Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of ␤-barrel pore-forming toxins (␤-PFTs). By contrast, other ␤-PFTs form homo-oligomeric pores; for example, the staphylococcal ␣-hemolysin (␣HL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site-specific chemical modification during single-channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the ␣HL pore. It is also pertinent to the mechanism of assembly of ␤-PFT pores and suggests new possibilities for engineering these proteins.

The genes encoding the F and S components of a leukocidin, LukF (HlgB) and LukS (HlgC), a pore-forming binary toxin, were amplified from the Smith 5R strain of Staphylococcus aureus both with and without sequences encoding 3'-hexahistidine tags. The His-tagged components were expressed in Escherichia coli and purified under nondenaturing conditions. In addition, the two unmodified proteins and the His-tagged versions were produced in an E. coli cell-free in vitro transcription and translation system. An SDS-stable oligomer of approximately 200 kDa appeared when both components were cotranslated in the presence of rabbit erythrocyte membranes. Hemolytic activity of the combined components against rabbit erythrocytes was measured for both in vitro- and in vivo-produced polypeptides, yielding similar HC(50) values of approximately 0.14 microg/mL. The pore-forming properties of the recombinant leukocidin were also investigated with planar lipid bilayers of diphytanoylphosphatidylcholine. Although leukocidins and staphylococcal alpha-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS [1 M KCl and 5 mM HEPES (pH 7.4)], which is more than 3 times greater than that of alpha-hemolysin measured under the same conditions. Therefore, if the leukocidin pore were a cylinder, its diameter would be almost twice that of alpha-hemolysin. In addition, the leukocidin pore is weakly cation selective and exhibits gating at low positive potentials, while alpha-hemolysin is weakly anion selective and gates only at high potentials. Taken together, these data suggest that the structure of the oligomeric pore formed by the leukocidin examined here has diverged significantly from that of alpha-hemolysin.

The gene encoding hemolysin II (HlyII) was amplified from Bacillus cereus genomic DNA and a truncated mutant, HlyII(⌬CT), was constructed lacking the 94 amino acid extension at the C terminus. The proteins were produced in an E. coli cell-free in vitro transcription and translation system, and were shown to assemble into SDS-stable oligomers on rabbit erythrocyte membranes and liposomes. The hemolytic activity of HlyII was measured with rabbit erythrocytes yielding an HC 50 value of 1.64 ng mL −1 , which is over 15 times more potent than staphylococcal ␣-hemolysin. HlyII(⌬CT) was about eight times less potent than HlyII in this assay. Limited proteolysis of the oligomers formed by HlyII and HlyII(⌬CT) on red cell membranes showed that the C-terminal extension is sensitive to digestion, while HlyII(⌬CT) is protease resistant and migrates with an electrophoretic mobility similar to that of digested HlyII. HlyII forms moderately anion selective, rectifying pores (I +80 /I −80 ‫ס‬ 0.57, 1 M KCl, pH 7.4) in planar lipid bilayers of diphytanoylphosphatidylcholine with a unitary conductance of 637 pS (1 M KCl, 5 mM HEPES, pH 7.4) and exhibits no gating over a wide range of applied potentials (−160 to +160 mV). In addition, it was demonstrated that HlyII forms a homoheptameric pore by using gel shift electrophoresis aided by a genetically encoded oligoaspartate tag. Although they share limited primary sequence identity (30%), these data confirm that HlyII is a structural and functional homolog of staphylococcal ␣-hemolysin.

Staphylococcal ␣-hemolysin (␣HL) is a ␤ barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, ␣HL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of ␣HL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of ␣HL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser 217 3 Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of ␣HL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of ␣HL monomers in solution.

Staphylococcal leukocidin (Luk) and ␣-hemolysin (␣HL) are members of the same family of ␤ barrel pore-forming toxins (␤PFTs). Although the ␣HL pore is a homoheptamer, the Luk pore is formed by the co-assembly of four copies each of the two distantly related polypeptides, LukF and LukS, to form an octamer. Here, we examine N-and C-terminal truncation mutants of LukF and LukS. LukF subunits missing up to nineteen N-terminal amino acids are capable of producing stable, functional hetero-oligomers with WT LukS. LukS subunits missing up to fourteen N-terminal amino acids perform similarly in combination with WT LukF. Further, the simultaneous truncation of both LukF and LukS is tolerated. Both Luk subunits are vulnerable to short deletions at the C terminus. Interestingly, the N terminus of the LukS polypeptide becomes resistant to proteolytic digestion in the fully assembled Luk pore while the N terminus of LukF remains in an exposed conformation. The results from this work and related experiments on ␣HL suggest that, although the N termini of ␤PFTs may undergo reorganization during assembly, they are dispensable for the formation of functional pores.