Structure-based discovery of a small-molecule inhibitor of methicillin-resistant Staphylococcus aureus virulence

Abstract

The rapid emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains poses a major threat to public health. MRSA possesses an arsenal of secreted host-damaging virulence factors that mediate pathogenicity and blunt immune defenses. Panton–Valentine leukocidin (PVL) and α-toxin are exotoxins that create lytic pores in the host cell membrane. They are recognized as being important for the development of invasive MRSA infections and are thus potential targets for antivirulence therapies. Here, we report the high-resolution X-ray crystal structures of both PVL and α-toxin in their soluble, monomeric, and oligomeric membrane-inserted pore states in complex with n-tetradecylphosphocholine (C₁₄PC). The structures revealed two evolutionarily conserved phosphatidylcholine-binding mechanisms and their roles in modulating host cell attachment, oligomer assembly, and membrane perforation. Moreover, we demonstrate that the soluble C₁₄PC compound protects primary human immune cells in vitro against cytolysis by PVL and α-toxin and hence may serve as the basis for the development of an antivirulence agent for managing MRSA infections.

Introduction

Infection with Staphylococcus aureus can cause severe and devastating illness and is one of the leading causes of death by any infectious agent in the United States (1, 2). S. aureus is notorious for its ability to acquire genetic determinants of antibiotic resistance and virulence that enhance fitness and pathogenicity (3, 4). Methicillin-resistant S. aureus (MRSA)² now accounts for >60% of S. aureus isolates in United States intensive care units, severely restricting antibiotic treatment options (2). MRSA also spreads rapidly among healthy individuals in the community, causing predominantly skin and soft tissue infections and life-threatening infections, including bacteremia, endocarditis, osteomyelitis, and necrotizing pneumonia (2). Disturbingly, MRSA can live in the biofilm state (5, 6), and it has long been recognized that biofilms increase resistance to antimicrobial agents and the host immune response (7). MRSA is currently treated with vancomycin, clindamycin, linezolid, and daptomycin (8), but resistance to these “last-resort” antibiotics has been reported (9–13). For these reasons, the World Health Organization identifies MRSA as one of six “high-priority” pathogens that pose an enormous threat to public health (14). Thus, new therapeutics with novel mechanisms of action are desperately needed to combat this high-threat pathogen.

USA300 is the most prevalent strain of MRSA in the United States and represents a growing threat in both community and healthcare settings (15). Its heightened virulence and severity are related to the production of a mixture of cytolytic pore-forming exotoxins that mediate virulence and impair host immune defenses (3, 16). The pharmacological targeting of these cytotoxins has been recognized as a promising new therapeutic approach to reducing morbidity and mortality associated with MRSA infection. Leukocidins and α-toxin, secreted by S. aureus as water-soluble, monomeric polypeptides, constitute the α-hemolysin subfamily of β-barrel pore-forming toxins (17). Five different bipartite leukocidins have been described, including Panton–Valentine leukocidin (PVL), leukocidin ED (LukED), two γ-hemolysins (HlgAB and HlgCB), and leukocidin AB (LukAB; also known as LukGH), each of which consists of two distinct polypeptides referred to as the S and F subunits (reviewed in Ref. 18). Their cellular tropism and species specificity are determined by the S subunits LukS-PV, LukE, HlgA, HlgC, and LukA (19–21). The S and F subunits and single-component α-toxin share a unique modular structure consisting of the amino latch and prestem regions and the β-sandwich and rim domains (see Fig. 1A) (22–26). The X-ray crystal structures of the membrane-inserted pore oligomer forms of α-toxin, HlgAB, and LukGH and of the membrane surface-bound prepore heterooctamer forms of HlgAB and HlgCB have been determined (27–30). These structures, and supporting biochemical and genetic data (26, 31–33), suggest that members of this subfamily share a common mechanism of cytolytic action (reviewed in Refs. 34 and 35). The cytolytic process begins with the binding of soluble toxin monomers to a cell surface receptor (21, 36). The membrane-bound monomers then associate to form a nonlytic, oligomeric prepore. Finally, the translocation of the prestem regions across the membrane results in the bilayer-spanning β-barrel pore structure and consequent membrane permeabilization and cell lysis.

Figure 1.

Structural basis for C₁₄PC binding to LukD. A, cocrystal structure of C₁₄PC bound to LukD shown from a side view perpendicular to the membrane plane, with the PCho moieties and the side chains of key binding site residues displayed as CPK spheres and in ball-and-stick format, respectively. The amino latch (magenta) and prestem (yellow) regions and the β-sandwich (green) and rim (blue) domains are indicated. The two binding sites are labeled, as are the β-strands that compose the rim domain and the Ω1 and Ω2 loops. B, 2F_o − F_c omit electron density map (green mesh) for the two PCho moieties at 1.5σ contour level. C, surface representation of the C₁₄PC-LukD complex viewed parallel to the membrane, with the PCho moieties shown in stick format with transparent CPK spheres. The locations of key binding site residues are indicated. D, close-up view of the two adjacent PCho binding pockets, with residues that make direct side-chain contacts with the PCho moieties shown in ball-and-stick format. Cation-π interactions are represented as green dotted lines. Hydrogen bonds and salt bridges are shown as pink dotted lines, and water molecules are shown as purple spheres. E, DSC thermograms of LukD in the absence (T_m = 51.0 °C) and presence (T_m = 52.8 °C) of PCho.

MRSA strains that harbor the phage-encoded PVL have been linked to highly virulent and severe community-acquired skin infections (37), as well as necrotizing pneumonia and lethal necrotizing fasciitis (38). The role of PVL production in the pathogenesis of MRSA was demonstrated in both a rabbit model of necrotizing pneumonia and humanized mouse models of skin infection and pneumonia (39–41). PVL induces leukocyte destruction and tissue necrosis through interaction with the complement receptors C5aR and C5L2 (42–45). PVL, in conjunction with HlgAB, contributes to MRSA biofilm-mediated killing of neutrophils (46). On the other hand, the chromosomally encoded α-toxin lyses epithelial and endothelial cells, red blood cells, lymphocytes, and monocytes by targeting its receptor, the metalloprotease ADAM10 (36, 47). The elevated expression of α-toxin in the USA300 clone and in historic human epidemic strains correlates with increased pathogenicity in mouse models of skin and soft tissue infection, pneumonia, and sepsis (48, 49). α-Toxin also plays a role in biofilm formation by clinical MRSA isolates (50). Moreover, LukED relies on the chemokine receptor CCR5 to kill T lymphocytes, macrophages, and dendritic cells, as well as CXCR1 and CXCR2 to kill leukocytes (19, 51). Inhibition of the interaction between LukED and CCR5 has been shown to block cytotoxicity and attenuate S. aureus infection in mice (19). Together, these cytotoxins can modulate phagocytic cell functions via their specific receptors and contribute to MRSA immune evasion and disease pathogenesis. As such, the discovery and development of new antivirulence agents that protect from the combined immune cytolytic activities of this subfamily of pore-forming toxins is of the utmost importance.

There is considerable evidence pointing to the role of phosphatidylcholine (PC) in the mechanism of pore formation by these toxins. PC is an absolute requirement for pore formation by α-toxin, HlgAB, and HlgCB and has been shown to inhibit their cytolytic effects (52–56). Particularly, crystallographic studies revealed the presence of single, highly conserved phosphocholine (PCho) binding sites on the rim domains of the monomeric F subunit HlgB and the α-toxin protomer in the heptameric pore complex (22, 57). These binding sites have been shown by mutational analysis to be required for membrane targeting and cytolytic function of the two toxins (32, 58). It is generally accepted that α-toxin and the F subunits LukD, LukF-PV, LukB, and HlgB also function in cell attachment through the engagement of their rim domains with the PC headgroup in the plasma membrane of target cells (52, 54, 57). In this report, we demonstrate that the soluble, monomeric, and oligomeric pore forms of both PVL and α-toxin deploy two distinct modes to recognize and bind the PC-containing membrane and suggest a novel molecular mechanism for PC-dependent pore formation by members of this subfamily. Furthermore, we find that the soluble compound n-tetradecylphosphocholine (C₁₄PC) effectively inhibits the cytolysis of primary human immune cells by PVL, α-toxin, and LukED in vitro, thus demonstrating the potential utility of this antivirulence agent alone or in combination with antibiotics against MRSA.

Results and discussion

C₁₄PC binds to the rim domain of LukD at two adjacent but distinct sites

To better understand the molecular basis for the recognition of PCho by the leukocidin F subunits, we determined the crystal structures of LukD with and without C₁₄PC at 1.5 and 1.75 Å resolution, respectively (Table 1). C₁₄PC was selected in the present study as a PC mimic for its high micellization efficiency due to low critical micelle concentration. The two protein structures are closely similar, with an r.m.s.d. for C_α atoms of 0.69 Å. The rim domain forms an antiparallel, three-stranded open-face β-sandwich toppled by two surface-exposed consecutive Ω loops (residues 180–194, Ω1 and 195–202, Ω2) (Fig. 1A). Two PCho moieties that bind to opposite sides of the Ω2 loop were unexpectedly discovered upon examination of the difference electron density map in the C₁₄PC-bound structure (Fig. 1B). The average B-factor for these two moieties is 22 Ų, and that for the surrounding solvent molecules and protein atoms is 21 Ų. The two binding sites are ∼16 Å apart (Fig. 1C). The PCho moiety at the first binding site (site 1) is lodged into a concave pocket similar to one in HlgB (PDB code 3LKF). This pocket is formed by two extended segments (residues 171–173 and 176–179, respectively) and the Ω1–Ω2 junction (residues 191–197) (Fig. 1, A and D). The quaternary ammonium group of the PCho moiety engages in a cation–π interaction with Trp¹⁷⁶ while forming a salt bridge to Glu¹⁹¹ (3.79 Å) (Fig. 1D). Its N-methyl and methylene groups are in van der Waal contact (<4.0 Å) with the main-chain atoms of Asn¹⁷³, Glu¹⁹¹, Leu¹⁹⁴, and Gly¹⁹⁵ and with the side chains of Asn¹⁷³, Trp¹⁷⁶, Tyr¹⁷⁹, and Glu¹⁹¹. Furthermore, the phosphate group is hydrogen-bonded through its O2 oxygen to the main-chain amide of Arg¹⁹⁷ (2.85 Å) on one side of the pocket opening, and the side-chain of this residue also wraps around the three other oxygens (Fig. 1D). In addition, three water molecules form hydrogen bonds to the O2 and O3 oxygens.

Table 1.

X-ray data collection and refinement statistics

	LukD, no ligand	LukD, C14PC-bound	LukF-PV, C14PC-bound	α-ToxinH35A, C14PC-bound	PVL, heterooctamer, C14PC-bound	α-Toxin, heptamer, C14PC-bound	α-ToxinH35A, heptamer, C14PC-bound
PDB code	6U33	6U2S	6U3F	6U3T	6U3Y	6U49	6U4P
Data collection	NSLS X4C	MacCHESS-F1	SSRL 9-2	SSRL 9-2	SSRL 9-2	SSRL 9-2	SSRL 9-2
Cell dimensions
a, b, c (Å)	123.6, 48.7, 66.7	149.2, 37.8, 77.9	49.3, 49.3, 266.9	136.2, 136.2, 166.0	139.1, 139.1, 246.8	150.4, 135.0, 132.5	151.2, 134.6, 130.7
α, β, γ (degrees)	90.0, 120.8, 90.0	90.0, 119.8, 90.0	90.0, 90.0, 120.0	90.0, 90.0, 90.0	90.0, 90.0, 90.0	90.0, 91.5, 90.0	90.0, 91.8, 90.0
Wavelength (Å)	0.979	0.977	0.979	0.979	0.979	0.886	0.979
Space group	C2	C2	P3221	I422	I422	C2	C2
Resolution (Å)	57.3–1.75 (1.78–1.75)a	67.6–1.50 (1.53–1.50)	89.0–1.78 (1.82–1.78)	43.8–2.80 (2.87–2.80)	38.7–2.04 (2.08–2.04)	132.4–2.35 (2.39–2.35)	37.8–2.50 (2.56–2.50)
Rmeas	0.055 (0.297)	0.054 (0.270)	0.035 (0.109)	0.135 (0.912)	0.098 (0.981)	0.101 (0.548)	0.061 (0.515)
I/σI	15.8 (3.7)	9.7 (4.4)	18.9 (20.5)	5.4 (2.4)	6.6 (2.1)	7.2 (2.1)	10.2 (2.4)
Completeness (%)	92.6 (58.7)	97.8 (84.7)	99.7 (98.2)	99.8 (99.1)	99.9 (98.7)	94.5 (96.6)	96.5 (98.1)
Multiplicity	3.0 (2.3)	3.2 (3.0)	9.4 (9.2)	12.7 (10.4)	12.8 (8.9)	2.8 (2.8)	3.4 (3.4)
CC½ (%)	98.4 (93.8)	98.9 (96.1)	99.9 (99.7)	95.6 (71.6)	95.6 (75.4)	92.5 (71.5)	96.8 (86.4)
No. of unique reflections	32,045 (1,505)	59,415 (3,824)	37,358 (2,428)	19,666 (1,262)	76,882 (3,741)	103,631 (7,554)	87,452 (6,406)
Refinement statistics
Resolution (Å)	57.3–1.75	67.6–1.50	89.0–1.78	43.8–2.80	38.7–2.04	132.4–2.35	37.8–2.50
	(1.79–1.75)	(1.54–1.50)	(1.83–1.78)	(2.86–2.80)	(2.09–2.04)	(2.41–2.35)	(2.56–2.50)
Rwork	0.175 (0.231)	0.162 (0.216)	0.179 (0.154)	0.197 (0.295)	0.182 (0.242)	0.194 (0.261)	0.212 (0.289)
Rfree	0.217 (0.286)	0.188 (0.245)	0.210 (0.195)	0.246 (0.381)	0.225 (0.269)	0.239 (0.314)	0.249 (0.372)
r.m.s.d.
Bond lengths (Å)	0.019	0.019	0.017	0.003	0.015	0.020	0.011
Bond angles (degrees)	1.9	1.9	2,4	1.0	2.2	2.3	2.0
Average B-factor (Å2)
Protein	15.2	14.3	21.5	51.4	33.1	26.5	51.4
C14PC		26.1	51.5	113.5	60.9	68.6	102.1
Water	34.1	39.3	37.6	59.4	47.5	40.1	60.0
Ion	35.7		47.8	119.5	70.8	73.8	120.0
Ramachandran plot
Favored (%)	95.6	95.5	95.5	94.4	96.9	96.0	94.0
Allowed (%)	4.4	4.5	4.5	5.6	3.1	4.0	6.0
Disallowed (%)	0	0	0	0	0	0	0

^a Values for the highest-resolution shell are shown in parentheses.

Immediately adjacent to site 1 is a novel second binding site (site 2), where the PCho moiety occupies a shallow surface pocket that is framed by the C-terminal half of the Ω2 loop (residues 198–202) and the β14–β15 loop (residues 257–260) and flanked by the side chains of Tyr⁷¹, Asn⁷², Trp²⁵⁶, and Trp²⁶¹ (Fig. 1, A and D). The quaternary ammonium group is sandwiched between the aromatic rings of Tyr⁷¹ and Trp²⁵⁶ through cation–π interactions, and the two indole rings of the latter residue and Trp²⁶¹ interact with each other in an edge-to-face fashion to engage the N-methyl and methylene groups, which also make contacts with the main-chain atoms of Ser¹⁹⁹, Ser²⁰⁰, and Ser²⁰¹ and with the side chain of Asn⁷² (Fig. 1D). The phosphate group is secured by a water-mediated hydrogen bonding interaction with the main-chain carbonyl of Ser²⁰⁰ (O₂–H₂O = 2.53 Å and H₂O–O = 2.76 Å), whose C_α and C_β atoms pack against the O1, O2, and O4 oxygens (Fig. 1D).

The highly complementary interactions between the two adjacent binding sites and the PCho moieties are ostensibly important for specific recognition and binding. The buried solvent-accessible surface area of PCho is 262 Ų at site 1 and 231 Ų at site 2, which correspond to ∼77 and 69% of the unbound PCho surface area, respectively. The side chains of the conserved Trp¹⁷⁶-Arg¹⁹⁷ and Ser²⁰⁰-Trp²⁵⁶-Trp²⁶¹ residues, seen below, that define site 1 and site 2, respectively, become more ordered upon binding to C₁₄PC. This side-chain flexibility could allow these two adjacent, largely preformed pockets to efficiently accommodate the PCho moieties that have distinct binding poses and residue interactions (Fig. 1, C and D). Consistent with this argument, in differential scanning calorimetry (DSC) experiments, LukD (10 μm) was found to unfold in a single cooperative transition, with a midpoint melting temperature (T_m) of 51.0 °C, whereas this T_m value was shifted to 52.8 °C in the presence of PCho (4 mm), representing the enhanced thermal stability that accompanies complex formation (Fig. 1E). Thus, our results suggest a revised mode of PC recognition and membrane targeting by the rim domain loops.

Binding mode of C₁₄PC to the rim domain of LukF-PV

To validate this binding mode, we co-crystallized LukF-PV with C₁₄PC and solved its structure at 1.78 Å resolution (Table 1). In effect, PCho moieties engage the aforementioned two adjacent binding pockets on the rim domain surface (Fig. 2, A and B). At site 1, the quaternary ammonium group of the PCho moiety forms both a cation–π interaction with Trp¹⁷⁶ and a salt bridge to Glu¹⁹¹ (3.84 Å); its N-methyl and methylene groups interact with both the main-chain atoms of Leu¹⁹⁴ and Gly¹⁹⁵ and the side chains of Asn¹⁷³, Trp¹⁷⁶, Tyr¹⁷⁹, Glu¹⁹¹, and Arg¹⁹⁷, and the phosphate group is held in place by a hydrogen bond between its O2 oxygen and the main-chain amide of Arg¹⁹⁷ (2.72 Å), along with the side chain of this residue lying against the O2 and O3 oxygens (Fig. 2B). At site 2, the quaternary ammonium group participates in a cation–π interaction with Trp²⁵⁶ (Fig. 2B). Further contacts are made between the N-methyl and methylene groups and both the main-chain atoms of Ser¹⁹⁹, Asn²⁰⁰, and Leu²⁰¹ and the side chains of Asn²⁰⁰, Trp²⁵⁶, and Trp²⁶¹. Polar interactions are also observed between the phosphate and both the main-chain atom of Asn²⁰⁰ and the side chain of Asn²⁰² (Fig. 2B).

Figure 2.

Structure of C₁₄PC-bound Luk-PV. A, 2F_o − F_c electron density map shown as green mesh around the two PCho moieties contoured at 1.5σ (site 1) and 1.0σ (site 2). B, molecular interactions in the C₁₄PC-LukF-PV complex, with the PCho moieties shown in stick format with transparent CPK spheres. The side chains of residues that make direct contacts with the PCho moieties are shown in ball-and-stick format. The two binding sites and the Ω1 and Ω2 loops are labeled. Cation-π interactions are represented as green dotted lines. Hydrogen bonds and salt bridges are shown as pink dotted lines. C, sequence alignment for members of the α-hemolysin toxin subfamily around the regions of the three PCho-binding sites (see “Results” for details). Four segments of the rim domain (residues 71–75, 170–180, 191–202, and 255–264) and two segments of the stem domain (residues 109–117 and 137–145) are delineated by spaces and numbered according to the mature LukF-PV protein. Conserved residues at the two binding sites on the rim domain are highlighted in red. Conserved residues that constitute the interprotomer binding sites on the PVL heterooctamer and the α-toxin heptamer are highlighted with a green and yellow background, respectively.

The solvent-accessible surface area of PCho buried by the LukF-PV interaction comprises 264 Ų (79%) at site 1 and 214 Ų (63%) at site 2. DSC measurements reveal that the T_m of LukF-PV increased from 50.3 to 52.3 °C when it was bound to PCho. We note that the PCho moiety at site 2 has considerably higher average B-factor and poorer electron density than that at site 1 (70 Ų as compared with 31 Ų), suggesting that the former moiety is less tightly bound and exhibits greater spatial or temporal disorder. In LukD, the aromatic side chain of Tyr⁷¹ contributes to the cation–π binding interaction at site 2 (see Fig. 1D), whereas the corresponding residue in LukF-PV (Thr⁷¹) cannot make this interaction (Fig. 2C), likely accounting for the lower-affinity binding site. The critical functional role of this affinity difference is highlighted by the observation that replacement of Thr⁷¹ with a tyrosine endows LukF-PV with the ability to bind human erythrocytes and acquire hemolytic activity when combined with the S subunit of HlgAB (33). Therefore, the elaborate structural features of the two distinct, adjacent PCho-binding sites on the leukocidin F subunits may be explained by a selective pressure for membrane PC itself acting as their cell surface receptor.

C₁₄PC binding by monomeric α-toxin^H35A

To discern the mechanism in the attachment of α-hemolysin subfamily members to host cells, we determined the 2.80 Å crystal structure of C₁₄PC in complex with the oligomerization-defective His³⁵→Ala mutant of α-toxin (α-toxin^H35A) (59) (Table 1). The asymmetric unit contains two nearly identical protein monomers (r.m.s.d. for C_α atoms of 0.44 Å), each bound to two PCho moieties (Fig. 3A). These moieties occupy the two adjacent binding pockets described above (Fig. 3B). At site 1, which is similar to that on the α-toxin protomer in the heptameric pore complex (57), the quaternary ammonium group of the PCho moiety makes a cation–π interaction with Trp¹⁷⁹ (Fig. 3C). Its N-methyl and methylene groups are surrounded by the main-chain atoms of Met¹⁹⁷ and Lys¹⁹⁸ and by the side chains of Asn¹⁷⁶, Gln¹⁷⁷, Trp¹⁷⁹, Tyr¹⁸², Gln¹⁹⁴, Met¹⁹⁷, and Arg²⁰⁰. Importantly, the O2 oxygen of the phosphate group establishes a strong hydrogen bond to the main-chain amide of Arg²⁰⁰ (2.64 Å) that also makes side-chain contacts with the O2 and O4 oxygens (Fig. 3C). At site 2, the quaternary ammonium group forms a cation–π interaction with Trp²⁶⁰, and the N-methyl and methylene groups interact with the main-chain atoms of Gly²⁰², Ser²⁰³, and Met²⁰⁴ and with the side chains of Ala⁷³, Asn⁷⁴, and Trp²⁶⁵ (Fig. 3B). The phosphate group is clearly visible in the electron density map, although the fine detail of the oxygens is not clear. There are contacts of 3.19 Å between the phosphate and Ser²⁰³ and of 3.62 Å between the phosphate and Trp²⁶⁰ (Fig. 3C). Upon binding to α-toxin^H35A, PCho buries 268 Ų (79%) and 203 Ų (61%) of its solvent-accessible surface area at site 1 and site 2, respectively. DSC analysis shows that the addition of PCho increased the T_m of α-toxin^H35A from 50.8 to 52.4 °C. We also observed that the average B-factor for the PCho moiety at site 2 is significantly higher than that at site 1 (112 Ų as compared with 75 Ų). As discussed in the preceding section, the decreased affinity of site 2 for PCho may arise from the presence of an alanine at position 73 (corresponding to LukD Tyr⁷¹) (Fig. 2C).

Figure 3.

The two adjacent PC-binding pockets on monomeric α-toxin^H35A. A, 2F_o − F_c electron density map (green mesh) for the two PCho moieties contoured at 1.2σ (site 1) and 1.0σ (site 2). B, surface representation of the C₁₄PC-α-toxin^H35A complex viewed parallel to the membrane, with the PCho moieties shown in stick format with transparent CPK spheres. The two binding sites are labeled. The locations of key binding site residues are indicated. C, close-up view of the two adjacent PCho-binding pockets, with the PCho moieties displayed in stick format with transparent CPK spheres. Residues that make direct side chain contacts with the PCho moieties are shown in ball-and-stick format. The Ω1 and Ω2 loops are labeled. Cation-π interactions are represented as green dotted lines. Hydrogen bonds are shown as pink dotted lines. D, close-up view of the interface between the two independent α-toxin^H35A monomers (blue and green, respectively) in the asymmetric unit. Hydrogen bonds and salt bridges near the His³⁵→Ala mutation site are depicted as pink dotted lines.

Closer examination of the positions and conformations of the two PCho moieties in the superimposed cocrystal structures of C₁₄PC with α-toxin^H35A, LukD, and LukF-PV revealed remarkable similarities. There are few differences in the positions of the five key binding site amino acid side chains (Trp¹⁷⁹, Arg²⁰⁰, Ser²⁰³, Trp²⁶⁰, and Trp²⁶⁵ in α-toxin; equivalent to Trp¹⁷⁶, Arg¹⁹⁷, Ser/Asn²⁰⁰, Trp²⁵⁶, and Trp²⁶¹ in LukD and LukF-PV) in these structures. The three Trp side chains provide two important anchor points for locating the PCho moieties in the two adjacent binding sites, and the Arg and Ser/Asn residues are critical determinants in the binding of the two phosphate groups. Evidently, PC recognition specificity is achieved by a combination of stacking and hydrogen-bonding interactions and van der Waals contacts. Our study shows that membrane PC serves as the common receptor for α-toxin and the leukocidin F subunits, in agreement with previous observations (52, 54, 57). The presence of the two adjacent PC-binding sites on the toxin monomer is consistent with the estimated cross-sectional areas of the PC-bound rim domain (∼150 Ų) and one PC molecule (∼70 Ų) (60).

Intermolecular contacts between the above two α-toxin^H35A monomers comprising the crystal asymmetric unit are formed by residues in the β-sandwich domain (Fig. 3D). Comparison of the conformation of these contact residues with their interprotomeric equivalents in the unliganded and C₁₄PC-bound heptamers of WT α-toxin (PDB code 7AHL; see Fig. 5) reveals no local conformational changes involving the main-chain or side-chain atoms. Superposition of the α-toxin^H35A dimer onto two adjacent promoters in the above two WT toxin heptamers yields overall C_α r.m.s.d. values of 0.99 and 0.95 Å, respectively, indicating their structural similarity. Dimer interfaces have similar buried surface area values, from 2,061 to 2,171 Ų. It is also important to note that the crystal structure of unliganded α-toxin^H35A (PDB code 4YHD) lacks the aforementioned intermolecular contacts between six independent monomers in the asymmetric unit. In this structure, both the amino latch and prestem regions have well-defined density with the exception of the six-residue prestem loop and pack against the β-sandwich core of the protein. By contrast, these two regions are apparently disordered in the C₁₄PC-bound structure. Our results suggest that α-toxin^H35A may be trapped in a PC-bound dimeric state, which may represent an on-pathway intermediate in the assembly of the heptameric pore complex.

Figure 5.

Structure of C₁₄PC bound to the α-toxin heptamer. A and B, ribbon representation of C₁₄PC in complex with α-toxin heptamer shown from the side (A) and cytoplasmic (B) views. The PCho moieties and the side chain of Trp¹⁷⁹ are shown as CPK spheres and in stick format, respectively. The amino latch region and the β-sandwich, rim, and stem domains are colored as in Fig. 1A. C, 2F_o − F_c electron density map (green mesh) contoured at 1.0σ for residues Asn¹⁷⁸, Trp¹⁷⁹, and Gly¹⁸⁰ and the three PCho moieties in a single protomeric unit. D, surface representation of the three partially overlapping PCho-binding pockets on a single protomeric unit viewed from the cytoplasmic side, with PCho moieties shown in stick format with transparent CPK spheres. The three binding pockets are labeled. The locations of key binding site residues are indicated. E, molecular interactions in the heptameric α-toxin-C₁₄PC complex, with the PCho moieties in a single protomeric unit displayed in ball-and-stick format. Residues that make direct side-chain contacts with the PCho moieties are shown in ball-and-stick format colored in blue for protomer A, in purple for protomer E, and in yellow for protomer F. The Ω1 and Ω2 loops are labeled. Cation-π interactions are represented as green dotted lines. Hydrogen bonds and salt bridges are shown as pink dotted lines. F, close-up view of interprotomer interactions in the triangle region in the crystal structure of the C₁₄PC-bound α-toxin^H35A heptamer. Protomers A, B, and C are colored blue, green, and yellow, respectively.

Given their expected importance in membrane targeting, the five key PC-binding site residues are highly conserved or invariant in both α-toxin and the leukocidin F subunits but are absent in the S subunits, with the exception of a histidine at position 176 in LukB (Fig. 2C). Of particular importance, LukB exists as a soluble heterodimeric complex with LukA (61). This finding is consistent with the central role of the conserved Trp¹⁷⁶ of the three other F subunits in their binding to the PC bilayer (22, 52, 54) (this study). We therefore propose that the binding of the F subunit to the PC-rich membrane is allosterically coupled to heterodimerization with its S subunit counterpart. Likewise, membrane binding by α-toxin, mediated by PC and/or ADAM10, irrevocably commits the monomers to dimerization. The remarkable high degree of conservation of the two adjacent PC-binding sites among α-toxin and the F subunits reflects a strong selective pressure on the ability of these two sites to help anchor toxin monomers to the cell surface and to form intermolecular contacts that prime the ensuing formation of the oligomeric, membrane-inserted pore complex.

In summary, the bivalent rim domain interaction with PC provides a mechanism by which soluble toxin monomers can recognize and target the PC-containing membrane, thereby promoting dimer-nucleated pore assembly. The relatively low affinity of PC-mediated binding may facilitate subsequent establishment of the final geometry of the oligomeric pore complex, which we discuss below. α-Toxin and the leukocidin S subunits also bind their cognate proteinaceous receptors (19–21, 47), and these interactions likely work in concert with the PC-targeting mechanism to modulate toxin binding, pore formation, and cytotoxicity. Finally, and most importantly, structural elucidation of the two conserved, adjacent PC-binding pockets on α-toxin and the leukocidin F subunits will guide the rational development of PC analogs as decoy receptors that prevent the cytotoxin from binding to susceptible cells.

Structure of the C₁₄PC-bound PVL heterooctamer

In light of previous studies suggesting that PC plays a crucial role in the assembly and function of the α-toxin heptamer (54, 57), we co-crystallized the LukS-PV and LukF-PV proteins with C₁₄PC in the presence of n-octyl-β-glucoside. The structure of the complex was solved at 2.04 Å resolution by molecular replacement (Table 1). The asymmetric unit contains one LukF-PV/LukS-PV heterodimer and a single LukS-PV molecule. The heterodimer interacts with three crystallographic 4-fold symmetry-related copies of itself to generate a heterooctamer (Fig. 4, A and B). In this β-barrel pore complex, four LukF-PV protomers (denoted A, C, E, and G) and four LukS-PV protomers (B, D, F, and H) are arranged in an alternating fashion around the central axis of the pore, in which the stem domain folds into an antiparallel β-barrel composed of 16 β-strands. We could not discern electron density corresponding to the bottom third of the stem domain in our structure. Two distinct interfaces between neighboring protomers involve residues that are distributed among the amino latch region and the β-sandwich and stem domains and bury 2,644 and 1,902 Ų of solvent-accessible surface area, respectively. The electron density map revealed clearly the presence of PCho moieties at three distinct binding sites on each of the four protomeric units of the PVL heterooctamer (Fig. 4, C and D). The two adjacent binding sites are essentially the same as those on the above-described toxin monomer, whereas the other, novel site lies at the interface between the rim domain of a LukF-PV protomer (e.g. protomer A) and the proximal stem domain regions of protomers G and H. The average B-factor for the three PCho moieties is significantly higher than that for the surrounding residues (60 Ų as compared with 31 Ų), possibly due to greater disorder and/or subunitary occupancy. Superposition of the PVL heterooctamer bound to C₁₄PC onto the unliganded HlgAB (PDB code 3B07) and LukGH (PDB code 4TW1) heterooctamers yields C_α r.m.s.d. values of 0.67 and 1.14 Å, respectively, suggesting that the PVL pore does not undergo large conformational changes upon binding to C₁₄PC.

Figure 4.

Crystal structure of C₁₄PC in complex with the PVL heterooctamer. A and B, ribbon representation of the C₁₄PC-bound PVL heterooctamer shown from the side (A) and cytoplasmic (B) views. The PCho moieties and the side chain of Trp¹⁷⁶ are displayed as CPK spheres and in stick format, respectively. The LukF-PV and LukS-PV subunits are colored green and yellow, respectively. The β-sandwich, rim, and stem domains are indicated. C, 2F_o − F_c omit electron density map contoured at 1.0σ shown as green mesh around the three PCho moieties in a single protomeric unit. D, surface representation of the three PCho-binding pockets on a single protomeric unit viewed from the cytoplasmic side, with the PCho moieties shown in stick format with transparent CPK spheres. The three binding sites are labeled. The locations of key binding site residues are indicated. E, close-up view of the PCho moieties in the three binding pockets on a single protomeric unit. The side chains of residues that make direct contacts with the PCho moieties are shown in ball-and-stick format colored in blue for protomer A, in magenta for protomer G, and in yellow for protomer H. The Ω1 and Ω2 loops are labeled. Cation-π interactions are depicted as green dotted lines. Hydrogen bonds and salt bridges are represented as pink dotted lines.

The three PCho-binding sites on a single protomeric unit are contained within a water-accessible crevice between the inner surface of the rim domain and the upper portion of the stem domain (Fig. 4, A and B). As noted above, the two adjacent sites correspond to those on the rim domain of monomeric LukF-PV (Fig. 4D; see Fig. 2B), differing only in the presence of more stabilizing molecular contacts at site 2 on the heterooctamer. Specifically, the quaternary ammonium group of the PCho moiety makes a cation–π interaction with Trp²⁵⁶, and the indole ring of this residue establishes an edge-to-face interaction with the indole ring of Trp²⁶¹ to pack against the N-methyl and methylene groups, which are also in contact with the main-chain atoms of Thr⁷¹, Ser¹⁹⁹, Asn²⁰⁰, and Leu²⁰¹ and with the side chain of Ile⁷² (Fig. 4E). At site 3, the aromatic ring of Tyr¹³⁷ of protomer H forms a cation–π interaction with the quaternary ammonium group and stacks against the N-methyl and methylene groups that are also lined with the side chain of Ile¹³⁵ of protomer H (Fig. 4E). Furthermore, the O3 oxygen of the phosphate group hydrogen-bonds to the main-chain amide of Gly¹⁷⁵ of protomer A (2.65 Å), and the O1 and O3 oxygens engage both the main-chain atoms of Asn¹⁷⁴ and Gly¹⁷⁵ of protomer A and the side chains of Met¹⁷² of protomer A and Gln¹¹² of protomer G (Fig. 4E). The solvent-accessible surface area of PCho buried upon complex formation is 258 Ų (77%) at site 1, 189 Ų (57%) at site 2, and 224 Ų (65%) at site 3.

Our results suggest that multivalent binding of the PVL heterooctamer to PC on the membrane surface leads to localized alterations in the lipid bilayer and thus promotes the insertion of amphipathic β-hairpins to produce the β-barrel piercing the bilayer. Critical residues Tyr¹³⁷ of LukS-PV and Gly¹⁷⁵ of LukF-PV at site 3 are invariant in the leukocidin S and F subunits, respectively (Fig. 2C), underscoring their functional importance. Furthermore, three similar PC-binding pockets also exist in protomers of the C₁₄PC-bound α-toxin heptamer described below.

Binding mode of C₁₄PC to the α-toxin heptamer

To evaluate the binding of the α-toxin heptamer to the PC headgroup in a membrane-mimicking environment, we determined the crystal structure of its complex with C₁₄PC at 2.35 Å resolution (Table 1). In this structure, three PCho moieties are bound to each of the seven protomeric units in the water-accessible crevice between the rim and stem domains (Fig. 5, A and B). The indole ring of Trp¹⁷⁹ mediates three-way interactions with these three moieties (Fig. 5C). Their conformations are clearly defined in three partially overlapping but distinct binding pockets of the crevice (Fig. 5D). One pocket corresponds to site 1 on the toxin monomer described above, whereas the other two are novel heptamer-specific binding sites (see below). The average B-factor for the three PCho moieties is 60 Ų, and for surrounding protein atoms, it is 33 Ų. The structure of the C₁₄PC-bound heptamer is very similar to that of the unliganded heptamer (PDB code 7AHL; r.m.s.d. for C_α atoms of 0.48 Å), with only minor changes in the positions of side chains involved in direct contact with C₁₄PC. The pairwise r.m.s.d. values among protomers A–G in the heptamer span a range from 0.13 to 0.17 Å for C_α atoms. The PCho moieties at each of the three binding sites have essentially identical conformation and orientation in each of the seven protomeric units, with average r.m.s.d. values of 0.34 Å for the first pocket, 0.32 Å for the second pocket, and 0.41 Å for the third pocket. For this reason, the following structural analysis of these binding pockets applies to all of the protomeric units.

The first pocket, defined by Trp¹⁷⁹ and Arg²⁰⁰, is the same as that on monomeric α-toxin^H35A (see Fig. 3), albeit the hydrogen bond between the phosphate group of the PCho moiety and the main-chain amide of Arg²⁰⁰ is considerably longer and weaker in the latter (Fig. 5E). The second pocket lined by all four residues on strand β12 of the rim domain snugly accommodates the PCho moiety (Fig. 5, D and E). It mediates a network of van der Waals contacts involving both the main-chain atoms of Gly¹⁸⁰ and Pro¹⁸¹ and the aromatic rings of Trp¹⁷⁹ and Tyr¹⁸², forming hydrogen bonds via its hydroxyl group toward the O3 oxygen of the phosphate group (2.69 Å) and via its O2 oxygen with the main-chain amide of Gly¹⁸⁰ (2.84 Å) while in the cis rotamer.

The third pocket is located at the interface between the rim domain of protomer A and the proximal stem domain regions of protomers E and F (Fig. 5E), in contrast to the other pockets that are constituted solely by residues from the rim domain. The third pocket is formed by residues Asn¹⁷⁸ and Trp¹⁷⁹ from the rim domain of protomer A; by Leu¹¹⁶ and Tyr¹¹⁸ from the stem domain of protomer E; and by Tyr¹¹², Ser¹¹⁴, Ile¹⁴², Gly¹⁴³, and His¹⁴⁴ from the stem domain of protomer F (Fig. 5E). The indole ring of Trp¹⁷⁹ is situated to produce a cation–π interaction with the quaternary ammonium group of the PCho moiety (Fig. 5E). The N-methyl and methylene groups participate in extensive contacts with the main-chain atoms of Gly¹⁴³ and Asn¹⁷⁸ and with the side chains of Tyr¹¹², Ser¹¹⁴, and Ile¹⁴². The PCho moiety is further stabilized by a hydrogen bond between the O3 oxygen of the phosphate group and the ND1 atom of His¹⁴⁴ (2.78 Å) and by contacts between the O1, O2, and O3 oxygens and the side chains of Leu¹¹⁶, Tyr¹¹⁸, and His¹⁴⁴ (Fig. 5E). The solvent-accessible surface areas buried upon binding of the PCho moieties to the first, second, and third pockets are 260 A² (76%), 207 A² (60%), and 285 A² (83%), respectively.

These results strengthen the hypothesis that multivalent binding of the PC bilayer by the α-toxin heptamer may help overcome the energetic barrier to deformation of the membrane during assembly of the β-barrel pore lining, thereby driving the conversion of the prepore to the transmembrane pore complex. Indeed, replacement of Trp¹⁷⁹ and Arg²⁰⁰ with alanines in α-toxin is known to lead to an arrested prepore state in which only the top half of the cytolytic β-barrel pore has formed (26). Together with analysis of intermediate stages of the α-toxin assembly process with engineered disulfide bonds (34), our study also suggests that the interaction between the α-toxin prepore and the PC headgroup may induce a large conformational change in the prestem region, which is essential for pore formation.

Structure of the α-toxin^H35A heptamer in complex with C₁₄PC

In the α-toxin pore structure, His³⁵ is located in the crucial interprotomeric contact region (27), and nonconservative replacements at this position (including H35A) have been shown to abolish heptamer formation and thus cytolytic activity and lethal toxicity (62–64). In light of our findings that the PC bilayer binding might promote both the oligomerization of α-toxin monomers and the structural rearrangements that accompany the prepore-to-pore conversion, we hypothesized that a high concentration of C₁₄PC could facilitate the assembly of the α-toxin^H35A pore complex. To directly test this hypothesis, we have determined the structure of the α-toxin^H35A heptamer crystallized in the presence of 25 mm C₁₄PC at 2.5 Å resolution (Table 1 and Fig. 5F); it is worth noting the use of 5 mm C₁₄PC for the crystallization of the α-toxin^H35A monomer (see Fig. 3 and “Experimental procedures”). In this mutant pore complex, PCho moieties bind in the first and second pockets described above on the rim domain of each protomer. In essence, the C₁₄PC-bound structures of the α-toxin^H35A and WT heptamers are nearly identical, with r.m.s.d. values of 0.04–1.42 Å over 2,051 C_α atoms. The positions and conformations of the two PCho moieties are also similar. However, C₁₄PC does not bind to the aforementioned interprotomer pocket on the α-toxin^H35A pore, whereas B-factors for this mutant pore are considerably higher than those for the WT one (24–201 Ų as compared with 13–73 Ų), consistent with the pronounced effect of the H35A mutation on cytotoxicity (59). These results support our hypothesis that the PC-rich membrane acts as a critical effector of oligomerization and pore formation by α-toxin.

In summary, despite their different subunit composition and stoichiometry, α-toxin and the leukocidins likely follow an evolutionarily conserved PC-dependent pore assembly pathway, involving the initial membrane binding of toxin monomers and membrane-dependent dimerization and oligomerization, followed by the prepore-to-pore transition and membrane perforation. It should be stressed that our crystallographic results demonstrate that the interactions between PCho and the oligomeric pore forms of α-toxin and PVL differ considerably. Importantly, atomic-level insight of the toxin oligomer-PC interactions obtained here will facilitate the development of PC analogs that inhibit pore formation and thus block the immune cytolytic effects of this subfamily of proteins.

Inhibition of the cytotoxicity of LukED, PVL, and α-toxin by C₁₄PC

The presence of the conserved PC binding sites in the leukocidins and α-toxin (see above) suggests that PC mimetic compounds may interfere with toxin-mediated killing of primary human immune cells. Therefore, flow cytometry experiments were conducted to first evaluate the ability of C₁₄PC to diminish the cytolytic activity of LukED in Jurkat cells expressing CCR5. This Jurkat cell line has been shown to be susceptible to the toxin (19). LukED at a concentration of 2.5 μg/ml resulted in ∼80% lysis of Jurkat cells within 1 h at 37 °C (Fig. 6A). We found that C₁₄PC inhibited the lysis in a concentration-dependent manner, with an IC₅₀ value between 15 and 25 μm (Fig. 6A). In sharp contrast, PCho did not show appreciable inhibitory activity up to 0.5 mm. We conclude that C₁₄PC produces effective toxin inhibition by presenting multiple copies of the PC headgroup on its micellar surface, in accordance with previous observations (54).

Figure 6.

C₁₄PC protects target cells from lysis by LukED. A, titration of the cytotoxicity of LukED by C₁₄PC. CCR5⁺ Jurkat cells were challenged with different concentrations of LukED in the absence and presence of C₁₄PC (6–100 μm). Cell viability was determined by flow cytometry and normalized to that in the medium control. Data are representative of at least three independent experiments, and values are expressed in the mean of triplicate measurements ± S.E. B, protective effect of C₁₄PC against LukED-mediated killing of primary human monocytes. PBMCs were challenged with LukED in the presence and absence of 50 μm C₁₄PC. Monocyte subsets were identified as CD3⁻CD14⁺ after gating on live PBMCs as determined by flow cytometry. Data are representative of two independent experiments. Percentages of cells in each quadrant are indicated. C, protective effect of C₁₄PC against LukED-mediated killing of CD8⁺ effector memory T cells (CCR7⁻CD45RO⁺ and CCR7⁻CD45RO⁻). PBMCs were challenged with LukED in the presence and absence of 50 μm C₁₄PC. Live CD3⁺CD8⁺ T cell subsets were gated and analyzed for the expression of CCR7 and CD45RO by flow cytometry. Data are representative of two independent experiments using blood from different donors. D and E, bar graphs showing inhibition of the LukED-induced lysis of monocytes (D) and CD8⁺ effector memory T cells (E) by 50 μm C₁₄PC. Error bars, S.E. F, protective effect of C₁₄PC against LukED-mediated killing of CD8⁺CCR5⁺ T cells. PBMCs were challenged with LukED in the presence and absence of 50 μm C₁₄PC. Live CD3⁺CD8⁺ T cell subsets were gated and analyzed for the expression of CCR5 and CD45RO by flow cytometry. Data are representative of two independent experiments. G, protective effect of C₁₄PC against LukED-mediated killing of NK cells. PBMCs were challenged with LukED in the presence and absence of 50 μm C₁₄PC. PBMCs were first gated on live CD3⁻HLA-DR⁻ cells, and the proportion of CXCR1⁺2B4⁺ NK cells was analyzed by flow cytometry. Data are representative of two independent experiments.

To investigate the protective effects of C₁₄PC on LukED-induced lysis of primary human leukocytes expressing CCR5 and CXCR1 in vitro, LukED at concentrations of 2.5 and 5 μg/ml was first preincubated with 50 μm C₁₄PC at 4 °C and was subsequently added to peripheral blood mononuclear cells (PBMCs) labeled with specific cell surface markers. After 1–1.5 h at 37 °C, the cells were stained with fixable viability dye eFluor 506 and analyzed by flow cytometry. Inhibition of LukED by C₁₄PC was assessed by determining the relative abundance of viable cells after challenge with the toxin or medium. As expected, CD14⁺ monocytes were significantly absent by 2.5 and 5 μg/ml of LukED (Fig. 6D), whereas pretreatment with 50 μm C₁₄PC produced a 70–90% protective effect against monocyte lysis (Fig. 6, B and D). Likewise, 50 μm C₁₄PC blocked the lysis of CD8⁺ effector memory T cells by 50–75% (Fig. 6, C and E) and of CD8⁺CCR5⁺ T cells by 50–95% (Fig. 6F). Moreover, 50 μm C₁₄PC also rescued 50–85% of NK cells (Fig. 6G), which are highly susceptible to LukED due to their surface expression of CXCR1 (19). These results demonstrate that C₁₄PC confers target cell protection by blocking the interaction between LukED and membrane PC.

We next sought to assess the ability of C₁₄PC to abrogate the cytolytic activities of PVL and α-toxin using the in vitro cell viability assay described above. The addition of 2 and 10 ng/ml PVL led to 85–95% lysis of monocytes after 1.5 h of incubation at 37 °C (Fig. 7, A and B). Pretreatment with 100 μm C₁₄PC suppressed the lysis by 90% (Fig. 7, A and B). Similarly, α-toxin at concentrations of 30 and 100 ng/ml caused 75–90% lysis of monocytes and ∼50% lysis of CD3⁺ T cells after incubation at 37 °C for 24 h, whereas 100 μm C₁₄PC caused 75–90% reduction of the lytic activity (Fig. 7, C and D). We conclude that C₁₄PC is a broad-spectrum small-molecule inhibitor of LukED, PVL, and α-toxin and that membrane PC contributes to the mechanism of their cytolytic action.

Figure 7.

C₁₄PC protects primary human monocytes from lysis by both PVL and α-toxin. A, protective effect of C₁₄PC against PVL-mediated killing of monocytes. PBMCs were challenged with PVL in the presence and absence of 100 μm C₁₄PC. Monocyte subsets were gated based on the forward scatter and side scatter parameters and then analyzed for the expression of CD14⁺ and CD3⁺, respectively. Cell viability was determined by flow cytometry and normalized to that in the medium control. Data are representative of two independent experiments using blood from different donors. Percentages of cells in each quadrant are indicated. B, bar graph showing inhibition of the PVL-induced lysis of monocytes by 100 μm C₁₄PC. Error bars, S.E. C, protective effect of C₁₄PC against α-toxin–mediated killing of monocytes. PBMCs were challenged with α-toxin in the presence and absence of 100 μm C₁₄PC. Monocyte subsets were identified and quantified as described in A. Data are representative of two independent experiments using blood from different donors. D, bar graph showing inhibition of the α-toxin–induced lysis of monocytes by 100 μm C₁₄PC. Error bars, S.E.

Implication for MRSA drug discovery

The high prevalence of highly pathogenic MRSA is creating a crisis in modern healthcare due to the limited therapeutic options available, the toll of severe disease and mortality it inflicts, and the enormous cost of inpatient care to which it contributes (3, 4). The ability of MRSA to form biofilms on necrotic tissues and medical devices is also an important virulence mechanism that complicates infections (5, 6). As antibiotic resistance continues to emerge, disarming the major virulence mechanisms of MRSA strains has the potential to become an alternative therapeutic approach aimed at limiting host tissue damage while aiding immune clearance. The α-hemolysin subfamily of cytotoxins represents a prime target for antivirulence drug development, owing to their critical roles in inactivating host immune defenses, destroying tissue barriers, and modulating inflammatory responses (3, 16). mAbs targeting α-toxin have been shown to prevent human lung cell injury in vitro and protect experimental animals against lethal S. aureus pneumonia (65). Several such mAbs are currently in clinical trials, including mAbs MEDI4893 and KBSA301 (Refs. 66–69). Given the variability of MRSA immune evasion determinants, such single-target drugs are most likely to be inadequate to achieve a therapeutic effect (68). Our structural elucidation of the two conserved, adjacent PCho-binding pockets on the rim domains of α-toxin and the leukocidin F subunits will guide the rational development of PC analogs that prevent cytotoxin assembly and pore formation in the susceptible cell membrane, thereby blocking the cytolytic effects of this subfamily of proteins. Using a combined structural biology and pharmacological approach, we have been able to demonstrate that C₁₄PC is a novel broad-spectrum inhibitor of PVL, LukED, and α-toxin in vitro. In light of the safety of miltefosine (hexadecylophosphocline, C₁₆PC), an oral drug used for the treatment of leishmaniasis (70), we expect that C₁₄PC will likewise be well-tolerated in humans. Considering its conserved mechanism of action and low production costs, C₁₄PC may provide the basis for the development of prophylactic and therapeutic agents that reduce the virulence of MRSA infection.

Experimental procedures

Chemicals

All chemicals used were of analytical grade. Unless otherwise indicated, chemicals were purchased from Sigma–Aldrich. Detergents were from Anatrace.

Cloning and protein purification

The full-length LukD (residues 1–301), LukE (residues 1–283), LukF-PV (residues 1–301), LukS-PV (residues 1-284), and α-toxin (residues 1–293) constructs, excluding their signal peptides, were subcloned individually into a modified pET3a vector (Novagen). Site-directed mutagenesis was carried out using the Kunkel method. All constructs were verified by DNA sequencing. E. coli BL21(DE3)pLysS cells transformed with each plasmid were grown at 37 °C in Luria-Bertani medium until the A₆₀₀ was between 0.6 and 0.8. IPTG was then added to a final concentration of 0.5 mm, and incubation was continued for 24 h at 16 °C. Cells were harvested by centrifugation; suspended in 50 mm sodium acetate buffer, pH 5.4, 25% sucrose, 5 mm EDTA, and 5 mm DTT; and lysed at 4 °C using an Avestin Emulsiflex C3 homogenizer. Inclusion bodies were isolated by centrifugation, washed twice with the same buffer, and subsequently incubated overnight at 4 °C in 50 mm sodium acetate buffer, pH 5.4, 5 mm DTT, and 6 m guanidine HCl or 8 m urea. Insoluble material was removed by centrifugation, and the protein solution was then dialyzed for 2 days at 4 °C against three changes of buffer A (50 mm sodium acetate, pH 5.4, and 1 mm EDTA). After removal of the insoluble material by centrifugation, the refolded recombinant toxin was loaded onto a CM-Sepharose CL-6B column equilibrated with buffer A and eluted using a linear gradient from 0 to 1 m NaCl. Fractions containing the recombinant toxin were pooled, dialyzed against buffer A, concentrated, and loaded onto a GE Mono S 5/50 GL equilibrated with buffer A, and the toxin was eluted using a linear gradient from 0 to 0.5 m NaCl. The toxin was further purified using size-exclusion chromatography on a GE Superdex 200 10/300 GL equilibrated with 50 mm sodium acetate, pH 5.4, 100 mm NaCl. Fractions containing the toxin were pooled, concentrated to ∼20 mg/ml, and stored at −80 °C until use. The concentration of the toxin in purified preparations was determined through UV absorbance measurements.

Crystallization

All crystallization experiments were performed at room temperature using the hanging-drop vapor diffusion method by mixing 1 μl of protein solution with an equal volume of precipitant solution. Crystals of LukD were grown from protein at 12 mg/ml in 10 mm sodium acetate, pH 5.4, and precipitant solution (20% PEG MME 2000, 10 mm NiCl₂, and 0.1 m Tris-HCl, pH 8.5). For data collection, the crystals were cryoprotected with 15% glycerol in the mother liquor and then flash-cooled in liquid nitrogen. The C₁₄PC-LukD complex was crystallized from protein at 10 mg/ml in 10 mm sodium acetate, pH 5.4, 10 mm C₁₄PC, and 30 mm n-octyl-β-d-glucoside (βOG) and precipitant solution (28% PEG 400, 0.2 m MgCl₂, and 0.1 m HEPES, pH 7.5). The crystals were flash-cooled by plunging directly into liquid nitrogen. Crystals of LukF-PV complexed with C₁₄PC were grown from protein at 10 mg/ml in 10 mm sodium acetate, pH 5.4, 10 mm C₁₄PC, and 30 mm βOG and precipitant solution (2.6 m ammonium sulfate, 5% PEG 400, and 0.1 m HEPES, pH 8.5). The crystals were flash-cooled in liquid nitrogen. The C₁₄PC–α-toxin^H35A complex was crystallized from protein at 10 mg/ml in 10 mm sodium acetate, pH 5.4, 5 mm C₁₄PC, 40 mm βOG, and 0.4 mm deoxy-Big CHAP and precipitant solution (1.5 m ammonium sulfate, 0.25 m potassium sodium tartrate, and 0.1 m sodium citrate, pH 6.0). The crystals were transferred into stabilizing solution (2.25 m ammonium sulfate, 5% glycerol, 20 mm C₁₄PC, and 0.1 m sodium citrate, pH 6.0) and then allowed to equilibrate against 3 m ammonium sulfate for 1 h at room temperature prior to flash-freezing in liquid nitrogen. The PVL heterooctamer in complex with C₁₄PC was crystallized from LukF-PV at 6.7 mg/ml and LukS-PV at 6.3 mg/ml in 10 mm sodium acetate, pH 5.4, 15 mm C₁₄PC, and 40 mm βOG and precipitant solution (0.16 m magnesium formate). The crystals were transferred into dehydrating solution (2.7 m ammonium sulfate, and 20 mm C₁₄PC) and then allowed to equilibrate against 3 m ammonium sulfate for 3 h at room temperature prior to flash-freezing in liquid nitrogen. Crystals of the α-toxin heptamer-C₁₄PC complex were grown from protein at 8 mg/ml in 10 mm sodium acetate, pH 5.4, 15 mm C₁₄PC, and 30 mm βOG and precipitant solution (2 m ammonium sulfate, 0.2 m potassium sodium tartrate, and 0.1 m sodium citrate, pH 6.0). The crystals were flash-frozen in liquid nitrogen. The α-toxin^H35A heptamer in complex with C₁₄PC was crystallized from protein at 10 mg/ml in 10 mm sodium acetate, pH 5.4, 25 mm C₁₄PC, and 40 mm βOG and precipitant solution (1.9 m ammonium sulfate, 0.25 m potassium sodium tartrate, and 0.1 m sodium citrate, pH 5.2). The crystals were flash-cooled in liquid nitrogen.

Data collection and structure determination

Diffraction data were collected at 100 K at beamline X4C at the National Synchrotron Light Source at Brookhaven National Laboratory, at the Cornell High Energy Synchrotron Source (CHESS) beamline F1, and at the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 9-2. The diffraction data were processed with HKL-2000 (71). Initial phases were determined by molecular replacement using Phaser (72) with respective models of HlgB (PDB code 1LKF), LukF-PV (1PVL), α-toxin^H35A (4YHD), the HlgAB heterooctamer (3B07), and the α-toxin heptamer (7AHL). Refinement was carried out in Refmac5 (73), alternating with manual rebuilding and adjustment in COOT (74). Coordinates for C₁₄PC were generated using LibCheck (75). TLS refinement was performed in Refmac5 (76). Crystallographic data and refinement statistics are summarized in Table 1.

Structural analyses

Model quality was judged using the programs Rampage, Procheck, and Sfcheck (77–79). Protein-ligand contacts for the toxin-C₁₄PC complex structures were analyzed using the program COOT (80). The r.m.s.d. values were calculated using the program SuperPose (81). Molecular and solvent-accessible surfaces were calculated with the AREAIMOL program (82) from the CCP4 suite (83). PyMOL (DeLano Scientific) was used to render structure figures.

Differential scanning calorimetry

Protein thermal stability was determined by differential scanning calorimetry (DSC) using a Nano-DSC model 602000 calorimeter (TA Instruments). Protein solutions in buffer A (20 mm sodium acetate, pH 5.8, and 50 mm NaCl) in the presence and absence of 4 mm PCho were subjected to a temperature increase of 1 °C/min from 0 to 100 °C under a pressure of 3 atm, and the evolution of heat was recorded as a differential power between reference (buffer A) and sample (10 μm protein in buffer A) cells. The resulting thermograms (after buffer subtraction) were used to derive thermal transition midpoints (T_m values). Fitting to the two-state scaled model provided in NanoAnalyze software was used to obtain a T_m value. The experiments were repeated two times with consistent results.

Isolation of human peripheral blood mononuclear cells

Blood samples were obtained from healthy, consenting donors as buffy coats (New York Blood Center) and leukopaks (AllCells, Alameda, CA). Human PBMCs were isolated from peripheral blood by density gradient centrifugation using Ficoll-Paque Plus (GE Life Sciences).

Cytolysis inhibition assay

Flow cytometry was used to assay permeabilization of the plasma membrane (pore formation) by LukED, PVL, and α-toxin in Jurkat cells and primary human immune cells as described previously (84). Briefly, C₁₄PC (6–100 μm) was preincubated individually with different concentrations of the LukD and LukF-PV F subunits and α-toxin in a V-bottom 96-well plate for 30 min at 4 °C. These mixtures were then added to prestained PBMCs and incubated with the cognate LukE and LukS-PV S subunits for 1–1.5 h and with α-toxin for 24 h in a humidified 5% CO₂ incubator at 37 °C. The cytotoxin-treated cells were stained with a viability dye and analyzed by FACS. IC₅₀ values were calculated using GraphPad Prism by fitting data to single-slope dose-response curves constrained to 0 and 100% values.

Staining and FACS analysis

PBMCs were differentially stained with specific cell surface markers prior to intoxication to identify distinct cell populations. Antibodies used for flow cytometric staining included CD3-Alexa 532 (clone UCHT1) (eBioscience, San Diego, CA), CD4-Brilliant Violet 570, CD8-Pacific Blue, CD45RO-APCCy7, CD14-Alexa 700, CD27-PeCy7, CD244 (2B4)-Percp Cy5.5, CXCR1-APC (Biolegend, San Diego, CA), CCR5-PE (BD Biosciences), and CCR7-FITC (R&D Systems, Minneapolis, MN). After intoxication, cells were collected, washed with PBS, and stained with fixable viability dye eFluor 506 (eBioscience). Data were acquired on a BD LSRFortessa X-20 instrument (BD Biosciences) using FACSDiva software and the iQue Screener PLUS (Intellicyt) using ForeCyt Software or SP6800 Spectral Analyzer (Sony Biotechnology, CA). Data analysis was performed using FlowJo software (TreeStar Inc, Ashland, OR). Statistical analysis was performed using GraphPad Prism 8 software.

Data availability

All data are contained within the article. The atomic coordinates and structure factors (codes 6U33, 6U2S, 6U3F, 6U3T, 6U3Y, 6U49, and 6U4P) have been deposited in the Protein Data Bank.

Author contributions

J. L., L. K., D. U., and M. L. data curation; J. L. software; J. L., L. K., D. U., and M. L. formal analysis; J. L. and M. L. validation; J. L. visualization; J. L., L. K., D. U., and M. L. methodology; J. L., D. U., and M. L. writing-original draft; V. J. T. and M. L. resources; D. U. and M. L. investigation; M. L. conceptualization; M. L. supervision; M. L. funding acquisition; M. L. project administration; M. L. writing-review and editing.