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Structure-Function Analysis of Heterodimer Formation, Oligomerization, and Receptor Binding of the Staphylococcus aureus Bi-component Toxin LukGH*

Open AccessPublished:November 03, 2014DOI:https://doi.org/10.1074/jbc.M114.598110
      The bi-component leukocidins of Staphylococcus aureus are important virulence factors that lyse human phagocytic cells and contribute to immune evasion. The γ-hemolysins (HlgAB and HlgCB) and Panton-Valentine leukocidin (PVL or LukSF) were shown to assemble from soluble subunits into membrane-bound oligomers on the surface of target cells, creating barrel-like pore structures that lead to cell lysis. LukGH is the most distantly related member of this toxin family, sharing only 30–40% amino acid sequence identity with the others. We observed that, unlike other leukocidin subunits, recombinant LukH and LukG had low solubility and were unable to bind to target cells, unless both components were present. Using biolayer interferometry and intrinsic tryptophan fluorescence we detected binding of LukH to LukG in solution with an affinity in the low nanomolar range and dynamic light scattering measurements confirmed formation of a heterodimer. We elucidated the structure of LukGH by x-ray crystallography at 2.8-Å resolution. This revealed an octameric structure that strongly resembles that reported for HlgAB, but with important structural differences. Structure guided mutagenesis studies demonstrated that three salt bridges, not found in other bi-component leukocidins, are essential for dimer formation in solution and receptor binding. We detected weak binding of LukH, but not LukG, to the cellular receptor CD11b by biolayer interferometry, suggesting that in common with other members of this toxin family, the S-component has the primary contact role with the receptor. These new insights provide the basis for novel strategies to counteract this powerful toxin and Staphylococcus aureus pathogenesis.

      Background

      LukGH is a member of the family of two-component bacterial toxins of Staphylococcus aureus that lyse human phagocytic cells.

      Results

      The crystal structure of LukGH and mutagenesis revealed the molecular basis for heterodimer formation in solution.

      Conclusion

      LukGH differs from other two-component leukocidins that interact only upon cell contact.

      Significance

      These data might assist with development of therapeutics that counteract Staphylococcus aureus pathogenesis.

      Introduction

      Staphylococcus aureus is a versatile microbe equipped with numerous virulence mechanisms that can transform this harmless colonizer into a powerful human pathogen causing a broad spectrum of diseases ranging from skin infections to severe deep tissue infections, pneumonia, bacteremia, and sepsis. The hallmark of S. aureus pathogenesis is the ability to avoid and survive the most important innate immune defense: phagocytic killing (
      • Spaan A.N.
      • Surewaard B.G.
      • Nijland R.
      • van Strijp J.A.
      Neutrophils versus Staphylococcus aureus: a biological tug of war.
      ,
      • Rigby K.M.
      • DeLeo F.R.
      Neutrophils in innate host defense against Staphylococcus aureus infections.
      ).
      The bi-component leukocidins of S. aureus are important virulence factors that attack human phagocytic cells, greatly contributing to immune evasion. A single S. aureus strain can express up to five different bi-component leukocidins: γ-hemolysins HlgAB and HlgCB, Panton-Valentine leukocidin (PVL or LukSF), LukED and LukGH (
      • Vandenesch F.
      • Lina G.
      • Henry T.
      Staphylococcus aureus hemolysins, bi-component leukocidins, and cytolytic peptides: a redundant arsenal of membrane-damaging virulence factors?.
      ,
      • Alonzo 3rd, F.
      • Torres V.J.
      The bicomponent pore-forming leucocidins of Staphylococcus aureus.
      ,
      • Aman M.J.
      • Adhikari R.P.
      Staphylococcal bicomponent pore-forming toxins: targets for prophylaxis and immunotherapy.
      ). These leukocidins belong to the family of structurally related pore-forming toxins (
      • Gouaux E.
      • Hobaugh M.
      • Song L.
      α-Hemolysin, gamma-hemolysin, and leukocidin from Staphylococcus aureus: distant in sequence but similar in structure.
      ). The mechanism of pore formation, mostly studied using γ-hemolysin and LukSF, is thought to be similar for all bi-component toxins and involves two separately secreted polypeptides, the S- and F-components, which are distinct in sequence and have a molecular mass between 32 and 38 kDa. Binding studies revealed that the S-component binds first and recruits the F-component to the surface of phagocytic cells, which is followed by oligomerization (
      • Colin D.A.
      • Mazurier I.
      • Sire S.
      • Finck-Barbançon V.
      Interaction of the two components of leukocidin from Staphylococcus aureus with human polymorphonuclear leukocyte membranes: sequential binding and subsequent activation.
      ). This step induces structural changes in the stem domains of both subunits that lead to membrane insertion and the formation of a β-barrel pore in the cell membrane, resulting in osmotic lysis (
      • Menestrina G.
      • Dalla Serra M.
      • Comai M.
      • Coraiola M.
      • Viero G.
      • Werner S.
      • Colin D.A.
      • Monteil H.
      • Prévost G.
      Ion channels and bacterial infection: the case of beta-barrel pore-forming protein toxins of Staphylococcus aureus.
      ). Biochemical and biophysical studies of LukSF and HlgAB (including the HlgAB crystal structure) revealed octameric structures formed by four alternating S- and four F-components (
      • Miles G.
      • Movileanu L.
      • Bayley H.
      Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore.
      ,
      • Jayasinghe L.
      • Bayley H.
      The Leukocidin pore: evidence for an octamer with four LukF subunits and four LukS subunits alternating around a central axis.
      ,
      • Yamashita K.
      • Kawai Y.
      • Tanaka Y.
      • Hirano N.
      • Kaneko J.
      • Tomita N.
      • Ohta M.
      • Kamio Y.
      • Yao M.
      • Tanaka I.
      Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components.
      ). By analogy, the other two leukocidins, LukED and LukGH are also thought to form similar structures.
      In the last two years, the cellular receptors of most leukocidins have been identified (reviewed in Refs.
      • Alonzo 3rd, F.
      • Torres V.J.
      The bicomponent pore-forming leucocidins of Staphylococcus aureus.
      ,
      • Aman M.J.
      • Adhikari R.P.
      Staphylococcal bicomponent pore-forming toxins: targets for prophylaxis and immunotherapy.
      , and
      • DuMont A.L.
      • Torres V.J.
      Cell targeting by the Staphylococcus aureus pore-forming toxins: it's not just about lipids.
      ). Importantly, all of these molecules play a significant role in immune regulation. LukSF binds complement receptors C5aR and C5LR, LukGH interacts with the C3R (Mac-1 complex, CD11b component), and LukED recognizes chemokine receptors CXCR1 and -2 and CCR5 on phagocytic cells and lymphocytes, respectively (
      • Spaan A.N.
      • Henry T.
      • van Rooijen W.J.
      • Perret M.
      • Badiou C.
      • Aerts P.C.
      • Kemmink J.
      • de Haas C.J.
      • van Kessel K.P.
      • Vandenesch F.
      • Lina G.
      • van Strijp J.A.
      The staphylococcal toxin Panton-Valentine leukocidin targets human C5a receptors.
      ,
      • DuMont A.L.
      • Yoong P.
      • Day C.J.
      • Alonzo 3rd, F.
      • McDonald W.H.
      • Jennings M.P.
      • Torres V.J.
      Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1.
      ,
      • Reyes-Robles T.
      • Alonzo 3rd, F.
      • Kozhaya L.
      • Lacy D.B.
      • Unutmaz D.
      • Torres V.J.
      Staphylococcus aureus leukotoxin ED targets the chemokine receptors CXCR1 and CXCR2 to kill leukocytes and promote infection.
      ,
      • Alonzo 3rd, F.
      • Kozhaya L.
      • Rawlings S.A.
      • Reyes-Robles T.
      • DuMont A.L.
      • Myszka D.G.
      • Landau N.R.
      • Unutmaz D.
      • Torres V.J.
      CCR5 is a receptor for Staphylococcus aureus leukotoxin ED.
      ). Based on competition studies, it is likely that HlgC uses the same receptor as LukS (
      • Gauduchon V.
      • Werner S.
      • Prévost G.
      • Monteil H.
      • Colin D.A.
      Flow cytometric determination of Panton-Valentine leucocidin S component binding.
      ).
      LukGH is the most recently discovered member of the bi-component leukocidin family. It is more distantly related to the other S. aureus leukocidins with only 30–40% amino acid sequence homology, whereas the other leukocidins have greater similarity to each other (60–80%).
      Most importantly, we and others discovered that LukGH is fundamentally different from the other bi-component toxins due to heterodimer formation in solution (
      • DuMont A.L.
      • Yoong P.
      • Liu X.
      • Day C.J.
      • Chumbler N.M.
      • James D.B.
      • Alonzo 3rd, F.
      • Bode N.J.
      • Lacy D.B.
      • Jennings M.P.
      • Torres V.J.
      Identification of a crucial residue required for Staphylococcus aureus LukAB cytotoxicity and receptor recognition.
      ). Therefore, strategies aimed at inhibiting the interaction of S- and F-components (for example, with monocomponent specific antibodies) are unlikely to be successful in preventing the formation of active toxin. Instead, steric interference with the binding of the LukGH complex to cells or oligomerization of dimers into pore complexes seem to be more promising strategies. For this, it is critical to determine at the molecular level how LukG and LukH heterodimerize. Here we describe the x-ray crystal structure of LukGH for the first time and determine the amino acid residues crucial for dimer formation, oligomerization, and cytolytic activity.

      DISCUSSION

      LukGH is one of the most potent of the S. aureus leukocidins toward human phagocytic cells. Its activity is comparable with the potency of LukSF. The lukGH genes are part of the core genome of S. aureus, whereas lukSF is carried by phages and only expressed by ∼5–10% of clinical isolates. Therefore, it is important to understand the mode of action of LukGH to counteract the lysis of human phagocytic cells by S. aureus.
      LukGH displays unique features compared with the other bi-component leukocidins. Based on the lower solubility of the subunits of LukGH and lack of binding of LukH or LukG to phagocytic cells, we hypothesized that LukGH forms a complex in solution even before contacting its target cell. Biolayer interferometry and intrinsic tryptophan fluorescence measurements confirmed the interaction of LukH and LukG in solution with an affinity in the low nanomolar range, and based on DLS analysis the size of the complex suggested formation of heterodimers. In addition, we could co-purify LukH with affinity tagged LukG from E. coli by co-expressing the two components with improved solubility. While we were generating these data, DuMont et al. (
      • DuMont A.L.
      • Yoong P.
      • Liu X.
      • Day C.J.
      • Chumbler N.M.
      • James D.B.
      • Alonzo 3rd, F.
      • Bode N.J.
      • Lacy D.B.
      • Jennings M.P.
      • Torres V.J.
      Identification of a crucial residue required for Staphylococcus aureus LukAB cytotoxicity and receptor recognition.
      ) reported co-purification of LukGH from S. aureus culture supernatants and showed that LukG and LukH could be cross-linked by glutaraldehyde.
      LukGH displays significant sequence variations in the different S. aureus strains, not observed with other bi-component toxins. The amino acid conservation among the most different forms is in the range of 82–88%. The sequence identity among the other S- and F-components within one strain is up to 81 (between LukS and HlgC) and 82% (between LukF and LukD), respectively. LukSF, LukED, and the γ-hemolysins clearly represent different toxin entities with different species specificity, receptors, and overlapping, but not identical, cell-type specificity (
      • Vandenesch F.
      • Lina G.
      • Henry T.
      Staphylococcus aureus hemolysins, bi-component leukocidins, and cytolytic peptides: a redundant arsenal of membrane-damaging virulence factors?.
      ,
      • Alonzo 3rd, F.
      • Torres V.J.
      The bicomponent pore-forming leucocidins of Staphylococcus aureus.
      ,
      • Aman M.J.
      • Adhikari R.P.
      Staphylococcal bicomponent pore-forming toxins: targets for prophylaxis and immunotherapy.
      ). Therefore, it was important to confirm that all LukGH variants expressed by S. aureus strains showing the most distant evolutionary relationship had comparable toxin activity toward human neutrophils and that LukGH from all strains formed heterodimers in solution.
      Our data suggested a different mode of action than described for other bi-component toxins. Therefore, we determined the crystal structure of USA300-type LukGH to compare with those reported for LukSF and HlgAB. Diffraction quality crystals were formed only in the presence of the hydrophobic solvent MPD, also reported for HlgAB (
      • Yamashita K.
      • Kawai Y.
      • Tanaka Y.
      • Hirano N.
      • Kaneko J.
      • Tomita N.
      • Ohta M.
      • Kamio Y.
      • Yao M.
      • Tanaka I.
      Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components.
      ). The structure of LukGH revealed an octamer arrangement that formed spontaneously under the crystallization conditions, indicating that the octamer is the most stable oligomeric form of LukGH at high protein concentrations in the presence of MPD. This is in good agreement with several previous reports showing that the pore formed by LukSF consists of four molecules of each of the S- and F-components (
      • Miles G.
      • Movileanu L.
      • Bayley H.
      Subunit composition of a bicomponent toxin: staphylococcal leukocidin forms an octameric transmembrane pore.
      ,
      • Jayasinghe L.
      • Bayley H.
      The Leukocidin pore: evidence for an octamer with four LukF subunits and four LukS subunits alternating around a central axis.
      ,
      • Yamashita K.
      • Kawai Y.
      • Tanaka Y.
      • Hirano N.
      • Kaneko J.
      • Tomita N.
      • Ohta M.
      • Kamio Y.
      • Yao M.
      • Tanaka I.
      Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components.
      ).
      The roles of the two interfaces in the octamer, interface 1 and 2, in dimer formation or oligomerization on the cell surface were explored by site-directed mutagenesis. The three electrostatic interactions (salt bridges) between the rim domains in interface 2 were disrupted by mutating either the negatively charged amino acid residues Glu171, Asp189, and Asp191 in LukG or the positively charged residues Arg215, Arg234, and Arg240 in LukH to Ala. These mutations resulted in lack of binding of LukG to LukH in solution and prevented the potent cytolytic activity seen with wild-type LukGH. The interface 2 mutant LukG2H and LukGH2 complexes were insoluble when co-expressed in E. coli cells, confirming our earlier observations of low solubility of the single components. These data suggest that interface 2 constitutes the binding surface in the dimer. Importantly, the charged residues involved in these interactions in the predicted dimer interface are not conserved between any of the other S- and F-components but are instead fully conserved between the LukGH sequence variants, suggesting that only LukGH has evolved toward heterodimerization in solution.
      In contrast, alanine mutations of the two salt bridges in interface 1 formed between the cap domains of LukG (Arg23, Lys217) and LukH (Asp75, Asp197) did not affect formation of soluble complexes in E. coli, dimerization in solution, or binding to target cells, but did eliminate toxin activity. These residues are conserved in all other S- and F-components. We therefore concluded that interface 1 is only required for the oligomerization of LukGH dimers on the cell surface.
      The finding that interface 1 mutants were able to form a dimer was further supported by their ability to inhibit LukGH activity, unlike interface 2 mutants. This inhibition is most likely due to competition for the receptor binding sites. Pull-down experiments with biotinylated LukGH and membrane preparations of differentiated HL-60 cells and PMNs identified the CD11b-CD18 complex as binding partner (
      • DuMont A.L.
      • Yoong P.
      • Day C.J.
      • Alonzo 3rd, F.
      • McDonald W.H.
      • Jennings M.P.
      • Torres V.J.
      Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1.
      ).4 DuMont et al. (
      • DuMont A.L.
      • Yoong P.
      • Day C.J.
      • Alonzo 3rd, F.
      • McDonald W.H.
      • Jennings M.P.
      • Torres V.J.
      Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1.
      ) reported direct interaction with CD11b and mapped the LukGH binding site to the CD11b I-domain by showing that a monoclonal antibody specific for this domain inhibited binding of LukGH to target cells. We therefore measured the interaction of the recombinant CD11b I-domain with the wild-type and mutated LukGH complexes. We found that interface 1 mutants bound to the purified I-domain of CD11b with an affinity similar to that of the wild-type complex, whereas interface 2 mutants displayed greatly diminished interaction.
      The decrease in LukGH potency in the presence of interface 1 mutant complexes is much greater than expected from a simple competition model. The concentration used for the mutant complex (12.75 nm) was in the range of the Kd value reported for the binding of LukGH to the full-length CD11b-CD18 complex (38 nm) that is ∼10-fold higher than the I domain of CD11b (
      • DuMont A.L.
      • Yoong P.
      • Day C.J.
      • Alonzo 3rd, F.
      • McDonald W.H.
      • Jennings M.P.
      • Torres V.J.
      Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1.
      ). However, the decrease in potency we detected was 10–100-fold. This indicates that toxin activity has a high dependence on receptor concentration, implying that cooperative binding to more than one receptor site is required for the formation of the LukGH pore. This is also supported by the fact that monovalent binding between LukGH and CD11b occurs in the low nanomolar range, whereas LukGH EC50 is at least 10-fold lower (∼100 pm, 10 ng/ml).
      Cell binding and receptor identification studies with the bi-component leukocidins suggested that it is the S-component of these toxins (LukS, LukE, and HlgC) that binds to phagocytic cells and their respective receptors, whereas HlgAB seems to contact its main target cells, red blood cells, via the F-component (HlgB) (
      • Spaan A.N.
      • Henry T.
      • van Rooijen W.J.
      • Perret M.
      • Badiou C.
      • Aerts P.C.
      • Kemmink J.
      • de Haas C.J.
      • van Kessel K.P.
      • Vandenesch F.
      • Lina G.
      • van Strijp J.A.
      The staphylococcal toxin Panton-Valentine leukocidin targets human C5a receptors.
      ,
      • DuMont A.L.
      • Yoong P.
      • Day C.J.
      • Alonzo 3rd, F.
      • McDonald W.H.
      • Jennings M.P.
      • Torres V.J.
      Staphylococcus aureus LukAB cytotoxin kills human neutrophils by targeting the CD11b subunit of the integrin Mac-1.
      ,
      • Reyes-Robles T.
      • Alonzo 3rd, F.
      • Kozhaya L.
      • Lacy D.B.
      • Unutmaz D.
      • Torres V.J.
      Staphylococcus aureus leukotoxin ED targets the chemokine receptors CXCR1 and CXCR2 to kill leukocytes and promote infection.
      ,
      • Alonzo 3rd, F.
      • Kozhaya L.
      • Rawlings S.A.
      • Reyes-Robles T.
      • DuMont A.L.
      • Myszka D.G.
      • Landau N.R.
      • Unutmaz D.
      • Torres V.J.
      CCR5 is a receptor for Staphylococcus aureus leukotoxin ED.
      ,
      • Kaneko J.
      • Ozawa T.
      • Tomita T.
      • Kamio Y.
      Sequential binding of Staphylococcal γ-hemolysin to human erythrocytes and complex formation of the hemolysin on the cell surface.
      ,
      • Nguyen V.T.
      • Kamio Y.
      • Higuchi H.
      Single-molecule imaging of cooperative assembly of γ-hemolysin on erythrocyte membranes.
      ). An interesting finding from our studies was the weak but detectable binding of LukH to the isolated CD11b I-domain (∼25-fold higher Kd relative to that of the LukGH dimer), not observed with LukG. The lack of binding to target cells by LukH and the absence of inhibition of LukGH cytolytic activity by LukH or LukH2 suggest that although the main contact point between the receptor and LukGH is most likely located in LukH, formation of the LukGH dimer is necessary for a sufficiently strong interaction to initiate oligomerization on the cell surface.
      Our data suggest that disruption of complex formation between LukG and LukH is unlikely to be an efficient strategy to prevent LukGH-associated toxicity, because the two components form a complex either inside bacteria or shortly after their secretion. A more promising approach seems to be to inhibit binding of the dimer to the CD11b receptor or oligomerization.
      These findings support the development of either small molecule or antibody therapeutics that inhibit LukGH toxin-mediated elimination of phagocytes and dendritic cells, both of which are crucial for effective innate and adaptive immune responses against S. aureus infections.

      Acknowledgments

      We thank the Campus Support Facility Unit for assisting in DLS and CD measurements. We acknowledge the work by the staff at beamline I911-3 (Lund, Sweden) and crystallization facility at the MAX IV Laboratory (Lund, Sweden). We thank Katharina Havlicek and Manuel Zerbs for technical help.

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