Abstract
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.
Highlights
Pore-forming toxins (PFTs)[2] exist in different orders of bacteria and eukaryotes and cause various human diseases (1)
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 Cytolysin A (ClyA)
Several reports have suggested that formation of the Cys-87–Cys-285 disulfide bond in ClyA prevents pore complex formation (13, 17, 20)
Summary
Materials—Chemicals of the highest available purity were purchased from Merck KGaA (Darmstadt, Germany) or SigmaAldrich. Preparation and Reduction of Oxidized ClyA—To generate the disulfide-bonded oxidized form of ClyA, ClyAred (40 M) 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). Air oxidation of the natural cysteine pair Cys-87/Cys-285 in the labeled ClyACys variant (4 M) 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. Number of measurements taken for the specified time; Time window length, size of time windows into which the cumulated measurements were divided; Burst min/max, lower and upper limit of number of photons in a burst to be considered for analysis; NA, not applicable
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