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* This work was supported by grants from the Swiss National Science Foundation (to R. G. and B. S.) and the National Center of Competence in Structural Biology (to R. G. and B. S.). The authors declare that they have no conflicts of interest with the contents of this article.
The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive.
The abbreviations used are: PFT, pore-forming toxin; ClyA, Cytolysin A; ClyAox, oxidized ClyA with Cys-87–Cys-285 disulfide bond; ClyAred, reduced ClyA with Cys-87 and Cys-286 in dithiol form; DDM, n-dodecyl-β-d-maltopyranoside; FRET, Förster resonance energy transfer; I, intermediate of protomer formation; M, ClyA monomer; Ni2+-NTA, nickel-nitrilotriacetic acid; O, soluble ClyA oligomer; P, ClyA protomer; OMV, outer membrane vesicle; RP-HPLC, reversed phase HPLC; TEV, tobacco etch virus; SO, small oligomer; LO, large oligomer; mOD, milli-optical density unit.
exist in different orders of bacteria and eukaryotes and cause various human diseases (
): The soluble, monomeric form of ClyA consists of a large tail domain with four long α-helices and one short α-helix (α-helices A, B, C, F, and G; residues 2–159 and 206–303) and a head domain (residues 160–205) with a central, hydrophobic β-hairpin (the “β-tongue,” residues 185–195) flanked by two short α-helices (α-helices D and E) (Fig. 1). The tail domain contains a conserved cysteine pair (Cys-87 and Cys-285 in α-helix B and G, respectively) that form a disulfide bond (
). The head domain of the monomer forms elongations of the flanking α-helices (C and F) of the tail domain, and the β-hairpin undergoes a β-to-α transition. The N-terminal α-helix (αA), which is part of the five-helix bundle of the tail domain in the monomer, swings around by 180°, elongating the flanking α-helix B and leaving a four-helix bundle in the protomer (Fig. 1). The remaining four α-helices of the tail domain rearrange to close the gap left by αA (
). The N-terminal half of αA forms the channel through the target cell membrane. The cysteine pair Cys-87/Cys-285 in the reduced protomer remains in spatial proximity with a Cα-Cα distance of 6.8 Å (Fig. 1), which would still enable the formation of a disulfide bond (
). The authors discovered two distinct, homo-oligomeric species of ClyA that are formed in the absence of detergent or membranes (termed soluble oligomers below). These oligomers were interpreted as prepores, similar to soluble, oligomeric intermediates in the pore formation of many β-PFTs (
Here we examined this alternative pore formation mechanism of ClyA further. We found no evidence for pore formation or efficient target cell lysis when these soluble oligomers were mixed with detergent or horse erythrocytes, respectively. In fact, the soluble ClyA oligomers showed only 1–2% hemolytic activity relative to ClyA monomers. In addition, no pores could be observed by electron microscopy after incubation of soluble oligomers with detergent (n-dodecyl-β-d-maltopyranoside (DDM)) under conditions where the majority of ClyA monomers assembled into dodecameric pore complexes. We therefore interpreted the soluble oligomers as off-pathway products of ClyA pore formation. This conclusion is supported by the finding that the kinetics of soluble oligomer formation coincided with loss in hemolytic activity.
In addition, we compared oligomer formation and hemolytic activity of the reduced, dithiol form of ClyA (ClyAred) with pore formation of the oxidized, disulfide form (ClyAox) to address contradictory reports in the literature, which had described ClyAox either as assembly-incompetent and inactive (
). In the present study, we show that ClyAox monomers have essentially the same specific hemolytic activity compared with monomeric ClyAred but form inactive, soluble oligomers 13–14 times faster than ClyAred upon incubation in the absence of detergent or membranes. The results provide a plausible explanation for previous, contradictory reports on the assembly competence of ClyAox.
Finally we also investigated the mechanism of the DDM-induced monomer-to-protomer formation of ClyAox with single-molecule Förster resonance energy transfer (FRET) measurements, demonstrating that ClyAox follows the same reaction pathway as ClyAred, in which an unstructured off-pathway intermediate (Iox) is formed (
). Due to the lower stability of Iox compared with Ired, assembly-competent protomers are even formed one order of magnitude faster from monomeric ClyAox compared with monomeric ClyAred.
Chemicals of the highest available purity were purchased from Merck KGaA (Darmstadt, Germany) or Sigma-Aldrich. Dithiothreithol (DTT), EDTA and β-mercaptoethanol were obtained from AppliChem. DDM was purchased from Anatrace. Brain total lipid extract was purchased from Avanti Polar Lipids, and horse erythrocytes were obtained from Oxoid AG.
Production and Purification of Reduced ClyA
To exclude any influence of an N- or C-terminal polyhistidine purification tag on the activity and assembly of ClyA, we introduced a TEV (tobacco etch virus) protease cleavage site between the N-terminal His6 tag and the natural ClyA sequence into the previously described ClyA expression plasmid derived from pET11a (
). All purification steps and the proteolytic cleavage were performed under reducing conditions with buffers containing 2 mm β-mercaptoethanol (when Ni2+-NTA columns were used) or 2 mm DTT. The yield of reduced wild-type ClyA (ClyAred) was 16 mg of ClyA/liter of bacterial culture. The concentration of ClyA was determined via its specific absorbance at 280 nm (30370 m−1cm−1 for ClyAred). The ClyAred monomer was stored in PBS buffer (20 mm potassium phosphate, pH 7.3, 150 mm NaCl, 0.1 mm EDTA) with 2 mm DTT at 4 °C and showed no oligomerization within 7 days.
Preparation and Reduction of Oxidized ClyA
To generate the disulfide-bonded oxidized form of ClyA, ClyAred (40 μm) was incubated in PBS buffer with 0.5 mm CuCl2 (i.e. 0.4 mm free CuCl2) as a catalyst of air oxidation for 3–4 h at 22 °C. These conditions guaranteed complete oxidation of the Cys-87/Cys-285 pair of ClyA (see below). Oxidized ClyA (ClyAox) was subsequently dialyzed at 4 °C against PBS containing 2 mm EDTA and subjected to gel filtration on a Superdex 200 column (GE Healthcare Life Sciences) equilibrated with PBS to separate oxidized monomers from oxidized oligomers. The absence of free thiols in the purified oxidized monomers was confirmed by Ellman's assay (
) under denaturing conditions (4.0 m guanidinium chloride, pH 8.0) and by analytical reversed phase HPLC (RP-HPLC) at 30 °C on a Zorbax SB300 C8 column (Agilent) using a water-acetonitrile gradient from 50 to 80% (v/v) acetonitrile in 0.1% trifluoroacetic acid (see Fig. 2A). The ClyAox monomer was stored in PBS buffer without DTT at 4 °C and showed no oligomerization within 1.5 days.
The reduction of ClyAox (monomer, oligomer, or assembled pore complex in 0.1% DDM) was performed in PBS with 100 mm DTT, pH 7.3, at 37 °C and quantified by RP-HPLC as described previously (
). The reduction of unfolded ClyAox was carried out in 50 mm MOPS-NaOH, 4.0 m guanidinium chloride, pH 7.3, with 20 mm DTT at 37 °C. Reactions were quenched after different time intervals by the addition of formic acid (12% final concentration), and samples were analyzed via RP-HPLC. The peaks corresponding to oxidized and reduced ClyA were integrated, and the data were evaluated according to pseudo first-order kinetics. The reaction between monomeric ClyAred (4 μm) and oxidized DsbAox (86 μm, corresponding to the periplasmic DsbA concentration in E. coli (
)) at pH 7.3 and 37 °C, was analyzed in the same way.
Purification and Oxidation of Förster Resonance Energy Transfer (FRET) Donor/Acceptor-labeled ClyA for Single-molecule Measurements
A ClyA variant with cysteine residues introduced at positions 56 and 252 (ClyACys) was expressed, purified, and labeled at Cys-56 with Alexa Fluor 488 and at Cys-252 with Alexa Fluor 594 as described previously (
). Air oxidation of the natural cysteine pair Cys-87/Cys-285 in the labeled ClyACys variant (4 μm) was performed by incubation in 50 mm KH2PO4/K2HPO4 pH 7.4, 150 mm NaCl, 10% (v/v) glycerol, and 0.5 mm CuCl2 for 5 h at 22 °C. Oligomers formed during this reaction were removed from the monomers by gel filtration on a Superdex 75 10/300 column (GE Healthcare Life Sciences) in PBS. The quantitative formation of the disulfide bonded Cys-87–Cys-285 was confirmed by reversed phase HPLC on an XTerra RP8 (30 × 4.6 mm) column (Waters) in 0.1% (v/v) trifluoroacetic acid using a gradient from 30 to 60% acetonitrile.
Oligomerization of ClyA
Oligomers of reduced or oxidized ClyA were formed in the absence of detergents or lipids by incubation at 37 °C (
). For preparative purposes, ClyAred (40 μm) was incubated overnight at 37 °C in PBS, pH 7.3, and subsequently subjected to gel filtration (Superdex 200) to separate the oligomer from the remaining monomer. In the case of ClyAox, a 4-h incubation at 22 °C (conditions of oxidation of ClyA) resulted in a sufficient amount of oligomeric ClyA (Fig. 2A). The kinetics of oligomerization of ClyAox and ClyAred in PBS at 4 °C or 37 °C were determined by quantification of the respective amount of monomeric or oligomeric ClyA via gel filtration on a ProSEC 300S column (Agilent) after different incubation times, starting with ClyA monomer at 5 μm concentration. The decrease in the monomer over time was fitted mono-exponentially, assuming a unimolecular rate-limiting step, which is consistent with previous findings that oligomer formation is concentration-independent at micromolar monomer concentrations (
To trigger pore formation from monomers, ClyA (2 μm in PBS) was incubated in 0.1% DDM for 1 h at 22 °C. Samples were adsorbed on glow-discharged 300-mesh carbon-coated copper grids (Quantifoil) and negatively stained with 2 mm uranyl acetate. Images were recorded by a KeenView CCD camera using a FEI Morgagni electron microscope operating at an acceleration voltage of 100 kV.
The kinetics of ClyA-dependent lysis of horse erythrocytes were measured as described previously (
) by following the decrease in optical density at 650 nm using a Varian Cary 100 spectrophotometer (Agilent). Horse erythrocytes at a density of 2 × 106 cells/ml in PBS, pH 7.3, were lysed at 37 °C by the addition of ClyA (final monomer concentrations of 2–100 nm). Reactions with ClyAred additionally contained 2 mm DTT. Hemolysis kinetics were evaluated by linearly fitting the data points in the middle of the lysis reaction between 35 and 75% of the initial optical density. The slope of the linear decrease in optical density was defined as the maximum lysis velocity. The linear dependence of the maximum lysis velocity on the concentration of ClyA was defined as the specific hemolytic activity of the different ClyA species (
The monomer-to-protomer transition and pore assembly of ClyAox and ClyAred (9 μm) in PBS at 22 °C was initiated by the addition of DDM (final concentration, 0.1% (w/v)) and followed via the CD signal change at 225 nm using a temperature-controlled J715 CD spectrometer (Jasco). Samples of reduced ClyA additionally contained 2 mm DTT. Before and after assembly, the CD spectra of ClyAox and ClyAred were recorded. The rate-limiting step of the monomer-to-protomer conversion was fitted according to a first-order reaction.
Single-molecule Measurements of ClyAox
Kinetic single-molecule FRET measurements were performed and evaluated essentially as described for the reduced ClyACys variant (
), and the later phase as well as the pore formation kinetics was constructed from repeated manual mixing measurements (Table 1). All measurements were done at a nominal concentration of labeled ClyACys of 300 pm in PBS with 0.001% (w/v) Tween 20 and 0.1% (w/v) DDM at 22 °C. For recording pore formation kinetics with FRET, excess unlabeled oxidized WT ClyA was added at 10 or 100 nm to achieve the concentrations required to observe the oligomerization of protomers.
TABLE 1Single-molecule FRET data sets collected for the kinetic analysis for ClyA protomer and pore formation
). The three populations in the transfer efficiency histograms were fitted with Gaussian distributions, once with free amplitudes and once with the amplitudes determined by the kinetic model. Due to the extensive overlap of the peaks, the position and width of the monomer peak function were fixed to the values from the first histogram measured in the microfluidic chip and to those of a reference measurement for the microfluidic mixing and manual mixing data, respectively. As the intermediate is not sufficiently populated in the manual mixing experiments, the position and width of the peak function of the intermediate were fixed to those of the fit of the microfluidic mixing data. The uncertainties of the fitted rate coefficients were estimated with a bootstrap analysis on the level of the individual photon bursts. New sets of photon bursts were synthesized by random sampling with replacement from the original data. One thousand synthetic data sets were created, and the resulting histogram time series were fitted as described above. The resulting distributions of the fit parameters are well described by normal distributions.
Soluble ClyA Oligomers Are Formed Faster by ClyAox Than by ClyAred and Are Assembly-incompetent
WT ClyA contains a single cysteine pair at positions 87 and 285 that is able to form a disulfide bond in both the monomer and the protomer (
). That study has revealed that the unimolecular formation of assembly-competent protomers (Pred) from monomers (Mred) is rate-limiting for the formation of dodecameric pore complexes at concentrations above ∼100 nm and is accompanied by the reversible formation of a monomeric off-pathway intermediate (Ired): 12 Ired ↔ 12 mred ↔ 12 Pred →→ (fast) →→ dodecameric pore complex.
To study the assembly properties of ClyAox, we first expressed and purified the authentic, untagged ClyA monomer under reducing conditions (2 mm β-mercaptoethanol or DTT) via chromatography of a ClyA variant with an N-terminal His6 tag on Ni2+NTA-agarose and chromatography on hydroxyapatite followed by specific cleavage of the N-terminal His6 tag by TEV protease. With this procedure, we obtained pure ClyAred (Thr-2–Val-303), for which Ellman's assay under denaturing conditions confirmed the presence of two thiol groups per polypeptide chain. ClyAox was obtained from ClyAred after incubation for 4 h with 0.4 mm free CuCl2 as a catalyst of air oxidation in PBS buffer, pH 7.3, at 22 °C in the absence of detergent. Ellman's assay revealed the quantitative formation of the Cys-87–Cys-285 disulfide bond. This was confirmed by a single peak in reversed phase HPLC runs, which allows the quantitative separation of ClyAox from ClyAred (Fig. 2B).
The preparation of ClyAox was immediately applied to gel filtration, which revealed three distinct species: the oxidized monomer (Mox) and a small (SOox) and large (LOox) oligomer of ClyAox with apparent molecular masses of 580 and 1180 kDa, respectively (Fig. 2A). All three ClyAox species were isolated and again analyzed for quantitative disulfide bond formation by reversed phase HPLC (Fig. 2B). The analysis confirmed the presence of the disulfide bond in Mox, SOox, and LOox and also showed that purified ClyAred stayed completely reduced, proving to be resistant to air oxidation in the absence of Cu2+ (Fig. 2B).
To test the ability of Mox, SOox, and LOox to form dodecameric pore complexes, all species were isolated by gel filtration (Fig. 2A) and incubated with 0.1% DDM to trigger pore formation under identical conditions (total monomer concentration of 2 μm, 1-h incubation time, 22 °C, and pH 7.3). Electron microscopy showed that only Mox readily assembled into pores complexes, whereas LOox and SOox showed no comparable pore formation activity (Fig. 2C, top panels).
Next, we found that ClyAred also formed small and large oligomers (SOred and LOred, respectively) in the absence of detergent with the same apparent molecular masses as SOox and LOox but with slower assembly kinetics. Fig. 2D shows the gel filtration profiles of the kinetics of spontaneous oligomerization of ClyAox and ClyAred at pH 7.3 and identical initial monomer concentrations of 5 μm, initiated by a temperature shift from 4 to 37 °C. The kinetics of oligomer formation, recorded via the decrease in the peak areas of the monomers, revealed oligomerization half-lives of 60 and 800 min at 37 °C for ClyAox and ClyAred, respectively (Fig. 2E and Table 2). Even at 4 °C, slow ClyA oligomerization occurred under these conditions with half-lives of 103 and 1410 h for ClyAox and ClyAred, respectively (Table 2). Together, these results show that the spontaneous formation of the species SO and LO is comparably slow and an intrinsic property of both ClyA redox forms ClyAox oligomerizes about 14 times faster than ClyAred at both 4 and 37 °C. Oligomerization is strongly favored at the physiological temperature of 37 °C. In addition, Fig. 2C demonstrates that Mox, in contrast to previous reports describing the inability of ClyAox to form pores (
Fig. 2D shows that the small oligomeric form SOred was much less populated than SOox during spontaneous oligomerization of ClyA in the absence of detergent, so that we could not separate SOred from the much larger LOox peak. However, we could isolate LOred by preparative gel filtration. Like LOox, LOred proved to be assembly-incompetent upon the addition of 0.1% DDM, whereas Mred efficiently assembled into pore complexes as shown previously (
) (Fig. 2C, lower panels). Thus, all oligomeric forms of ClyA that formed in the absence of detergent and could be isolated proved to be assembly-incompetent in vitro in the presence of DDM in a time frame in which monomeric ClyA was quantitatively incorporated into pores.
The Hemolytic Activity of Oligomeric Forms of Oxidized and Reduced ClyA Is Decreased 100-Fold Relative to the Respective Monomeric Forms
We next tested the ability of the purified oligomeric forms LOox, SOox, and LOred to form active pores in target cells. To this end, we compared their lytic activity toward horse erythrocytes with the hemolytic activity of the monomers Mox and Mred. Fig. 3A shows the hemolysis kinetics at pH 7.3 and 37 °C initiated by mixing erythrocyte suspensions (2 × 106 cells/ml) with LOox, SOox, LOred, Mox, or Mred at identical total monomer concentrations of 10 nm. The reaction was recorded via the decrease in optical density at 650 nm as a measure of erythrocyte lysis. Whereas Mox and Mred proved to be highly active and, after a lag time of about 100 s, caused complete lysis within 200 s, the oligomers LOox, SOox, and LOred showed a strongly reduced activity, with lysis half-lives above 2000 s (Fig. 3A). For quantification of the specific hemolytic activities of all purified ClyA species, we used the recently established linear dependence of the maximum lysis velocity (see “Experimental Procedures” for the definition) for ClyA concentration in the range of 1 to 100 nm ClyA monomer (
). Fig. 3B and Table 3 show that freshly prepared Mox and Mred had high specific hemolytic activity, with Mox even 1.2 times more active than Mred (1.86 ± 0.14 mOD s−1 nm−1 and 1.51 ± 0.07 mOD s−1 nm−1, respectively). In contrast, the specific activities of the oligomers of ClyAred and ClyAox were dramatically reduced to 0.9% (LOox and LOred) and 1.4% (SOox) of the activity of Mred (Fig. 3B and Table 3). These results demonstrate that the spontaneous formation of ClyA oligomers in the absence of detergent or membranes inhibits the formation of active pores in target cells. This finding provides strong evidence that the species LO and SO of both ClyA redox forms are off-pathway products of pore formation, reminiscent of oligomeric off-pathway species in the α-PFT equinatoxin II from the sea anemone Actinia equina (
) that neither back-react rapidly to active monomers nor integrate into membranes and then become functional pores. The minute hemolytic activity of the oligomers detected in Fig. 3B may result from a very slow dissociation of inactive oligomers to active monomers upon dilution to the low ClyA concentration ranges (1–100 nm) used in the hemolysis assays.
TABLE 3Specific hemolytic activities of the different forms of oxidized and reduced ClyA
). To test whether the contradictory data in the literature might be the result of the comparably rapid decrease in active monomers in preparations of ClyAox (Fig. 2E), we tested whether the decrease in the concentration of assembly-competent monomers upon oligomerization of ClyAox and ClyAred coincided with a loss of hemolytic activity. For this purpose, ClyAox and ClyAred were again incubated under the conditions described in Fig. 2E, which favor oligomerization (initial monomer concentration 5 μm, 37 °C, and pH 7.3). Samples were taken after different time intervals and analyzed for hemolytic activity as described above. Fig. 4 shows the recorded hemolysis profiles and the deduced kinetics of loss of hemolytic activity of ClyAox and ClyAred, which declined within a factor of 1.6 with the same half-lives as the corresponding concentrations of Mox and Mred (see Fig. 2E and Table 2). At 4 °C, the half-lives of hemolytic activity loss of ClyAox and ClyAred were about 50-fold longer compared with those recorded at 37 °C (2.6 and 16.9 days, respectively (Table 2)), showing that storage of ClyAox at 4 °C for 2 weeks leads to practically complete loss of activity. These results thus provide a plausible explanation for the fact that previous experiments had been interpreted such that ClyAox is assembly-incompetent (
ClyAox Not Only Forms Annular Pore Complexes but Even Assembles Faster Than ClyAred
To identify potential differences in the assembly mechanisms of ClyAox and ClyAred, we first compared the kinetics of the monomer-to-protomer transition and the kinetics of pore formation of ClyAox and ClyAred in the presence of 0.1% DDM by far-UV CD spectroscopy. In the case of ClyAred, DDM triggered a rapid CD signal intensity increase, corresponding to the population of the off-pathway intermediate Ired, followed by formation of the protomer Pred with a more negative CD signal at 225 nm compared with Mred, whereas the oligomerization of Pred to pore complexes remained spectroscopically silent (
). We observed similar biphasic CD kinetics for ClyAox at 225 nm (Fig. 5, A and B). However, the initial increase in the CD signal was less pronounced for ClyAox (hinting at a lower transient population of the off-pathway intermediate), and the second, rate-limiting step of protomer formation proceeded 7.1 times faster (Fig. 5B). The CD spectra and CD kinetics (Fig. 5, A and B), together with the fact that oxidized and reduced monomers assembled to intact pore complexes in 0.1% DDM (Fig. 2C), thus provided a first hint that ClyAox and ClyAred formed assembly-competent protomers via the same three-state reaction mechanism (I ⇆ M ⇆ P) (
) and that ClyAox formed protomers faster than ClyAred.
To get more quantitative information on the conformational states of ClyAox populated during DDM-induced protomer formation, we next investigated the kinetics of Pox from Mox by single-molecule FRET experiments as described previously for ClyAred (
). To this end, we used a ClyAox variant labeled with the FRET pair Alexa Fluor 488 and Alexa Fluor 594 at cysteine residues introduced at position 56 and 252, respectively, showing distinct conformation-specific transfer efficiencies (〈E〉) for Mox, Iox, and Pox (
) (Fig. 1). We used subnanomolar concentrations of ClyA that prevented protomer assembly, so that only the four unimolecular reactions in the scheme (Iox ⇆ Mox ⇆ Pox) were observed. The time course of the transfer efficiency (〈E〉) histograms was fully consistent with the expected conversion of Mox (〈E〉 = 0.42) to Pox (〈E〉 = 0.67) with the transient population of the off-pathway intermediate Iox (〈E〉 = 0.20) (Fig. 5C). Compared with ClyAred (
) (Fig. 5D, dotted lines), the formation of Pox was however about one magnitude faster than Pred and virtually completed within 200 s (Fig. 5D), and the intermediate Iox was less populated. Compared with Ired, shown to have molten globule-like characteristics (
) (Fig. 5, C and E), indicating a more compact conformation of Iox in comparison with Ired, likely due to the covalent linkage of helices B and G by the Cys-87–Cys-285 disulfide bond. Table 2 compares the microscopic rate constants obtained for the three-state mechanism of protomer formation of ClyAox and ClyAred. The results are that the intermediate Iox is 0.5 kJ/mol less stable than Mox, whereas Ired proved to be 4.4 kJ/mol more stable than Mred (
) (Fig. 5F). Overall, ClyAox formed the protomer 7–8 times faster than ClyAred upon the addition of DDM (Fig. 5, B and D), mainly because of a much lower population of the off-pathway intermediate, I (maximum, 30% Iox after 10 s (Fig. 5D)) and, to a smaller extent, because of about a two times faster formation of P from M (Table 2).
Finally, we also compared the kinetics of protomer association and pore assembly of ClyAox and ClyAred by single-molecule FRET experiments at higher protein concentrations (10 nm), which allowed oligomerization to intact pores. This was achieved by adding an excess of the corresponding unlabeled ClyA redox form to donor/acceptor-labeled ClyA as described previously (
), Pox was converted to a state with lower transfer efficiency (〈E〉 = 0.53 versus 0.67 for Pox) representing a mixture (Oox) of annular pores and emerging, incomplete pores that cannot be distinguished spectroscopically (Fig. 5E) (
). These results clearly confirmed the assembly competence of Pox as demonstrated by electron microscopy (see Fig. 2C) and hemolysis experiments (Fig. 3). Moreover, the results showed that the assembly of Pox and Pred occurred within comparable time frames (Fig. 5E). In summary, the single-molecule FRET measurements showed that the DDM-induced monomer-to-protomer transition of ClyAox follows the same kinetic model as observed for ClyAred.
The Disulfide of ClyAox Is Not Susceptible to Reduction Independently of the Oligomerization State
The in vivo role of periplasmic ClyAox has remained enigmatic, in particular because several groups have reported that ClyAox has a lower intrinsic hemolytic activity than ClyAred (
), a finding that agrees with the observation that strains deficient in the periplasmic dithiol oxidase DsbA, which introduces disulfide bonds into periplasmic proteins, show ClyA-dependent hemolytic activity, whereas respective wild-type strains are hemolytically inactive (
). To investigate the possibility of a regulated change in the ClyA redox state in vivo, we tested the accessibility of the disulfide bond of ClyAox for reduction by DTT. Fig. 6A shows that Mox, LOox, and oxidized pore complexes were reduced only extremely slowly and remained more than 90% oxidized after 3 h even in 100 mm DTT. In contrast, unfolded Mox was completely reduced within 5 min by 20 mm DTT under the same conditions. The deduced rate constants of reduction (see legend to Fig. 6A) revealed that the tertiary structure of Mox, LOox, and Pox in pore complexes protected the Cys-87–Cys-285 disulfide bond 105-106-fold against reduction relative to unfolded Mox. The reduction of ClyAox by disulfide exchange at physiologically relevant rates would thus require unfolding, which is also consistent with the inaccessibility to solvent of the Cys-87/Cys-285 cysteine pair in the structures of the monomer and protomer (Fig. 6C). Finally, we also recorded the kinetics of oxidation of Mred by DsbA. This reaction also proved to be extremely slow at physiological concentrations of DsbAox (Fig. 6B). The results indicate that the formation of ClyAox from folded ClyAred in the periplasm via disulfide exchange with DsbA would require the unfolding of Mred.
The Hemolytic Activity and Pore Formation Competence of ClyAox
In the present study, we addressed two controversies in the literature on the assembly mechanism of ClyA. The first controversy is related to contradictory reports on the ability of ClyAox to form pore complexes. Several reports have suggested that formation of the Cys-87–Cys-285 disulfide bond in ClyA prevents pore complex formation (
). A loss of assembly competence of ClyAox was proposed based on the findings that ClyA in OMVs secreted by cytotoxic E. coli strains is present in the reduced form and that deletion of the periplasmic dithiol oxidase DsbA, which oxidizes ClyA in the periplasm, restores hemolytic activity (
). In addition, treatment of ClyA with excess Cu(phenanthroline)2, an established reagent for the oxidation of cysteine pairs in vitro, led to a loss of hemolytic activity that could be partially recovered by treatment with DTT, and a ClyA variant in which both cysteines were replaced by serine residues stayed active independently of the redox conditions (
We were able to resolve this controversy with our present study, in which we compared the mechanism of DDM-induced assembly of ClyAox and ClyAred in detail in a quantitative manner. The key proved to be the important recent discovery by Fahie et al. (
) that ClyA shows a tendency toward spontaneous formation of larger oligomers in the absence of detergents, in particular at elevated temperatures. We thus compared ClyAox and ClyAred for their ability to form oligomers spontaneously. For this purpose, we first established the quantitative absence and presence of the Cys-87–Cys-285 disulfide bond in our ClyA preparations (Fig. 2B), and we then prepared ClyAox under mild oxidative conditions by using catalytic concentrations of Cu2+ ions for Cu2+-catalyzed air oxidation. We fully confirmed the formation of two main oligomeric species for ClyAred reported previously (
). In addition, however, we showed that ClyAox forms soluble oligomer 13–14 times faster than ClyAred, that oligomer formation is favored at elevated temperatures (Table 2), and that all soluble oligomeric ClyA forms are hemolytically inactive (see below and Table 3). Thus, the formation of assembly-incompetent oligomers in preparations of ClyAox provides a plausible explanation for the interpretations of previous studies that ClyAox is hemolytically inactive. Specifically, we have shown that storage of ClyAox at 4 °C for 3 days already causes a loss of 50% of its hemolytic activity. Therefore, monomers of ClyAox, freshly isolated from gel filtration chromatography, should be used immediately for assembly studies or kept frozen. We also suggest that ClyAred should be prepared and stored in the presence of reducing agents such as DTT to prevent air oxidation.
We analyzed the kinetic mechanism of DDM-induced pore formation for monomeric ClyAox and ClyAred in detail, which clearly revealed that ClyAox is not only fully assembly-competent and forms pores with even 20% higher specific hemolytic activity than pores of ClyAred (Fig. 3B), but also follows the same reaction mechanism in which the formation of assembly-competent protomers is the rate-limiting step of pore formation at total monomer concentrations above ∼100 nm. Protomer formation also is accompanied by the formation of a molten globule-like off-pathway intermediate. Notably, our single-molecule FRET experiments showed that the intermediate Iox is less stable than Ired and therefore much less populated during protomer formation, so that assembly-competent protomers Pox even form about one order of magnitude faster than protomers of ClyAred (Pred) (Fig. 5). The formation of the Cys-87–Cys-285 disulfide bridge between helixes B and G thus accelerates protomer formation by decreasing the population of off-pathway intermediates during DDM-triggered pore formation.
ClyA in the periplasm of E. coli strains containing DsbA has been reported to be oxidized and to exhibit no cytotoxic activity (
). It remains to be established whether ClyAox also assembles to inactive oligomers in the periplasm, analogous to its spontaneous assembly to oligomers in vitro. A recent study reported the co-localization of DsbA and ClyA in OMVs (
), where the reduced form is the main ClyA redox species. Our experiments show that the Cys-87/Cys-285 pair in folded ClyAred monomers cannot be oxidized efficiently by DsbA (Fig. 6B). This makes it unlikely that ClyAox in OMVs needs to be activated by an unknown reducing agent, as proposed previously (
). An alternative explanation for the presence of ClyAred in OMVs could be that ClyAred is the predominant ClyA species in OMVs because it folds rapidly so that DsbA cannot access the Cys-87/Cys-285 pair and the ClyA molecules that stay in the periplasm are oxidized by molecular oxygen under aerobic conditions. Unraveling the translocation mechanism by which ClyA enters the periplasm in a Sec-independent manner appears to be essential for answering this open question.
Are Soluble ClyA Oligomers Really Intermediates of an Alternative Pore Formation Pathway?
), together with the present data on the kinetic mechanism of DDM-induced pore formation of ClyA, clearly establishes a reaction pathway in which the unimolecular formation of protomers from monomers, with the transient population of a molten globule-like off-pathway intermediate, is the rate-limiting step of pore assembly at ClyA concentrations above ∼100 nm. Here, we also examined the alternative ClyA assembly mechanism proposed by Fahie et al. (
), in which soluble ClyA oligomers, formed in the absence of detergent or membranes, represent prepores that can integrate into membranes and form hemolytically active pore complexes. We purified soluble oligomers of ClyAox and ClyAred by gel filtration, but could detect neither a significant population of annular complexes by electron microscopy after addition of detergent (Fig. 2C) nor hemolytic activity above 1.4% relative to that of the reduced monomer after mixing the oligomers with erythrocytes (Fig. 3B). The interpretation of ClyA oligomers as prepore complexes by Fahie et al. (
) is based on findings that the oligomers showed hemolytic activity in end point hemolysis assays (see below) and single-channel conductance activity after reconstitution into planar lipid bilayers. So why did the work of Fahie et al. (
) is the fact that the dependence of hemolytic activity of ClyA oligomers on ClyA concentration was not investigated. Erythrocytes were incubated for 15 min at a total ClyA concentration of 0.8 μm, and the amount of hemoglobin released after 15 min by ClyA oligomers and monomers was compared and used as a measure of hemolytic activity (
). Our Fig. 3, however, shows that the amount of lysed cells at a single time point is not a quantitative measure of hemolytic activity. Instead, the hemolytic activity of ClyA can be quantified accurately only via the dependence of maximum lysis velocity (or the inverse lag time of hemolysis (
)) on ClyA concentration (Fig. 2B). Therefore, determination of the specific hemolytic activity of ClyA requires the recording of hemolysis kinetics at different ClyA concentrations. Fig. 3A shows that the oligomers LOred, LOox, and SOox, already at concentrations of 10 nm, cause significant hemolysis after an incubation time of 1 h, for example, although their specific hemolytic activity relative to the monomer is only about 1%. In fact, ClyA monomers are highly active toxins, and the incubation of erythrocytes with only 10 nm ClyA monomers leads to complete lysis within 200 s (Fig. 3A). The experiments by Fahie et al. (
) were however performed at 80-fold higher ClyA concentrations, and ClyA activity was deduced from the amount of lysed cells after a single time point (15 min). Fig. 3 shows that we would have detected similarly high hemolytic activity in our preparation of purified ClyA oligomers if we had performed our experiments under the same conditions, i.e. at ClyA concentrations of 800 nm.
So what is the reason underlying the low apparent hemolytic activity of ClyA oligomers detected by us and Fahie et al.? A rough estimation based on the concentration dependence of ClyA activity (Fig. 3B) shows that even a minute fraction of about 0.1% of monomers spontaneously dissociating from the oligomers and reassembling into active pores via the classical assembly pathway can readily explain the hemolytic and single-channel conductance activity of oligomers detected at the high ClyA oligomer concentrations used by Fahie et al. (
). Nevertheless, we cannot completely exclude the possibility that ClyA oligomers react directly to active pores after insertion into erythrocyte membranes, but we believe that this reaction is far too slow to be physiologically relevant.
R. G., D. R., and B. S. designed the research. S. B. and D. R. prepared protein samples and performed the experiments. D. R., S. B., B. S., and R. G. analyzed and interpreted the data. D. R. and R. G. wrote the manuscript with the help of the other authors.
We thank Hiang Dreher and Helene Fäh-Rechsteiner for excellent technical support, Daniel Nettels for discussion and help with data analysis, and Bengt Wunderlich for help in the initial stages of the microfluidic mixing experiments. Electron micrographs were recorded using the equipment of the Scientific Center for Optical and Electron Microscopy (SCOPEM) at ETH Zurich.
van der Goot F.G.
Bacterial pore-forming toxins: the (w)hole story?.