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Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly*

  • Daniel Roderer
    Correspondence
    To whom correspondence should be addressed: Dept. of Structural Biochemistry, Max Planck Inst. of Molecular Physiology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany. Tel.: 49-231-1332312;
    Affiliations
    From the Institute of Molecular Biology and Biophysics ETH Zurich, Otto-Stern-Weg 5, CH-8093 Zurich, Switzerland and
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  • Stephan Benke
    Affiliations
    the Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
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  • Benjamin Schuler
    Affiliations
    the Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
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  • Rudi Glockshuber
    Affiliations
    From the Institute of Molecular Biology and Biophysics ETH Zurich, Otto-Stern-Weg 5, CH-8093 Zurich, Switzerland and
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  • Author Footnotes
    * 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.
Open AccessPublished:January 12, 2016DOI:https://doi.org/10.1074/jbc.M115.700757
      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.

      Introduction

      Pore-forming toxins (PFTs)
      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 (
      • Gonzalez M.R.
      • Bischofberger M.
      • Pernot L.
      • van der Goot F.G.
      • Frêche B.
      Bacterial pore-forming toxins: the (w)hole story?.
      ). Some of the most potent bacterial toxins are PFTs, such as anthrax toxin (
      • Blaustein R.O.
      • Koehler T.M.
      • Collier R.J.
      • Finkelstein A.
      Anthrax toxin: channel-forming activity of protective antigen in planar phospholipid bilayers.
      ) and cytolysin from Vibrio cholerae (
      • Olson R.
      • Gouaux E.
      Crystal structure of the Vibrio cholerae cytolysin (VCC) pro-toxin and its assembly into a heptameric transmembrane pore.
      ). A common feature of all PFTs is the conversion from a soluble, monomeric form into a membrane-embedded oligomeric pore complex (
      • Gonzalez M.R.
      • Bischofberger M.
      • Pernot L.
      • van der Goot F.G.
      • Frêche B.
      Bacterial pore-forming toxins: the (w)hole story?.
      ). The membrane-spanning region of the pore can be formed either by α-helices or β-strands; therefore, PFTs are classified as α-PFTs and β-PFTs (
      • Parker M.W.
      • Feil S.C.
      Pore-forming protein toxins: from structure to function.
      ).
      The 34-kDa PFT Cytolysin A (ClyA, also termed Hemolysin E (HlyE)) is an α-PFT existing in various Escherichia coli and Salmonella enterica strains (
      • del Castillo F.J.
      • Leal S.C.
      • Moreno F.
      • del Castillo I.
      The Escherichia coli K-12 sheA gene encodes a 34-kDa secreted haemolysin.
      • Ludwig A.
      • von Rhein C.
      • Bauer S.
      • Hüttinger C.
      • Goebel W.
      Molecular analysis of cytolysin A (ClyA) in pathogenic Escherichia coli strains.
      ,
      • Huang L.J.
      • Cui J.
      • Piao H.H.
      • Hong Y.
      • Choy H.E.
      • Ryu P.Y.
      Molecular cloning and characterization of clyA genes in various serotypes of Salmonella enterica.
      ,
      • Oscarsson J.
      • Westermark M.
      • Löfdahl S.
      • Olsen B.
      • Palmgren H.
      • Mizunoe Y.
      • Wai S.N.
      • Uhlin B.E.
      Characterization of a pore-forming cytotoxin expressed by Salmonella enterica serovars typhi and paratyphi A.
      ,
      • von Rhein C.
      • Hunfeld K.P.
      • Ludwig A.
      Serologic evidence for effective production of cytolysin A in Salmonella enterica serovars typhi and paratyphi A during human infection.
      • von Rhein C.
      • Bauer S.
      • López Sanjurjo E.J.
      • Benz R.
      • Goebel W.
      • Ludwig A.
      ClyA cytolysin from Salmonella: distribution within the genus, regulation of expression by SlyA, and pore-forming characteristics.
      ). The structure of the annular, dodecameric pore complex of E. coli ClyA has been solved to atomic resolution (Protein Data Bank ID: 2WCD) (
      • Mueller M.
      • Grauschopf U.
      • Maier T.
      • Glockshuber R.
      • Ban N.
      The structure of a cytolytic α-helical toxin pore reveals its assembly mechanism.
      ). The pore subunit (protomer) shows major structural differences when compared with the soluble ClyA monomer (Protein Data Bank ID: 1QOY) (
      • Wallace A.J.
      • Stillman T.J.
      • Atkins A.
      • Jamieson S.J.
      • Bullough P.A.
      • Green J.
      • Artymiuk P.J.
      E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy.
      ): 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 (
      • Wallace A.J.
      • Stillman T.J.
      • Atkins A.
      • Jamieson S.J.
      • Bullough P.A.
      • Green J.
      • Artymiuk P.J.
      E. coli hemolysin E (HlyE, ClyA, SheA): X-ray crystal structure of the toxin and observation of membrane pores by electron microscopy.
      ,
      • Atkins A.
      • Wyborn N.R.
      • Wallace A.J.
      • Stillman T.J.
      • Black L.K.
      • Fielding A.B.
      • Hisakado M.
      • Artymiuk P.J.
      • Green J.
      Structure-function relationships of a novel bacterial toxin, hemolysin E: the role of αG.
      • Eifler N.
      • Vetsch M.
      • Gregorini M.
      • Ringler P.
      • Chami M.
      • Philippsen A.
      • Fritz A.
      • Müller S.A.
      • Glockshuber R.
      • Engel A.
      • Grauschopf U.
      Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
      ). During pore formation, ClyA undergoes major structural rearrangements involving more than 50% of all of its amino acids (
      • Mueller M.
      • Grauschopf U.
      • Maier T.
      • Glockshuber R.
      • Ban N.
      The structure of a cytolytic α-helical toxin pore reveals its assembly mechanism.
      ). 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 (
      • Mueller M.
      • Grauschopf U.
      • Maier T.
      • Glockshuber R.
      • Ban N.
      The structure of a cytolytic α-helical toxin pore reveals its assembly mechanism.
      ). 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 (
      • Fass D.
      Disulfide bonding in protein biophysics.
      ). The formation of the assembly-competent protomer is the rate-limiting step (
      • Eifler N.
      • Vetsch M.
      • Gregorini M.
      • Ringler P.
      • Chami M.
      • Philippsen A.
      • Fritz A.
      • Müller S.A.
      • Glockshuber R.
      • Engel A.
      • Grauschopf U.
      Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
      ) and the prerequisite (
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      ) for pore formation.
      Figure thumbnail gr1
      FIGURE 1.Ribbon diagrams showing the positions of the natural cysteine residues 87 and 285 in the ClyA monomer (left) and protomer (middle) in the context of the homododecameric ClyA pore (right). The sulfur atoms of both residues are shown as yellow spheres. Two additional cysteine residues were introduced for site-specific labeling with Alexa Fluor 488 (green star) at position 56 and Alexa Fluor 594 (red star) at position 252 for monitoring the monomer-to-protomer transition by FRET. The FRET efficiencies for both conformational states, calculated based on the respective Cα-Cα distances, are indicated. The α-helix G containing Cys-285 is colored blue, and segment 81–90 of α-helix B containing Cys-87 is depicted in red. The β-tongue of the ClyA monomer and the respective residues in the ClyA protomer are shown in yellow. This figure was prepared using PyMOL (
      • DeLano W.L.
      ).
      Recently, a new study on ClyA activation and pore assembly proposed an alternative mechanism of pore assembly (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ). 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 (
      • Degiacomi M.T.
      • Iacovache I.
      • Pernot L.
      • Chami M.
      • Kudryashev M.
      • Stahlberg H.
      • van der Goot F.G.
      • Dal Peraro M.
      Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism.
      ,
      • Petosa C.
      • Collier R.J.
      • Klimpel K.R.
      • Leppla S.H.
      • Liddington R.C.
      Crystal structure of the anthrax toxin protective antigen.
      ).
      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 (
      • Atkins A.
      • Wyborn N.R.
      • Wallace A.J.
      • Stillman T.J.
      • Black L.K.
      • Fielding A.B.
      • Hisakado M.
      • Artymiuk P.J.
      • Green J.
      Structure-function relationships of a novel bacterial toxin, hemolysin E: the role of αG.
      ,
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ,
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ) or as assembly competent (
      • Eifler N.
      • Vetsch M.
      • Gregorini M.
      • Ringler P.
      • Chami M.
      • Philippsen A.
      • Fritz A.
      • Müller S.A.
      • Glockshuber R.
      • Engel A.
      • Grauschopf U.
      Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
      ). 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 (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). 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.

      Experimental Procedures

      Materials

      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 (
      • Eifler N.
      • Vetsch M.
      • Gregorini M.
      • Ringler P.
      • Chami M.
      • Philippsen A.
      • Fritz A.
      • Müller S.A.
      • Glockshuber R.
      • Engel A.
      • Grauschopf U.
      Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
      ). ClyA production in E. coli was carried out at 20 °C for 15 h, as described (
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      ). ClyA was purified from the soluble fraction of the cell extract by Ni2+-NTA affinity chromatography followed by chromatography on hydroxyapatite as described (
      • Eifler N.
      • Vetsch M.
      • Gregorini M.
      • Ringler P.
      • Chami M.
      • Philippsen A.
      • Fritz A.
      • Müller S.A.
      • Glockshuber R.
      • Engel A.
      • Grauschopf U.
      Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
      ). The N-terminal His6 tag of ClyA was then cleaved by recombinant His6-tagged TEV protease (
      • Finder V.H.
      • Vodopivec I.
      • Nitsch R.M.
      • Glockshuber R.
      The recombinant amyloid-β peptide Aβ1–42 aggregates faster and is more neurotoxic than synthetic Aβ1–42.
      ), and the resulting wild-type ClyA (residues Thr2-Val 303) was obtained in the flow-through of a second Ni2+-NTA affinity chromatography as described (
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      ). 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 (
      • Ellman G.L.
      A colorimetric method for determining low concentrations of mercaptans.
      ) 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.
      Figure thumbnail gr2
      FIGURE 2.Analysis of spontaneous oligomerization of ClyAox and ClyAred in the absence of detergent and competence of pore formation of purified ClyA oligomers. A, preparative gel filtration at pH 7.3 of oxidized ClyA after Cu2+-catalyzed air oxidation of ClyAred. Besides the oxidized monomer (Mox), a large (LOox) and a small (SOox) oligomeric species could be detected. The apparent molecular masses (in kDa) of LOox and SOox and the masses of calibration standard proteins are indicated. Mox eluted at an apparent molecular mass of 40 kDa. The height of 100 milliabsorbance units (mAU) is indicated. B, reversed phase HPLC analysis of ClyA after incubation of ClyAred at pH 7.3 and 22 °C for 3.5 h in the presence or absence of 0.4 mm free CuCl2, showing that ClyAred remained resistant to air oxidation in the absence of CuCl2, whereas it was oxidized quantitatively in the presence of CuCl2. Samples were separated on an analytical C8 column in 0.1% trifluoroacetic acid and eluted with a water/acetonitrile gradient. Due to the denaturing conditions, oligomeric ClyA dissociated so that only a single peak was detected in each run. The HPLC retention times corresponding to ClyAred and ClyAox are indicated. C, negative stain electron micrographs of samples of the purified ClyAox species LOox, SOox, and Mox (see A) and the isolated ClyAred species LOred and Mred (see D) (2 μm total monomer concentration in each sample) after incubation with 0.1% DDM for 1 h at 22 °C. The results show that only Mox and Mred were able to form intact pore complexes. Scale bar: 100 nm. D, comparison of the spontaneous oligomerization propensity of Mox (black lines) and Mred (red lines) (5 μm each) at 37 °C in PBS buffer, pH 7.3. Purified Mox or Mred was incubated for 0, 1, or 5 h at 37 °C, and the reaction products were separated at 22 °C in PBS on an Agilent 300S gel filtration column. The results show that Mox has a higher oligomerization tendency than Mred and that both redox species can form large and small oligomers. E, kinetics of spontaneous oligomerization of Mox and Mred (5 μm each) at 37 °C in PBS buffer, pH 7.3, analyzed via the decrease in the monomer peak. Data were normalized to the initial peak area of the monomer and were fitted to a first-order decay (solid lines), yielding apparent rate constants of 0.69 ± 0.02 h−1 for ClyAox and 0.052 ± 0.002 h−1 for ClyAred, respectively.
      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 (
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      ). 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 (
      • Crespo M.D.
      • Puorger C.
      • Schärer M.A.
      • Eidam O.
      • Grütter M.G.
      • Capitani G.
      • Glockshuber R.
      Quality control of disulfide bond formation in pilus subunits by the chaperone FimC.
      )) 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 (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). 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 (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ). 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 (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ).

      Negative Stain Transmission Electron Microscopy

      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.

      Hemolysis Kinetics

      The kinetics of ClyA-dependent lysis of horse erythrocytes were measured as described previously (
      • Rennie R.P.
      • Freer J.H.
      • Arbuthnott J.P.
      The kinetics of erythrocyte lysis by Escherichia coli haemolysin.
      ) 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 (
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      ).

      Circular Dichroism (CD) Spectroscopy

      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 (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). The measurements were recorded on a modified MicroTime 200 instrument (PicoQuant) using a setup for pulsed interleaved excitation (PIE (
      • Müller B.K.
      • Zaychikov E.
      • Bräuchle C.
      • Lamb D.C.
      Pulsed interleaved excitation.
      )) with two excitation pulses for the donor dye alternating with a single acceptor pulse. The initial phase of protomer formation was measured in a microfluidic mixer (
      • Wunderlich B.
      • Nettels D.
      • Benke S.
      • Clark J.
      • Weidner S.
      • Hofmann H.
      • Pfeil S.H.
      • Schuler B.
      Microfluidic mixer designed for performing single-molecule kinetics with confocal detection on timescales from milliseconds to minutes.
      ), 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
      Total ClyA concentrationTotal measurement timeRepeats usedTime window lengthBurst min/max
      s
      100 pm60 s (microfluidics)NANA40/300
      100 pm10 min216080/250
      100 pm30 min918080/250
      100 pm60 min290080/250
      10 nm15 min83080/250
      10 nm60 min430080/250
      100 nm5 min243080/250
      100 nm15 min136080/250
      100 nm30 min430080/250
      100 nm60 min260080/250
      The model fitting of the protomer formation was performed as described for the reduced variant (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). 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.

      Results

      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 (
      • Eifler N.
      • Vetsch M.
      • Gregorini M.
      • Ringler P.
      • Chami M.
      • Philippsen A.
      • Fritz A.
      • Müller S.A.
      • Glockshuber R.
      • Engel A.
      • Grauschopf U.
      Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
      ), consistent with the small Cα-Cα distances between the cysteines of 5.2 and 6.8 Å in the structures of the monomer and the protomer, respectively (
      • Fass D.
      Disulfide bonding in protein biophysics.
      ) (Fig. 1). Both ClyA redox forms have been described in vivo. Although ClyA lacks an N-terminal signal sequence (
      • del Castillo F.J.
      • Moreno F.
      • del Castillo I.
      Secretion of the Escherichia coli K-12 SheA hemolysin is independent of its cytolytic activity.
      ), it is secreted to the periplasm where it accumulates in its oxidized form (
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ,
      • Ludwig A.
      • Bauer S.
      • Benz R.
      • Bergmann B.
      • Goebel W.
      Analysis of the SlyA-controlled expression, subcellular localization, and pore-forming activity of a 34-kDa haemolysin (ClyA) from Escherichia coli K-12.
      ). In contrast, the assembled ClyA pore complexes that are exported by the bacteria to the extracellular medium in outer membrane vesicles (OMVs) are composed of reduced ClyA protomers (
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ). The mechanisms that underlie the secretion of ClyA into the periplasm and its assembly in OMVs are still unknown.
      The DDM-induced assembly of ClyA had been characterized in detail for the disulfide-free form of ClyA (ClyAred) (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). 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 (
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ) or its lack of cytotoxicity (
      • Atkins A.
      • Wyborn N.R.
      • Wallace A.J.
      • Stillman T.J.
      • Black L.K.
      • Fielding A.B.
      • Hisakado M.
      • Artymiuk P.J.
      • Green J.
      Structure-function relationships of a novel bacterial toxin, hemolysin E: the role of αG.
      ,
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ), readily assembles into annular pore complexes.
      TABLE 2Apparent rate constants of the decay of hemolytic activity of Mred and Mox as a consequence of spontaneous oligomerization in PBS in the absence of detergent or membranes
      ClyAredClyAox
      Decrease of hemolytic activities and monomer depletion
      ClyA was incubated at 5 μm monomer concentration.
      Decrease of hemolytic activity at 37 °C (h−1)
      See Fig. 4C.
      8.5 ± 0.7·10−25.3 ± 0.3·10−1
      Monomer depletion at 37 °C (h−1)
      See Fig. 2E.
      5.2 ± 0.2·10−26.9 ± 0.2·10−1
      Decrease in hemolytic activity at 4 °C (h−1)
      Data not shown.
      1.7 ± 0.1·10−31.1 ± 0.1·10−2
      Monomer depletion at 4 °C (h−1)
      Data not shown.
      4.9 ± 0.5·10−46.7 ± 0.2·10−3
      Kinetics of I ↔ M ↔ P determined by single-molecule FRET in DDM at 22 °C
      kMI (s−1)3.0 ± 0.3·10−1
      Benke et al. (21).
      1.2 ± 0.1·10−1
      Rate constants obtained by single-molecule FRET (see Fig.5D).
      kIM (s−1)5.0 ± 1.0·10−2
      Benke et al. (21).
      1.5 ± 0.1·10−1
      Rate constants obtained by single-molecule FRET (see Fig.5D).
      kMP (s−1)1.7 ± 0.6·10−2
      Benke et al. (21).
      4.7 ± 0.3·10−2
      Rate constants obtained by single-molecule FRET (see Fig.5D).
      kPM (s−1)4.7 ± 0.5·10−4
      Benke et al. (21).
      8.1 ± 0.2·10−3
      Rate constants obtained by single-molecule FRET (see Fig.5D).
      a ClyA was incubated at 5 μm monomer concentration.
      b See Fig. 4C.
      c See Fig. 2E.
      d Data not shown.
      e Benke et al. (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ).
      f Rate constants obtained by single-molecule FRET (see Fig.5D).
      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 (
      • Eifler N.
      • Vetsch M.
      • Gregorini M.
      • Ringler P.
      • Chami M.
      • Philippsen A.
      • Fritz A.
      • Müller S.A.
      • Glockshuber R.
      • Engel A.
      • Grauschopf U.
      Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
      ,
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      ) (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 (
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      ). 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 (
      • Rojko N.
      • Kristan K.Č.
      • Viero G.
      • Žerovnik E.
      • Maček P.
      • Dalla Serra M.
      • Anderluh G.
      Membrane damage by an α-helical pore-forming protein, equinatoxin II, proceeds through a succession of ordered steps.
      ) 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.
      Figure thumbnail gr3
      FIGURE 3.Competence of pore complex formation in the presence of membranes of the different association states of ClyAred and ClyAox. A, hemolytic activity of M, SO, and LO at identical total monomer concentrations of 10 nm. The purified species Mox, SOox, LOox, Mred, and LOred (10 nm monomer each) were mixed at 37 °C and pH 7.3 with horse erythrocytes (2 × 106 cells/ml). Erythrocyte lysis was followed via the decrease in optical density at 650 nm. Due to its low population, the species SOred could not be purified and separated from LOred by preparative gel filtration. Assays containing reduced ClyA additionally contained 2 mm DTT. B, quantification of the specific hemolytic activity of Mox, SOox, LOox, Mred, and LOred via the linear dependence of maximum lysis velocity on ClyA concentration (see “Experimental Procedures” and Ref.
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      for experimental details), showing that oligomeric species essentially lack hemolytic activity. The maximum lysis velocity (slope of OD650 decrease at the half-life of lysis) was plotted against the respective total monomer concentration. The following specific hemolytic activities (in mOD s−1 nm−1) were obtained: Mox, 1.86 ± 0.14; Mred, 1.51 ± 0.07; LOox, 0.013 ± 0.002; LOred, 0.013 ± 0.001; SOox, 0.021 ± 0.005 (indicated errors represent S.D. from three independent measurements).
      TABLE 3Specific hemolytic activities of the different forms of oxidized and reduced ClyA
      SampleSpecific hemolytic activity at 37 °CSpecific activity of Mred
      mOD s−1 nm−1%
      Mref1.51 ± 0.07100
      Mox1.86 ± 0.14123
      LOred0.013 ± 0.0010.9
      LOox0.013 ± 0.0020.9
      SOox0.021 ± 0.0051.4
      The high hemolytic activity of Mox contradicts the previously reported assembly incompetence of oxidized ClyA (
      • Atkins A.
      • Wyborn N.R.
      • Wallace A.J.
      • Stillman T.J.
      • Black L.K.
      • Fielding A.B.
      • Hisakado M.
      • Artymiuk P.J.
      • Green J.
      Structure-function relationships of a novel bacterial toxin, hemolysin E: the role of αG.
      ,
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ,
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ). 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 (
      • Atkins A.
      • Wyborn N.R.
      • Wallace A.J.
      • Stillman T.J.
      • Black L.K.
      • Fielding A.B.
      • Hisakado M.
      • Artymiuk P.J.
      • Green J.
      Structure-function relationships of a novel bacterial toxin, hemolysin E: the role of αG.
      ,
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ,
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ).
      Figure thumbnail gr4
      FIGURE 4.The loss of hemolytic activity of ClyAox and ClyAred coincides with the decrease in ClyA monomer concentration during spontaneous ClyA oligomerization at 37 °C in the absence of detergent or lipids. A and B, hemolysis kinetics initiated by the addition of ClyAox (A) or ClyAred (B) after different times of oligomerization to horse erythrocytes. The purified monomers Mox and Mred were incubated at 37 °C and pH 7.3 at a concentration of 5 μm for the indicated time intervals an diluted 500-fold with horse erythrocytes (final concentrations, 2 × 106 cells/ml; 10 nm total monomer concentration), and the decrease in cell density was followed via the decrease in the optical density at 650 nm. C, kinetics of the decrease in hemolytic activity of ClyAox and ClyAred at 37 °C and pH 7.3. The maximum lysis velocity as a measure of hemolytic activity (see B) was plotted against incubation time. The decay in hemolytic activity yielded apparent rate constants of 0.53 ± 0.03 h−1 for ClyAox and 0.085 ± 0.007 h−1 for ClyAred, respectively (solid lines).

      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 (
      • Eifler N.
      • Vetsch M.
      • Gregorini M.
      • Ringler P.
      • Chami M.
      • Philippsen A.
      • Fritz A.
      • Müller S.A.
      • Glockshuber R.
      • Engel A.
      • Grauschopf U.
      Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
      ). 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) (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ) and that ClyAox formed protomers faster than ClyAred.
      Figure thumbnail gr5
      FIGURE 5.Both oxidized and reduced ClyA monomers assemble via the same mechanism into functional pore complexes after the addition of the detergent DDM. A, far-UV CD spectra of ClyAred and ClyAox monomers (solid lines) and after assembly to pores in 0.1% DDM at pH 7.3 (dashed lines), demonstrating the increased α-helix content of the protomers in the assembled pore complexes. The total monomer concentration in each sample was 9 μm. B, reaction of Mox (gray symbols) and Mred (red symbols) to assembly-competent protomers, recorded via the change in the far-UV CD signal at 225 nm. Reactions were initiated by the addition of DDM (manual mixing). The gray and red squares represent the CD signals of monomeric ClyAox and ClyAred, respectively, in the absence of DDM. The rate-limiting step of monomer-to-protomer transition was fitted monoexponentially, resulting in apparent rates of protomer formation of 9.64 ± 0.02 × 10−3 s−1 and 1.35 ± 0.01 × 10−3 s−1 for ClyAox and ClyAred, respectively. C and D, single-molecule FRET measurements of the kinetics of the monomer-to-protomer transition of ClyAox labeled with Alexa Fluor 488 and 594. Protomer formation was initiated by the addition of 0.1% DDM, and the reactions were performed at low ClyA concentrations (300 pm), where oligomerization to pore complexes does not occur (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). CD signal intensities are shown as mean residue molar ellipticity (MRW). C, transfer efficiency histograms for the monomer-to-protomer transition of ClyAox in DDM measured by microfluidics and manual mixing. Each line represents one histogram normalized to a total area of 1. Line colors indicate the reaction time after addition of DDM. The order of the colors is indicated on the color bar on the right. Iox, off-pathway intermediate; Mox, monomer; Pox, protomer; r.e.f., relative event frequency. D, time course of the populations of Mox, Iox, and Pox after initiation of protomer formation by the addition of DDM. Top panel, kinetic model with four rate constants used to fit the kinetic data. Middle panel, sum of squared residuals (SSR) of the kinetic model fit (line) and the free population fit (dots) to the individual histograms. Bottom panel, population time courses for Mox, Iox, and Pox (blue, green, and red squares, respectively) fitted to the model with the off-pathway intermediate indicated at the top (solid lines). The dotted lines indicate the previously determined kinetics of protomer formation of ClyAred under identical conditions and fitted to the same reaction mechanism (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). E, dependence on ClyA subunit concentration of the kinetics of DDM-induced pore complex formation of ClyAred and ClyAox recorded by single-molecule FRET experiments. Conditions were identical to those described in C and D, but reactions were performed at 10 nm ClyA subunit concentration allowing oligomerization of assembly-competent protomers and pore complex formation. The higher protein concentrations were achieved by mixing 300 pm FRET-labeled ClyA with excess unlabeled WT ClyA with the same redox state. Emerging and final pore complexes of ClyAox (left panel) show identical transfer efficiencies of ∼0.53 and appear as a single peak, (Oox)n (orange) as observed for ClyAred (right panel). The transfer efficiencies of Mox, Iox, and Pox (0.42, 0.20, and 0.67, respectively) and of Mred, Ired, and Pred (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ) are also indicated. Transfer efficiency histograms were recorded after different reaction times, with color codes indicated in the bars at the top right of each panel. F, schemes of free energies (in kJ/mol) of M, I, and P of ClyAred and ClyAox in 0.1% DDM at pH 7.3 and 22 °C. The free energies of Mox and Mred were assumed to be identical. Iox is 0.5 kJ/mol less stable than Mox, whereas Ired is 4.4 kJ/mol more stable than Mred. Pox is only 4.3 kJ/mol less stable than Mox, whereas Pred is 8.9 kJ/mol more stable than Mred.
      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 (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). 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 (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ) (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 (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ) (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 (
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      ,
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ), Iox showed an increased transfer efficiency (〈E〉 = 0.20 versus 0.12) (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ) (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 (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). However, Pox was only 4.3 kJ/mol more stable than Mox, whereas Pred is 8.9 kJ/mol more stable than Mred (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ) (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 (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ) (see also “Experimental Procedures” and the legend to Fig. 5E for the details). As observed for ClyAred (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ), 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) (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ). 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 (
      • Atkins A.
      • Wyborn N.R.
      • Wallace A.J.
      • Stillman T.J.
      • Black L.K.
      • Fielding A.B.
      • Hisakado M.
      • Artymiuk P.J.
      • Green J.
      Structure-function relationships of a novel bacterial toxin, hemolysin E: the role of αG.
      ,
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ,
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ), 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 (
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ). 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.
      Figure thumbnail gr6
      FIGURE 6.The natural cysteine pair Cys-87/Cys-285 of ClyA is buried and resistant to reduction and oxidation via disulfide exchange. A, kinetics of the reduction of Mox, LOox, and assembled pores of ClyAox by 100 mm DTT at 37 °C and pH 7.3, and identical total monomer concentrations of 5 μm (the reaction with assembled pores also contained 0.1% DDM). As a control, unfolded ClyA in 4 m guanidinium chloride was reduced at 37 °C and pH 7.3 with 20 mm DTT. The reactions were stopped after different times by the addition of formic acid (16% final concentration), and oxidized and reduced ClyA was analyzed via RP-HPLC (see B). The decrease in the fraction of ClyAox was fitted according to a pseudo first-order reaction, resulting in rate constants of 1.1 ± 0.1 m−1 s−1 for unfolded ClyA, 1.9 ± 1.1 × 10−5 m−1 s−1 for Mox, 1.2 ± 0.2 × 10−5 m−1 s−1 for LOox, and 1.9 ± 0.2 × 10−6 m−1 s−1 for the oxidized ClyA pore complex. B, oxidation of monomeric ClyAred (5 μm) by 86 μm DsbAox (in vivo concentration in E. coli (
      • Crespo M.D.
      • Puorger C.
      • Schärer M.A.
      • Eidam O.
      • Grütter M.G.
      • Capitani G.
      • Glockshuber R.
      Quality control of disulfide bond formation in pilus subunits by the chaperone FimC.
      )) at 37 °C and pH 7.3. The decrease in the fraction of ClyAred was fitted according to a pseudo first-order kinetics, yielding a rate constant of 4.3 ± 0.1 × 10−2 m−1 s−1 for the oxidation of ClyAred by DsbAox. C, surface representation of the ClyA monomer (left) and the ClyA protomer (right) in the region around the cysteine pair Cys-87/Cys-285, showing that the sulfur atoms of Cys-87 and Cys-285 are buried in both structures. The positions of α-helixes B (red) and G (blue) and the C termini are indicated. The sulfur atoms of Cys-87 and Cys-285 are shown as yellow spheres. This figure was illustrated in PyMOL (
      • DeLano W.L.
      ).

      Discussion

      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 (
      • Atkins A.
      • Wyborn N.R.
      • Wallace A.J.
      • Stillman T.J.
      • Black L.K.
      • Fielding A.B.
      • Hisakado M.
      • Artymiuk P.J.
      • Green J.
      Structure-function relationships of a novel bacterial toxin, hemolysin E: the role of αG.
      ,
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ,
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ). 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 (
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ). 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 (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ). Based on these results, it was speculated that formation of the Cys-87–Cys-285 disulfide bond prevents the conformational transition to the assembly-competent protomers (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ). All of these data were however in contrast to previous findings that ClyAox forms active pores upon the addition of DDM in vitro and that both ClyA redox forms show comparable hemolytic activity (
      • Eifler N.
      • Vetsch M.
      • Gregorini M.
      • Ringler P.
      • Chami M.
      • Philippsen A.
      • Fritz A.
      • Müller S.A.
      • Glockshuber R.
      • Engel A.
      • Grauschopf U.
      Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.
      ).
      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. (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ) 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 (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ). 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 (
      • Atkins A.
      • Wyborn N.R.
      • Wallace A.J.
      • Stillman T.J.
      • Black L.K.
      • Fielding A.B.
      • Hisakado M.
      • Artymiuk P.J.
      • Green J.
      Structure-function relationships of a novel bacterial toxin, hemolysin E: the role of αG.
      ,
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ). 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 (
      • Kim J.Y.
      • Doody A.M.
      • Chen D.J.
      • Cremona G.H.
      • Shuler M.L.
      • Putnam D.
      • DeLisa M.P.
      Engineered bacterial outer membrane vesicles with enhanced functionality.
      ), 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 (
      • Wai S.N.
      • Lindmark B.
      • Söderblom T.
      • Takade A.
      • Westermark M.
      • Oscarsson J.
      • Jass J.
      • Richter-Dahlfors A.
      • Mizunoe Y.
      • Uhlin B.E.
      Vesicle-mediated export and assembly of pore-forming oligomers of the enterobacterial ClyA cytotoxin.
      ). 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?

      Our previous work (
      • Benke S.
      • Roderer D.
      • Wunderlich B.
      • Nettels D.
      • Glockshuber R.
      • Schuler B.
      The assembly dynamics of the cytolytic pore toxin ClyA.
      ), 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. (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ), 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. (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ) 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. (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ) and our present data lead to opposite conclusions on the role of soluble ClyA monomomers?
      We believe that the main reason for the interpretation by Fahie et al. (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ) 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 (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ). 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 (
      • Roderer D.
      • Benke S.
      • Müller M.
      • Fäh-Rechsteiner H.
      • Ban N.
      • Schuler B.
      • Glockshuber R.
      Characterization of variants of the pore-forming toxin ClyA from Escherichia coli controlled by a redox switch.
      )) 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. (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ) 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. (
      • Fahie M.
      • Romano F.B.
      • Chisholm C.
      • Heuck A.P.
      • Zbinden M.
      • Chen M.
      A non-classical assembly pathway of Escherichia coli pore-forming toxin cytolysin A.
      ). 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.

      Author Contributions

      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.

      Acknowledgments

      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.

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