ligated into the BamHI–HindIII site within the multiple cloning site of pET 30(a).

For mutagenesis, amplified alpha-toxin gene was ligated into BamHI- and HindIII-digested PUC19 vector (Takara, Tokyo, Japan). Mutagenesis of the tryptophan-rich region in alpha-toxin was performed using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA; Table 1). Pairs of complementary oligonucleotides were used to construct mutant alpha-toxin molecules as shown in Table 2. In all cases, oligonucleotides were designed to preserve the amino acid sequence, except for the desired substitution. Nucleotide sequences of the mutants were verified by DNA sequencing. After digestion of the mutated plasmid with BamHI and HindIII, the fragments were ligated into BamHI- and HindIII-digested pET 30(a). Escherichia coli strain BL21 (DE3; Novagene) was transformed with pET 30(a) carrying wild-type and mutant alpha-toxin genes.

Table 1. Mutants generated in this experiment
MutantsAmino acid sequences
302312
  1. Amino acids mutated in alpha-toxin are underlined.

N302AAGYSEWDWKWV
E306RNGYSRWDWKWV
W307ANGYSEADWKWV
W307FNGYSEFDWKWV
D308RNGYSEWRWKWV
W309ANGYSEWDAKWV
W309FNGYSEWDFKWV
K310ENGYSEWDWEWV
K310RNGYSEWDWRWV
W311ANGYSEWDWKAV
W311FNGYSEWDWKFV
V312ANGYSEWDWKWA
W307A/W309A/W311ANGYSEADAKAV
W307F/W309F/W311FNGYSEFDFKFV
Table 2. Primers for site-direct mutagenesis
MutantsPrimers
  1. Mutations are underlined.

N302AForward 5′-AGTCATAAGAATATTGCTGGATATTCAGAATGG-3′
 Reverse 5′-CCATTCTGAATATCCAGCAATATTCTTATGACT-3′
E306RForward 5′-ATTAATGGATATTCACGATGGGATTGGAAATGG-3′
 Reverse 5′-CCATTTCCAATCCCATCGTGAATATCCATTAAT-3′
W307AForward 5′-AATGGATATTCAGAAGCGGATTGGAAATGGGTA-3′
 Reverse 5′-TACCCATTTCCAATCCGCTTCTGAATATCCATT-3′
W307FForward 5′-AATGGATATTCAGAATTCGATTGGAAATGGGTA-3′
 Reverse 5′-TACCCATTTCCAATCGAATTCTGAATATCCATT-3′
D308RForward 5′-GGATATTCAGAATGGCGTTGGAAATGGGTAGAT-3′
 Reverse 5′-ATCTACCCATTTCCAACGCCATTCTGAATATCC-3′
W309AForward 5′-TATTCAGAATGGGATGCGAAATGGGTAGATGAG-3′
 Reverse 5′-CTCATCTACCCATTTCGCATCCCATTCTGAATA-3′
W309FForward 5′-TATTCAGAATGGGATTTCAAATGGGTAGATGAG-3′
 Reverse 5′-CTCATCTACCCATTTGAAATCCCATTCTGAATA-3′
K310EForward 5′-TCAGAATGGGATTGGGAATGGGTAGATGAGAAA-3′
 Reverse 5′-TTTCTCATCTACCCATTCCCAATCCCATTCTGA-3′
K310RForward 5′-TCAGAATGGGATTGGAGATGGGTAGATGAGAAA-3′
 Reverse 5′-TTTCTCATCTACCCATCTCCAATCCCATTCTGA-3′
W311AForward 5′-GAATGGGATTGGAAAGCGGTAGATGAGAAATTT-3′
 Reverse 5′-AAATTTCTCATCTACCGCTTTCCAATCCCATTC-3′
W311FForward 5′-GAATGGGATTGGAAATTCGTAGATGAGAAATTT-3′
 Reverse 5′-AAATTTCTCATCTACGAATTTCCAATCCCATTC-3′
V312AForward 5′-TGGGATTGGAAATGGGCAGATGAGAAATTTGGT-3′
 Reverse 5′-ACCAAATTTCTCATCTGCCCATTTCCAATCCCA-3′
W307A/W309A/W311A
Forward 5′-AATGGATATTCAGAAGCGGATGCGAAAGCGGTAGATGAGAAATTT-3′
Reverse 5′-AAATTTCTCATCTACCGCTTTCGCATCCGCTTCTGAATATCCATT-3′
W307F/W309F/W311F
Forward 5′-AATGGATATTCAGAATTCGATTTCAAATTCGTAGATGAGAAATTT-3′
Reverse 5′-AAATTTCTCATCTACGAATTTGAAATCGAATTCTGAATATCCATT-3′

Preparation of recombinant alpha-toxin and mutants

The growth and harvesting of E. coli BL21 (DE3) expressing polyhistidine-tagged wild-type and various mutant alpha-toxin derivatives were performed as described previously [12]. Cells were pelleted, suspended in B-PER (Pierce, Rockford, IL, USA) and digested for 20 min at room temperature with 0.2 mg/mL lysozyme, supplemented with 0.5% (v/v) protease inhibitor cocktail (Sigma Chemical., St Louis, MO, USA), followed by sonication at 4°C. Lysates were clarified by centrifugation at 27,200 g for 15 min at 4°C. The recombinant proteins were purified from supernatant by Ni-NTA (Qiagen GmbH, Hilden, Germany) affinity chromatography according to the manufacturer's instructions. The recombinant alpha-toxin and mutants were stored at 4°C until use. Protein purity was clarified by SDS–PAGE [22] with a 12.5% resolving gel.

Cytotoxicity assay

Vero cells were inoculated into a 96-well plate at a density of 2 × 105 cells/mL. Cells were grown to confluence in Dulbecco's modified Eagle's medium (Sigma Chemical) supplemented with 10% FCS at 37°C under 5% CO2. After removal of medium and washing with PBS, the toxin diluted with cell culture medium was added to each well. The plates were incubated for 1 hr at 37°C under 5% CO2. Cell Titer 96 Aqueous One Solution Reagent (Promega, Madison, WI, USA) was added and incubated for a further 1 hr at 37°C under 5% CO2. Absorbance of each well was measured at 490 nm. The data are presented as percent viability to determine the concentration of toxin causing 50% cell death (EC50) as described previously [23].

Phosphatidylinositol-specific phospholipase C treatment

Vero cells (3 × 107/mL) were treated with PI-PLC (0.5 U/mL; EMD Biosciences, Darmstadt, Germany) for 2 hr at 37°C in PBS and centrifuged as described previously [24]. Aliquots of cells and supernatants were used for SDS–PAGE and toxin overlay assay.

Preparation of detergent-insoluble fraction

Vero cells were scraped from 25 cm2 flasks with a rubber policeman and harvested by centrifugation at 1000 g for 5 min. After washing, cells were suspended in 1 mL of cold lysis buffer consisting of 10 mM Tris–HCl buffer (pH 7.0) containing 150 mM NaCl, 1% Triton X-114 (Pierce) and 0.1% protease inhibitor cocktail. After allowing them to stand for 1 hr on ice, the detergent-insoluble fractions were separated from the supernatants (the detergent-soluble fractions) by centrifugation at 15,000 g for 15 min, and finally resuspended in 1 mL of PBS.

Electrophoresis and toxin overlay assay

SDS–PAGE was carried out in 5–20% gradient gels (ATTO, Tokyo, Japan). After electrophoresis, detergent-soluble and -insoluble fractions from Vero cells were blotted onto PVDF membranes. After blotting, the membranes were blocked with 5% skim milk in PBS for 1 hr at room temperature. After washing three times with PBS-0.01% Tween 20, the membranes were incubated for 1 hr at room temperature in the presence of 10 µg/mL wild-type or mutant alpha-toxin in 0.5% skim milk. This was followed by washing and incubation for a further 1 hr at room temperature with 5 µg/mL affinity-purified rabbit anti-alpha-toxin IgG [25] in 0.5% skim milk. The membranes were treated for 30 min at room temperature with goat anti-rabbit IgG (H + L) conjugated with peroxidase (1:3000 dilution; Cappel, West Chester, PA, USA) in 0.5% skim milk. After washing, the membranes were developed in 20 mL of PBS containing 0.05% 3′3-diaminobenzidine (Dojin Laboratories, Kumamoto, Japan) and 0.02% H2O2.

Determination of protein concentrations

Protein concentrations were determined by the method of Bradford [26] with bovine gamma globulin as a standard.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Construction, purification and characterization of mutant toxins

To evaluate the roles of the tryptophan-rich region in the C-terminal domain in the cytotoxic effect of alpha-toxin, we constructed several mutant toxins by individually replacing tryptophan and some residues surrounding tryptophan with other amino acids (Table 1). We individually replaced tryptophan (W307, W309, and W311) with phenylalanine (W307F, W309F, and W311F), which is hydrophobic and also has an aromatic side chain. These tryptophans were also replaced with alanine to create loss of an aromatic side chain and substitution by its minimal side chain (W307A, W309A, and W311A). We simultaneously replaced all three tryptophans with phenylalanine or alanine (W307F/W309F/W311F and W307A/W309A/W311A).

We also performed the following mutations for the amino acid residues surrounding the tryptophans. Because some of the amino acids adjacent to the three tryptophan residues carry electrical charges, we changed the charge in each amino acid residue. We changed two residues, E306 and D308, from acidic to basic amino acids by replacement with arginine (E306R and D308R). We replaced the residue K310 with glutamic acid in order to change from basic to acidic type (K310E). We also substituted the residue V312 with alanine to maintain hydrophobicity and no electric charge (V312A). We constructed mutant toxins in which we replaced residue N302, the most amino-terminal domain side in the tryptophan-rich region, with alanine (N302A). Wild-type and mutant alpha-toxins were expressed in E. coli BL21 and purified by affinity chromatography. SDS–PAGE detected every purified mutant toxin at the expected positions and each of their secondary structures was similar to that of wild-type toxin according to far-ultraviolet (190–260 nm) circular dichroism spectral analysis (data not shown).

Effects of amino acid substitution in tryptophan rich region on cytotoxicity

As shown in Table 3, the cytotoxic activities (EC50) of mutant toxins were compared with that of wild-type toxin. We found that the EC50 of W307F/W309F/W311F and W307A/W309A/W311A were >640 ng/mL, indicating that the cytotoxic activity of alpha-toxin decreased remarkably to below the limit of detection. The mutants of W307A, W309A and W311A also had marked reduction of cytotoxic activity. Although replacements of W307 and W311 with phenylalanine decreased the cytotoxic activities (207 and 113 ng/mL), they did not completely abolish them. Interestingly, replacement of W309 with phenylalanine did not greatly reduce cytotoxic activity. The mutant of W309F retained the same activity as the wild type. In the case of amino acid substitutions surrounding the three tryptophan residues, only D308R caused a decrease in cytotoxic ability (127 ng/mL). The cytotoxic activities of E306R, K310E, K310R, V312A and N302A did not change in comparison with that of the wild type.

Table 3. EC50 of wild-type and mutant alpha-toxins
MutantsEC50 (ng/mL)
  1. EC50 refers to concentration of wild-type and mutant alpha-toxins required for 50% cytotoxicity.

Wild type29.7
N302A21.2
E306R30.3
W307A>640.0
W307F207.0
D308R127.1
W309A>640.0
W309F25.8
K310E36.4
K310R25.5
W311A>640.0
W311F113.2
V312A25.5
W307A/W309A/W311A>640.0
W307F/W309F/W311F>640.0

Binding of mutant alpha-toxins to glycosylphosphatidylinositol-anchored proteins

To determine whether the tryptophan-rich region plays an important role in the binding of alpha-toxin to cell membranes, we used a toxin overlay assay to examine the binding activities of mutant toxins to detergent-insoluble proteins from Vero cells. After lysis with 1% Triton X-114, we separated Vero cells into detergent-soluble and -insoluble fractions by centrifugation. As shown in Figure 2a, we observed a specific band with a molecular mass of about 34 kDa in the detergent-insoluble fraction using a toxin overlay assay with wild-type alpha-toxin. In previous studies, we reported that alpha-toxin selectively binds to GPI-anchored proteins detected in the detergent-insoluble fractions from various cell lines [12, 25]. Since treatment with PI-PLC enables easy liberation of toxin-binding molecules without changing the molecular size, this molecule in the detergent-insoluble fraction from Vero cells possesses the characteristics of GPI-anchored proteins (Fig. 2a). We examined bindings of mutant alpha-toxin for the detergent-insoluble fraction by toxin overlay assay (Fig. 2b). We detected specific toxin-binding bands in some mutant alpha-toxins (N302A, E306R, W309F, K310E, K310R and V312A) that retained the same amount of cytotoxic activities as the wild type (Table 3). The bands reacting to the detergent-insoluble fraction disappeared for W307A, W309A and W311A, whose cytotoxic activities decreased to below the limit of detection (Fig. 2b). In addition, the mutants W307F, D308R, and W311F showed slightly less cytotoxic activities than did the wild-type toxin (Table 3). These results indicate that the low cytotoxic activities of these mutant toxins are due, at least in part, to decreases in their binding capabilities to the GPI-anchored protein (Fig. 2b).

image

Figure 2. Binding of alpha-toxins to detergent-insoluble proteins. (a) Detection of wild-type alpha-toxin binding to cell fractions treated with detergent and PI-PLC by a toxin overlay assay. insol, detergent-insoluble fraction; sol, detergent-soluble fraction; sup, proteins liberated by treatment with PI-PLC. The arrow head points to GPI-anchored protein. (b) Detection of mutant alpha-toxins binding to detergent-insoluble proteins. M, protein standard marker.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Cytotoxic and binding activities for Vero cells entirely disappeared in the mutant of alpha-toxin in which we simultaneously or individually replaced W307, W309, and W311 with alanine. When we simultaneously replaced three tryptophan residues with phenylalanine, which is hydrophobic and also possesses an aromatic side chain, toxic activity of the mutant toxin disappeared entirely. To examine which tryptophan is the most important residue for toxicity, we prepared several mutants in which we replaced individual tryptophans with alanine or phenylalanine (Table 3 and Fig. 2b). Individual mutation of W307A, W309A or W311A results in abolition of cytotoxic and binding activity, as described in a previous paper [16]. In perfringolysin O, one of the CDCs, mutant toxins produced by replacing three individual tryptophans with phenylalanine in the tryptophan-rich motif have significantly reduced hemolytic and binding activities [21]. In alpha-toxin, the mutants of W307F and W311F show remarkable decrease in cytotoxic activity. In contrast, the mutant W309F has the same cytotoxic and binding activities for Vero cells as the wild-type toxin. These results suggest that full toxic activity requires the amino acid residues at positions 307 and 311 to be tryptophan. It seems that it is important for position 309 to possess an aromatic side chain like tryptophan and phenylalanine rather than a hydrophobic residue.

Because the residues E306, D308 and K310 adjacent to the three tryptophan residues in the tryptophan-rich region carry electric charges, we constructed mutants with different electric charges at these amino acid residues (E306R, D308R and K310E). We observed reduction in cytotoxic activity and disappearance of binding activity only in the D308R mutant, the electric charge of which we changed from acidic to basic (Table 3 and Fig. 2b). These results strongly suggest that it is essential for alpha-toxin's toxic activity that residue 308 be aspartic acid or an acidic amino acid.

Binding of mutant alpha-toxins to the detergent insoluble fraction correlates closely with cytotoxic activities. We detected mutants W307F, D308R and W311F, in which cytotoxic activities are decreased, as faint bands in a toxin overlay assay (Fig. 2b). This indicates that the weak cytotoxic activities of these mutants are due to attenuation of the affinity of mutant alpha-toxin to the GPI-anchored protein. Because WDW_W is the most important sequence in the tryptophan-rich region, hydrophobicity and electrical charge in the side chain of these four amino acids would affect cytotoxic activity.

Researchers have shown that the cytotoxic mechanisms and primary structure of C. septicum alpha-toxin are similar to those of Aeromonas hydrophila aerolysin [6, 8]. Although the receptor of aerolysin on cell membranes is also a GPI-anchored protein [24], N-glycan on GPI-anchored proteins is required for efficient binding of aerolysin; however, binding of alpha-toxin is independent of N-glycan [27]. Aerolysin has a tryptophan-rich region (GEVKWWDWNWT) that is similar to that of alpha-toxin near the C-terminus, and possesses the same sequence in this region, WDW_W, which should be an important sequence for binding of alpha-toxin to cell receptors. With the exception of WDW_W, the amino acid sequence in the tryptophan-rich region of alpha-toxin does not exhibit identity with that of aerolysin. Therefore, this difference may determine whether N-glycan is indispensable for binding of alpha-toxin and aerolysin to GPI-anchored proteins.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology in Japan.

DISCLOSURE

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

The authors have no conflicts of interest associated with this study.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
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