All major cholesterol-dependent cytolysins use glycans as cellular receptors

Eight major CDCs use glycans as cellular receptors to potentiate cytotoxicity and bind glycans and cholesterol independently.

Abstract

Cholesterol-dependent cytolysins (CDCs) form pores in cholesterol-rich membranes, but cholesterol alone is insufficient to explain their cell and host tropism. Here, we show that all eight major CDCs have high-affinity lectin activity that identifies glycans as candidate cellular receptors. Streptolysin O, vaginolysin, and perfringolysin O bind multiple glycans, while pneumolysin, lectinolysin, and listeriolysin O recognize a single glycan class. Addition of exogenous carbohydrate receptors for each CDC inhibits toxin activity. We present a structure for suilysin domain 4 in complex with two distinct glycan receptors, P₁ antigen and αGal/Galili. We report a wide range of binding affinities for cholesterol and for the cholesterol analog pregnenolone sulfate and show that CDCs bind glycans and cholesterol independently. Intermedilysin binds to the sialyl-TF O-glycan on its erythrocyte receptor, CD59. Removing sialyl-TF from CD59 reduces intermedilysin binding. Glycan-lectin interactions underpin the cellular tropism of CDCs and provide molecular targets to block their cytotoxic activity.

INTRODUCTION

The common feature of the cholesterol-dependent cytolysins (CDCs) is the ability to form pores in membranes. The multistep process of pore formation by the CDCs is as follows: Soluble CDC monomers bind to the cellular membrane, the monomers then interact with each other to form an oligomeric prepore structure consisting of 34 to 50 monomers, and then the prepore structure collapses vertically to penetrate into the membrane, becoming a transmembrane β barrel pore (1, 2).

The importance of membrane cholesterol in CDC function has been known since 1939, when it was reported that preincubation of the CDC streptolysin O (SLO; from Streptococcus pyogenes) with free cholesterol inhibited hemolytic activity (3). Later work with listeriolysin O (LLO; from Listeria monocytogenes) showed that preincubation with cholesterol inhibited oligomerization of the toxin in cellular membranes, preventing pore formation and blocking hemolytic and cytolytic activity. However, it is notable that cholesterol does not interfere with membrane binding of LLO or its ability to induce cytokine expression in macrophages (4, 5), indicating that other cellular receptors may exist. Intermedilysin (ILY; from Streptococcus intermedius) requires cholesterol for insertion of the pore into the membrane (6) but uses human CD59 (hCD59) as a cellular receptor (7).

Several proteinaceous receptors for membrane lipid-dependent, pore-forming toxins from Staphylococcus aureus have recently been identified (8). Furthermore, we have shown that the CDCs SLO and pneumolysin (Ply; from Streptococcus pneumoniae) require binding to glycan cellular receptors on red blood cells (RBCs) for hemolytic activity (9). In the current study, we examine all major CDCs for glycan binding properties using a comprehensive glycan array and investigate the role of specific glycans as functional cellular receptors for CDCs.

RESULTS

All CDCs screened by glycan array have lectin activity

All major CDCs [Ply, SLO, perfringolysin O (PFO), vaginolysin (VLY), lectinolysin (LLY), LLO, suilysin (SLY), and ILY] were screened for lectin activity using an array of 402 unique glycan structures to identify potential glycan receptors. The His-tagged CDCs were detected with a fluorescent antibody complex. All of the CDCs screened showed glycan binding to a unique set of glycan structures (Fig. 1 and data S1). Some toxins showed highly specific binding; for example, LLY bound a single structure, while other CDCs such as SLO and PFO bound to a broad range of glycans. The binding profile of each CDC is detailed below.

Fig. 1. Glycan array analyses of glycan binding by CDCs.

Red indicates binding above background. Blue indicates binding observed here and previously (9). White indicates that the structure was not bound. Classes of glycans arrayed are indicated to the left of the figure. A complete list of glycan structures on the array and the mean fold fluorescence increase above background of three replicate glycan array experiments are shown in data S1. GAGs, glycosylaminoglycans.

To further investigate the glycan array results using an independent method and to characterize the kinetics of the interactions of the CDCs with their glycan targets, we used surface plasmon resonance (SPR). Recombinant His-tagged CDCs (see Materials and Methods and fig. S1) were immobilized, and free glycans were flowed over the proteins. These data showed high-affinity glycan interactions for all CDCs tested (Table 1 and table S1). CDCs have a conserved, four-domain structure (10). Ply binds to its glycan receptor, sialyl-Lewis X (sLex), via domain 4 (D4) (9). To investigate whether D4 mediates glycan binding in other CDCs, we expressed and purified D4 of LLO, ILY, and SLY and examined these domains for glycan binding by SPR. The glycan binding we observed with the whole toxins was recapitulated by the recombinant domain 4 from LLO, ILY, SLY, and Ply (9) and indicated that glycan binding via D4 may be a common feature of all CDCs (Table 1).

Table 1. A subset of SPR results for CDCs against glycans plus cholesterol and PS.

Glycans, cholesterol, and PS were tested at concentrations listed in data S2 and the Materials and Methods. Data shown are the mean K_Ds from at least three independently run SPR analyses. NCDI, no concentration dependent binding observed up to the maximum concentration tested; n.t., not tested, sample was not run against that toxin. α1-3Gal tri, Galili (αGa1) antigen trisaccharide; sTF, Neu5Acα2-3Ga1β1-3Ga1NAc; sLeC, Neu5Acα2-3Ga1β1-3G1cNAc; α2-6sLN, Neu5Acα2-6LacNAc; BGA tri, blood group A trisaccharide; BGA type 4, blood group A type IV pentasaccharide; BGB type 4, blood group B type IV pentasaccharide; BGH di, blood group H antigen disaccharide; BGH type 4, blood group H antigen type IV tetraose; PS, pregnenolone sulfate (sodium salt). For the full range of glycans tested, error, and more than two significant figures, see table S1.

	Ply	SLO	PFO	VLY	LLY	LLY (−LD)	LLO	LLO D4	SLY	SLY D4	ILY	ILY D4
GT2	n.t.	NCDI	n.t.	n.t.	n.t.	n.t.	6.7 nM	10 nM	n.t.	n.t.	n.t.	n.t.
α1-3Gal tri	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.	258 nM	547 nM	NCDI	NCDI
P1 Antigen	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.	n.t.	359 nM	342 nM	NCDI	NCDI
LNnT	n.t.	0.93 nM	2.8 μM	NCDI	NCDI	n.t.	NCDI	NCDI	n.t.	n.t.	4.9 μM	n.t.
TF	n.t.	n.t.	46 μM	n.t.	NCDI	n.t.	n.t.	n.t.	n.t.	n.t.	60 μM	n.t.
sTF	n.t.	n.t.	13 μM	n.t.	NCDI	n.t.	n.t.	n.t.	NCDI	NCDI	599 nM	568 nM
sLeC	n.t.	1.4 μM	1.6 μM	NCDI	16 μM	n.t.	n.t.	n.t.	NCDI	NCDI	16 μM	4.9 μM
α2-6sLN	n.t.	n.t.	23 μM	633 nM	1.6 μM	1.8 μM	n.t.	n.t.	n.t.	n.t.	NCDI	NCDI
sLeX	1.3 μM	3.1 μM	3.9 μM	n.t.	n.t.	n.t.	n.t.	n.t.	NCDI	NCDI	2.8 μM	4.9 μM
BGA tri	n.t.	1.3 μM	5.1 μM	n.t.	NCDI	n.t.	n.t.	n.t.	8.7 nM	4.4 nM	9.9 μM	4.9 μM
BGA type 4	n.t.	105 nM	150 μM	n.t.	NCDI	n.t.	n.t.	n.t.	5.7 μM	9.2 μM	NCDI	NCDI
BGB type 4	n.t.	0.12 nM	32 μM	n.t.	NCDI	n.t.	n.t.	n.t.	8.4 μM	6.4 μM	NCDI	n.t.
BGH di	n.t.	603 nM	NCDI	n.t.	NCDI	n.t.	n.t.	n.t.	19 μM	26 μM	NCDI	n.t.
BGH type 4	n.t.	2.6 μM	NCDI	n.t.	NCDI	n.t.	n.t.	n.t.	n.t.	n.t.	NCDI	n.t.
Cellobiose	NCDI	NCDI	34 μM	NCDI	NCDI	n.t.	NCDI	NCDI	NCDI	NCDI	NCDI	NCDI
Cholesterol	1 μM	189 nM	296 nM	NCDI	NCDI	NCDI	NCDI	NCDI	NCDI	NCDI	NCDI	NCDI
PS	0.4 nM	52 nM	n.t.	n.t.	n.t.	997 nM	n.t.	214 nM	1.8 μM	n.t.	588 nM	NCDI

SLO: Human blood group B antigen type IV pentasaccharide can inhibit SLO hemolytic activity

SLO is a major virulence factor of group A Streptococcus (S. pyogenes) (11). Although SLO bound to a broad range of glycans in the array studies, SPR analysis showed that the highest affinity interactions were detected for the ABO blood group antigens, with the highest affinity observed for the group B type IV pentasaccharide with a dissociation constant (K_D) of 116 pM (Table 1), several fold higher than the previously identified ligand, lacto-N-neotetraose (9).

We next investigated whether these high-affinity glycan ligands could inhibit SLO hemolytic activity. SLO lysis of human RBCs was assessed in the presence of three of the synthetic ABO blood group antigen structures (Fig. 2A). Despite the broad range of glycan binding we observed with SLO on the glycan array, the blood group B type IV pentasaccharide was able to completely inhibit SLO hemolytic activity, consistent with a single glycan binding site on SLO. Differential blood group antigen inhibition of SLO hemolytic activity was in proportion to the binding affinities determined by SPR (Fig. 2A).

Fig. 2. Analysis of the hemolytic activity of CDCs in the presence of candidate glycan cellular receptors.

(A to F) The hemolytic activity of the CDCs at the hemolytic dose 100 (HD₁₀₀) or HD₅₀ against 1% (v/v) human group O RBCs was determined after preincubation with phosphate-buffered saline, or with the indicated concentration of candidate glycan receptor, or with the same concentration of cellobiose, which served as a common, nonbinding glycan control. For the analysis of the impact of sTF and TF on ILY binding to RBCs (G), RBCs were fixed to the wells of a 96-well plate, and the ability of 2 mM sTF, 2 mM TF, or 2 mM cellobiose to block the binding of 25 ng of ILY was assessed. The HD₁₀₀ of SLO was also assessed in the presence of blood group B type IV pentasaccharide and pregnenolone sulfate (PS) (sodium salt) (H), individually and in combination (see fig. S7G for determination of HD₁₀₀ of SLO and fig. S7, I and J for hemolytic assays in the presence of a range of concentrations of blood group B type IV pentasaccharide and PS). Results presented are a representative assay conducted with a single blood donor and are shown as the mean of triplicate samples, with error bars showing ±1 SD from the mean. Multiple independent assays (minimum of three) were performed all showing the same trend. The percentage values for each data point used to generate bar graphs are shown in table S4A. The structure of the glycan receptor and the K_D of the CDC for the glycan as determined by SPR are shown above the appropriate bar in each histogram. Statistical significance was determined using a two-tailed unpaired Student’s t test. **P < 0.005, ***P < 0.0005, and ****P < 0.0001. P values for t tests comparing hemolytic activity of CDCs without and with glycan/PS are shown in table S4B. B pentaose, blood group B type IV pentasaccharide; A pentaose, blood group A type IV pentasaccharide; H tetraose, blood group H antigen type IV tetraose; sLeC, sialyl-Lewis C; α2-6SLN, Neu5Acα2-6 LacNAc; P1, P₁ antigen; Xeno, Xeno antigen/Galili epitope; A-tri, blood group A trisaccharide; sTF, sialyl-TF.

We investigated the susceptibilities of the different human blood groups to SLO, but we did not observe any obvious differences in the hemolytic activity of SLO against human groups O, A, and B RBCs. The blood group B type IV antigen has not been detected on RBCs from group B donors (12), which would explain this observation. There may be other cell types with high levels of the blood group B type IV antigen that are highly sensitive to SLO-mediated cytolysis.

PFO: PFO has broad lectin activity, with a preference for structures containing the sialic acid, Neu5Ac

PFO is one of two toxins produced by Clostridium perfringens that are considered to be essential for the development of gas gangrene in humans (13). However, paradoxically, PFO is not essential for disease in a mouse model (14). As the receptor biology of PFO is not well understood, it is not known whether the human-specific role for PFO in disease is due to differences in receptor expression. Glycan array analysis showed that PFO bound to a broad range of glycans (Fig. 1), with SPR measurements indicating the highest affinity binding to sialylated structures (Table 1). PFO showed a binding preference to N-acetylneuraminic acid (Neu5Ac) sialylated structures compared to N-glycolylneuraminic acid (Neu5Gc) sialylated structures. For example, SPR demonstrated that PFO has a 72-fold higher affinity for Neu5Acα2-3Galβ1-3GlcNAc (N-acetylglucosamine) (sLeC) than the Neu5Gc equivalent structure (table S1). Humans do not express Neu5Gc on normal tissues due to an inactive CMAH (Cytidine monophosphate-N-acetylneuraminic acid hydroxylase) enzyme, therefore a Neu5Ac preference may reflect adaptation to the human host. The type of sialic acid linkage (α2-3 compared to α2-6) also influences binding preference, as PFO had higher affinity for Neu5Acα2-3LeC compared to the corresponding α2-6–linked structure (Table 1). Overall, sLeC was the highest affinity PFO glycan ligand identified in our SPR analysis. The presence of 2 mM sLeC significantly inhibited PFO hemolytic activity (Fig. 2B), suggesting a role for glycan binding on the RBC surface in mediating PFO hemolytic activity.

VLY: VLY hemolytic activity can be inhibited with the glycan Neu5Acα2-6LacNAc

VLY is the CDC produced by Gardnerella vaginalis, a facultative anaerobic bacterium found in most of women with bacterial vaginosis. VLY is one of the few known virulence factors of G. vaginalis (15). Like SLO and PFO, VLY is bound to a broad range of glycans (Fig. 1). SPR analysis confirmed an interaction between VLY and the glycan Neu5Acα2-6LacNAc, with a K_D of 633.4 nM (Table 1). The presence of 100 μM Neu5Acα2-6LacNAc inhibited the hemolytic activity of VLY against human RBCs (Fig. 2C).

LLY: LLY binds to a single glycan

Streptococcus mitis produces the CDC LLY, also referred to as human platelet aggregation factor. In addition to the four-domain structure that is common to CDCs, LLY has an extra 162–amino acid N-terminal lectin domain (16). This N-terminal lectin domain was reported to have specificity for Lewis Y and Lewis B glycans; this specificity was demonstrated by probing a glycan microarray with a fluorescently labeled version of the LLY lectin domain in the absence of LLY domains 1 to 4 (17). We analyzed the full-length His-tagged LLY and found that, unlike the other CDCs, LLY only bound to a single glycan structure on the array (Fig. 1).

SPR analysis showed that LLY can bind to the terminal structure of this biantennary glycan, Neu5Acα2-6LacNAc, and to sLeC, which has the same structure but with an α2-3–linked terminal Neu5Ac (Table 1). LLY hemolytic activity was inhibited with 2 mM sLeC (Fig. 2D). SPR analysis was also performed with LLY lacking the N-terminal lectin domain, LLY-LD. This protein recapitulated full-length LLY binding to Neu5Acα2-6LacNAc, confirming that the previously identified N-terminal lectin domain has no role in the lectin activity observed here. Consistent with our observation, removal of the N-terminal lectin domain from LLY does not considerably alter the hemolytic activity of this toxin (17), and the species-dependent hemolytic activity is dictated by D4 of LLY (18).

LLO: LLO binds to carbohydrate structures found on the C-series gangliosides

LLO is an essential virulence factor produced by the intracellular pathogen L. monocytogenes (19). LLO is unique among the CDCs, as it displays pH-dependent behavior, with maximal hemolytic and cytolytic activity at pH 5.5 (20). Glycan array analysis with LLO revealed binding to the C-series ganglioside structures. We assessed the glycan binding of LLO at pH 7.0 and 5.5 and found that interactions with the C-series ganglioside structures were pH dependent (Table 1 and table S1). The highest affinity LLO ligand is the glycan structure found on the C-series ganglioside GT2 at pH 5.5. The correspondence of the pH optima of LLO cytolytic activity with the highest binding affinity for the glycan receptor identified in this study supports the role of engagement of LLO with these receptors to potentiate cytolytic activity. Free GT2 glycan blocked LLO-mediated hemolytic activity against human RBCs (Fig. 2E), and flow cytometry analysis of LLO binding showed that 2 mM free GT2 inhibited association of the toxin with the RBC surface (fig. S2, A and B). These data are consistent with previous studies on SLO and Ply (9), indicating that blocking access to cellular glycan receptors prevents CDCs binding to the target cell surface.

SLY: SLY engages structurally distinct glycan receptors via multiple glycan binding sites

Streptococcus suis is a major swine pathogen and causes a variety of diseases. It is also recognized as an emerging human pathogen, particularly in Southeast Asia, where it is a primary cause of bacterial meningitis. S. suis produces the CDC SLY (21).

Glycan array analysis of SLY showed binding to both terminals α1-3 and α1-4 Gal structures lacking a branched fucose moiety (Fig. 1). SPR analysis of SLY revealed high-affinity binding to Galα1-3Galβ1-4GlcNAc (αGal/Galili epitope, nonhuman), to the human P blood group antigen P₁, and to the human ABO blood group A trisaccharide (Table 1). The αGal/Galili epitope is abundantly expressed on glycoconjugates of nonprimate mammals, including pigs, and is not expressed in humans, apes, and Old World monkeys. Antibodies against the αGal epitope are the most abundant naturally occurring antibodies in humans and are responsible for xenograft rejection of pig organs in humans (22). SPR analysis of purified D4 of SLY showed that all lectin activity resided in D4 (Table 1).

Blocking of SLY hemolytic activity was attempted using three distinct high-affinity glycan ligands. Individually or in combinations of two, these glycans did not inhibit SLY hemolytic activity when tested up to 2 mM. However, a combination of all three glycans at 2 mM completely blocked SLY hemolytic activity against human RBCs (Fig. 2F). These findings suggest that SLY may have multiple binding sites that correspond to these structurally distinct (see Fig. 2F) glycan receptors.

Ply: sLeX glycosylated Mac-1 is a cellular receptor for Ply on human phagocytes

sLeX is a cellular receptor for Ply on human RBCs (9). Others report mannose as a receptor and a crystal structure of Ply with monosaccharide mannose has been reported (23). We did not observe mannose as a high-affinity ligand for Ply (K_D = 338 mM; table S1). In previous work, we demonstrated that Ply cytotoxicity for human phagocytic cells could not be blocked with exogenous sLeX (9) and hypothesized the potential for alternative receptors. S. aureus toxin LukAB uses the I-domain of the CD11b component of Mac-1(Macrophage-1 antigen) as a cellular receptor (24). The I-domain of human CD11b has a single glycosylation: an N-linked glycan terminating in sLeX (25). We hypothesized that Ply may also engage glycosylated CD11b as a cellular receptor and used SPR analysis with Ply flowed over immobilized human Mac-1. Ply has a high-affinity interaction with Mac-1 (K_D = 6.62 nM) (Fig. 3A). When the sLeX N-glycan of Mac-1 (fig. S3) was enzymatically removed, the affinity of Ply for Mac-1 was reduced approximately fourfold (K_D = 27.8 nM; Fig. 3A), indicating a key role for the glycan in the Ply-receptor interaction. Ply interaction with unglycosylated recombinant human I-domain was not significantly different from deglycosylated Mac-1 (K_D = 22.3 nM, P = 0.4013; Fig. 3A), demonstrating the I-domain mediates the polypeptide interaction with Ply. To confirm the role of CD11b as a receptor for Ply in the intoxication of human phagocytic cells, S. pneumoniae expressing either wild-type Ply or a mutant, nontoxic version of Ply (Ply L460D) were tested for cytotoxicity against THP-1 cells with and without short hairpin RNA (shRNA) knockdown of CD11b expression. The reduction in surface expression of CD11b on THP-1 cells has been confirmed by flow cytometry (26). We observed a ~30% reduction in cytotoxicity with the pneumococcus expressing wild-type Ply when CD11b was knocked down in THP-1 cells (Fig. 3B). There was no cytotoxicity observed for the Ply L460D mutant strain (Fig. 3B), indicating that the effect we observed by knocking down CD11b was Ply-dependent.

Fig. 3. Analysis of the interaction of Ply with sLeX-glycosylated Mac-1 and cytotoxicity for THP-1 cells with and without CD11b.

(A) SPR measurements for the affinity of Ply for Mac-1, Mac-1 with the N-linked sLeX glycan removed using peptide N-glycosidase (PNGase) (Mac-1 + PNGase), and the CD11b I-domain showing sLeX enhanced binding to Mac-1 (P = 0.0163) and that glycan-independent binding of Ply to Mac-1 occurs via the I-domain as no difference in binding was observed between Mac-1 lacking sLeX and recombinant human I-domain (P = 0.4031). NCDI, no concentration dependent interaction detected at the concentrations tested. A graphical representation of the Mac-1 complex is shown under the SPR K_D values: CD18 = yellow, CD11b = blue, and I-domain = orange, with sLeX glycosylation of I-domain structure shown. (B) Cytotoxicity of S. pneumoniae D39 expressing wild-type Ply (D39) or a nontoxic version of Ply (Ply460D and L460D) for THP-1 cells with control shRNA (shRNA control) or THP-1 CD11b shRNA knockdown cells (shRNA CD11b). A multiplicity of infection of 2.5 of S. pneumoniae cells was used. Results are shown as the mean of duplicate, independent assays (each assay consisting of triplicate samples), with error bars showing ±1 SD from the mean. Statistical significance was determined using a two-tailed unpaired Student’s t test. *P < 0.05.

ILY: O-linked glycans are crucial components of the CD59 receptor for ILY on human erythrocytes

ILY is a major virulence factor for S. intermedius, a causative agent of brain and liver abscesses in humans (27). ILY uses the ~20 kDa glycophosphatidyl-inositol (GPI)–anchored membrane glycoprotein hCD59 as a receptor. hCD59 has a single N-glycosylation site (Asn18) and multiple O-glycosylation sites (28). Glycan array analysis identified ILY binding to the glycan structure Neu5Acα2-3Galβ1-3GalNAc, also known as sialyl-TF (Thomsen–Friedenreich) (sTF; Fig. 1), one of the most abundant O-linked glycans present on human erythrocyte CD59 (28). These findings prompted us to investigate whether sTF on hCD59 was contributing to the interaction of ILY with this cellular receptor.

SPR analysis showed that the K_D of the interaction between ILY and sTF was 599 nM (Table 1). ILY also bound to the asialo version of sTF (TF). The affinity of ILY for TF was almost 100-fold lower than for sTF, indicating that the sialic acid residue of sTF is important for ILY binding. Moreover, the recognition of sialic acid–terminated structures by ILY showed a marked preference for Neu5Ac over Neu5Gc, indicating human adaptation of this CDC (table S1). We found that recombinant D4 of ILY recapitulated the lectin activity of full-length ILY (Table 1). We observed that sTF can significantly inhibit the binding of ILY to human RBCs (Fig. 2G), showing that this O-linked glycan is crucial in hCD59’s role as a cellular receptor for ILY.

Previous studies have shown that the N-linked glycan on hCD59 is not involved in the binding of ILY (7), and our studies here confirm these findings (fig. S4A). To further investigate the role of the O-linked sTF on hCD59 in ILY binding, we treated solubilized RBC membrane proteins with α2-3 neuraminidase to remove the sialic acid from sTF or with both α2-3 neuraminidase and O-glycosidase to remove the entire sTF structure from hCD59. The untreated and treated RBC membrane proteins were probed with ILY labeled with biotin (Fig. 4 and fig. S4B). We found that the removal of the sialic acid from the O-linked glycan or removal of the entire O-linked glycan from hCD59 reduced the binding of ILY, indicating that O-linked sTF is a crucial component of this glycoprotein receptor for ILY.

Fig. 4. Analysis of the impact, on ILY binding, of removing the sialic acids and O-linked glycans from RBC hCD59.

Solubilized RBC membrane proteins were treated with α2-3 neuraminidase (N) to remove the sialic acids from hCD59, or with both α2-3 neuraminidase and O-glycosidase (N + O) to remove O-linked glycans from hCD59, or with enzyme buffer only under the same conditions (buffer); then were probed with biotin labeled ILY (ILY). Western blotting with anti-CD59 polyclonal antibody confirmed that ILY was binding to hCD59 (anti-CD59). M, molecular weight marker. See fig. S4B for complete far Western/Western blot images and Coomassie-stained gel.

The structural basis of CDC-glycan receptor interactions

X-ray crystal structures are available for all CDCs investigated in this study [reviewed in (29)]. To identify the structural basis of glycan binding to the CDCs observed in our analyses, crystal soaks and cocrystallization studies were attempted with the highest affinity ligands in Table 1. No cocrystals could be obtained, and crystal soaks resulted in no obvious glycan binding. All high-affinity glycan binding observed in SPR analysis is via D4 of the CDCs tested (Table 1). We purified D4 from multiple toxins (Ply, LLO, ILY, SLO, and SLY) to attempt nuclear magnetic resonance (NMR) as an alternative approach to study the CDC-glycan interactions. Only D4 of SLY was sufficiently soluble for assignment of the two-dimensional (2D) spectra.

SLY: Monitoring the binding of two glycan receptors to SLY D4 with ¹H-¹⁵N HSQC NMR experiments

To study the binding interaction between SLY D4 and the αGal/Galili epitope and the P₁ antigen, initially, the backbone chemical shifts (¹H_N, ¹⁵N, ¹³C_α, and ¹³C_β) of the SLY D4 protein were assigned (fig. S5 and table S2) using double-labeled (¹³C/¹⁵N) protein and triple resonance NMR experiments. Then, we monitored the binding of the αGal/Galili epitope and the P₁ antigen to SLY D4 using ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) NMR spectroscopy in the presence of ligands shown in Fig. 5. The spectrum of SLY D4 in complex with the αGal/Galili epitope (Fig. 5A) revealed substantial signal intensity changes for Arg⁴⁶⁵ and Asp⁴⁶⁹ compared to the uncomplexed apo-SLY D4 spectra, suggesting that this glycan binds to a high-affinity site with a slow off rate (30). In addition, ¹⁵N and ¹H chemical shift perturbations (CSPs) were observable for Ala³⁹⁸ and Asp³⁹⁴, suggesting a lower affinity binding site with a faster off rate. ¹H-¹⁵N HSQC NMR spectra of SLY D4 in complex with P₁ antigen (Fig. 5B) revealed signal intensity changes for Asp³⁹⁴ and Ser³⁹⁶, also suggesting a high-affinity site and slow off rate. No CSPs were observed in the SLY D4/P₁ antigen complex HSQC spectra. Control NMR experiments with cellobiose, a nonbinding glycan, showed neither CSPs nor signal intensity changes (fig. S6A). Our NMR results of the ¹H-¹⁵N HSQC titration experiments (Fig. 5, A and B) are in excellent agreement with an unbiased molecular docking study of αGal/Galili that identified a potential αGal-specific binding site. The Galili epitope and the P₁ antigen were both docked into the two potential sites. Arg⁴⁶⁵/Asp⁴⁶⁹ and Asp³⁹⁴/Ser³⁹⁶ were identified as potential αGal binding sites with high ligand occupancy (Fig. 5C). The methyl protons of the N-acetamido group of the reducing GlcNAc residue are involved in strong hydrophobic interactions with the Asp⁴⁶⁹. Similarly, the C6 of GlcNAc with two geminal protons (H6 and H6′) are engaging hydrophobically with Thr⁴⁶⁶. Arg⁴⁶⁵ plays a crucial role in coordinating the hydroxyl groups at C6 of the GlcNAc and Gal residues. This bound conformation also demonstrates clearly that only an α(1,3)Gal linkage can be accommodated as the hydroxyl group at C4 of Gal points directly to Tyr⁴⁸⁸. Glycans with α(1,4)Gal linkage would encounter steric hindrance with Tyr⁴⁸⁸ and hence do not bind at this αGal/Galili binding site. A similar important role is played by Tyr⁴⁹² that holds the terminal Gal unit in place. Molecular docking of the P₁ antigen (Fig. 5D) showed that the methyl protons of the N-acetamido group of the reducing GlcNAc residue are involved in strong hydrophobic interactions with the Trp⁴⁸³. Thr⁴⁸¹, Asp³⁹⁴, and Ser³⁹⁶ are all involved in strong hydrophobic interactions coordinating the GlcNAc and Gal residues. The α(1,4)Gal linkage is easily coordinated by this P₁ binding site, and no steric hindrances could be observed. In summary, molecular docking studies revealed the most favorable binding affinity for the P₁ antigen when bound to the Asp³⁹⁴/Ser³⁹⁶ site and for the αGal/Galili epitope when coordinated by the Arg⁴⁶⁵/Asp⁴⁶⁹ site; this corresponds with the signal intensity changes observed in the ¹H-¹⁵N HSQC NMR data. From these results, we conclude that SLY D4 has two binding sites—one with a high affinity and α(1,3)-Gal specificity (the αGal/Galili binding site) and another with a preference for α(1,4)-Gal linkages (the P₁ binding site), as shown in Fig. 5 (C and D). Our experiments also suggest that the P₁ binding site has a low affinity for α(1,3)-Gal–containing glycans.

Fig. 5. NMR, molecular dynamics and site-directed mutagenesis, confirmed structures of SLY D4 engaged with two distinct glycan receptors.

Sections of ¹H-¹⁵N HSQC NMR CSP spectra of SLY D4 in the presence of αGal/Galili antigen (A) and P₁ antigen (B) at a protein:ligand ratio of 1:10. Signals of apo-SLY D4 are shown in blue and SLY D4 in complex with glycans are shown in red. Full ¹H-¹⁵N HSQC NMR CSP spectra are shown in fig. S6 (B and C). Amino acids that showed an intensity change or CSP are highlighted in yellow in the SLY D4 structure. Unbiased docking experiments of SLY D4 with αGal/Galili antigen (C) and P₁ antigen (D) represent an energetically favored bound conformation. Both modeled structures are in excellent agreement with ¹H-¹⁵N HSQC NMR titration experiments (see Fig. 5, A and B). Binding residues identified in molecular docking experiments only are highlighted in cyan. Coordinating amino acids are shaded in green. Dotted yellow bars represent hydrogen bonds. Green bars represent strong hydrophobic interactions, whereas orange bars represent weaker hydrophobic interactions. For an enlarged version of (C) and (D) showing the key coordinating residues, see fig. S6D. See table S3 for SPR analysis of site-directed mutants of key coordinating residues highlighted in (A) and (B). The whole SLY toxin crystal structure is shown in blue (D1), purple (D2), yellow (D3), and gray (D4) in (A) (Protein Data Bank code 3HVN). ppm, parts per million.

Site-directed mutagenesis was performed in the full-length SLY protein on residues identified in NMR experiments and molecular modeling analyses. Mutagenesis was also performed on Thr⁴⁸¹, which was identified as a key residue for binding to the P₁ antigen in the molecular modeling analyses only. The following SLY mutants were analyzed by SPR: R465A, D469A, D394A, S396A, and T481A (table S3). SLY R465A showed an approximately 15-fold reduction in binding to the Galili epitope. Binding to the P₁ antigen was not greatly affected, with a slight increase in affinity observed. The SLY D469A mutant also had decreased binding to the Galili epitope (approximately fivefold) and an increase in binding to the P₁ antigen (approximately twofold) compared to the wild-type protein. The SLY D394A mutant showed a twofold reduction in binding to the P₁ antigen with a similar affinity to wild-type SLY for the Galili epitope, while the SLY S396A mutant showed an approximate fourfold reduction in binding to the P₁ antigen with no significant difference in binding to the Galili epitope compared to the wild-type protein. The SLY T481A mutant had an approximate 11-fold reduction in binding to the P₁ antigen with no negative impact on binding to the Galili epitope.

The relationship between high-affinity glycan receptors and cholesterol

To investigate the relationship between these newly discovered glycan receptors and cholesterol, we conducted SPR measurements over a range of concentrations of cholesterol, including sub–critical micellar concentrations (CMCs) [25 to 40 nM at 25°C; (31)], to provide a like-for-like comparison with glycan receptors using this sensitive label-free biophysical approach. The data shown in Table 1 (also table S1) indicate that Ply, SLO, and LLO have high-affinity interactions with cholesterol, whereas the other CDCs had no detectable concentration-dependent interaction with cholesterol up to 10 μM.

We conducted SPR competition studies between cholesterol and the respective glycan receptors for Ply, SLO, and LLO (table S1 and fig. S7, A to E). These studies demonstrated that there is no competition between the binding of cholesterol and the high-affinity glycan receptors, indicating that the cholesterol and glycans bind to Ply, SLO, and LLO independently.

We screened 3141 drugs, small molecules, and dyes against Ply and SLO by SPR to identify drugs that can block CDC action. In this screen, we discovered that pregnenolone binds to both CDCs (data S2, AC-i and AC-ii). Pregnenolone is a steroid hormone synthesized from cholesterol (fig. S7F) (32). Pregnenolone sulfate (PS) is a soluble form of pregnenolone. The K_D of Ply for PS and pregnenolone is not significantly different (both at ~400pM; see table S1). SPR analysis showed that PS binds with high affinity to Ply, SLO, and LLO and with lower affinity to PFO, LLY, and SLY (Table 1 and table S1). Competitive SPR analysis of Ply, SLO, and LLO with cholesterol and PS showed that PS can compete directly with cholesterol, indicating that they bind to the same site on these CDCs. PS could not compete for binding of the high-affinity glycan receptors to Ply, SLO, and LLO, indicating that PS and the glycans bind these CDCs at independent sites (fig. S7).

Investigating the relative contributions of engagement of CDCs with glycan receptors and cholesterol in the initiation of the multistep process of CDC hemolytic activity is difficult to resolve because of the relative insolubility of cholesterol compared to the solubility of glycan receptors. In previous studies, cholesterol has typically been provided at concentrations higher than the CMC (5, 27, 33) and often in the form of a liposome or extruded membrane (34, 35), thus differentiating between a CDC binding to cholesterol and the subsequent pore formation steps (oligomerization/prepore, vertical collapse, and transmembrane pore) is difficult. Our observation that PS can act as a soluble cholesterol analog provides an opportunity to test the independent roles of glycans and membrane cholesterol as RBC surface receptors in hemolytic assays. As an exemplar, we used SLO, which has high-affinity interactions with both a glycan (blood group B type IV pentasaccharide, K_D = 116pM) and cholesterol (K_D = 52.3 nM) in hemolytic assays. We showed that the high-affinity glycan receptor, blood group B type IV pentasaccharide, and PS can both inhibit SLO hemolytic activity (fig. S7, I and J). When these molecules are provided in combination, a synergistic inhibitory effect is observed (Fig. 2H). These data further support the idea that the glycan receptor and PS are binding to distinct sites on SLO and also that they are likely blocking sequential steps in SLO-mediated hemolysis.

DISCUSSION

Our findings reveal that lectin activity is a widespread feature of CDCs and is mediated via D4. These data suggest that glycoprotein and/or glycolipid receptors present on the cellular membrane contribute to the cell and tissue tropism of the CDCs. In some cases, the glycan targets that we have identified offer an explanation for the cell tropism of the toxin and thus the pathologies caused by the bacterial species producing the CDC. For example, PLY is known to target human neutrophils and macrophages (36), and its glycan target, sLeX, is highly expressed on these cell types (37). In another example, the C-series gangliosides are expressed in the human brain during embryonic development (38) and are expressed on neural progenitor cells in the adult human brain (39). Listeriosis can result in meningitis and has one of the highest mortality rates of any bacterial meningitis (40). The potential tropism of LLO for stem cells bearing these receptors, which are required for repairing neuronal damage, may play a role in this disease pathology.

The range of glycans bound by the CDCs varies widely. Ply and LLY only bind to one class of glycan structure, while others, such as PFO, bind to a broad range of glycan structures. Our studies with SLY have shown that D4 of SLY has multiple glycan binding sites, allowing this toxin to accommodate different types of glycan targets. This does not appear to be the case for SLO, which has very broad lectin activity for a related set of galactose-containing structures. Regardless of this broad lectin activity, its highest affinity ligand, blood group B type IV pentasaccharide, with a remarkable K_D of 116 pM (Table 1) can completely block SLO hemolytic activity at the hemolytic dose 100 (HD₁₀₀). These data suggest that SLO may have a single glycan binding site that can no longer interact with any other glycan structures when occupied by its highest affinity ligand.

We demonstrate that Ply uses Mac-1 as a cellular receptor to partly mediate killing of human phagocytic cells and that engagement of Mac-1 is influenced by binding to a sLeX N-glycan present on the I-domain of CD11b. These data are supported by the observation that purified Ply can pull down CD11b from a lysate of primary human monocyte-derived dendritic cells, reported in a recent study focusing on the identification of a noncytotoxic cellular receptor for Ply, the mannose receptor C type 1 (41). Similarly, we have shown that hCD59 is a compound cellular receptor for ILY, comprising a polypeptide and O-linked glycans that have distinct and essential roles in the efficient engagement of ILY. The potential O-glycosylation sites of human erythrocyte CD59 have been identified as Thr⁵¹ and Thr⁵² (28). In support of our findings, the binding site of a monoclonal antibody that can completely block ILY binding to human RBCs has been mapped to the amino acid residue directly next to the two identified O-glycosylation sites of hCD59 (Arg⁵³) (7). Note that the residues of hCD59 to which ILY binds are amino acids 42 to 58 (7), encompassing the O-glycosylation sites.

While it is accepted that the CDCs interact with membrane cholesterol to form functional pores, cholesterol acting as the sole cellular receptor for these toxins does not fully explain cellular tropism. For example, mouse RBCs are far more resistant to SLO hemolytic activity than human and rabbit RBCs, despite RBCs from these species having similar cholesterol content (42). For ILY, which does not use cholesterol as a receptor (9), the proposed role of cholesterol in pore formation is to anchor the oligomerized toxin to the membrane surface as it disengages from hCD59 during prepore to transmembrane pore conversion (6, 43, 44). Here, we demonstrate that the hCD59 interaction involves ILY lectin activity. On the basis of our data, showing independent binding of glycans and cholesterol by Ply, SLO, and LLO, we propose that the role of glycoconjugate receptors in CDC pore formation is as follows: There is an initial interaction of the soluble CDC monomer via D4 with a glycan receptor on the cell surface, directing and accumulating the toxin monomer to its target cell, followed by disengagement from the glycan receptor, and lastly an interaction with the membrane during the transition from the prepore to the transmembrane pore. This proposed role for glycan receptors as an initial point of contact for the toxin to host cells before the pore-formation process is further supported by the synergistic inhibitory effect we observed with the exogenous cholesterol analog, PS, and exogenous glycan receptor in blocking SLO hemolytic activity, suggesting that two crucial, independent steps are being inhibited. These data are consistent with the model for CD59 as the receptor for ILY described in previous work by others (6, 43, 44). In addition to this newly defined role for glycoconjugate receptors in directing CDCs to particular target cells as the first step in the pore formation process, these receptors may also play an important role in the tropism of the well-described sublytic effects of CDCs on host cells (45).

We report a wide spectrum of binding affinities between CDCs and cholesterol. Ply, SLO, and LLO have high-affinity interactions. PFO, LLY, and SLY have no detectable interactions with cholesterol but did recognize the soluble cholesterol analog, PS, presumably due to its greater solubility at higher concentrations. Moreover, consistent with other reports (7), we observe that ILY binds neither cholesterol nor PS. Regardless of the lack of a direct high-affinity interaction between the CDCs and cholesterol, this sterol is important for pore formation for all CDCs, perhaps by imparting biophysical properties to the membrane that are highly conducive to the latter steps in the multistep, pore-formation process. Note that all of the glycan receptors identified in this study typically exist as glycoproteins, glycolipids, and GPI-anchored glycoproteins that are frequently associated with the periphery of cholesterol-rich lipid rafts. The classic reagent used to define the periphery of a lipid raft is cholera toxin, which targets the glycolipid GM1 (Ganglioside-monosialic acid 1) (46). Therefore, the identification of glycan receptors for all major CDCs and their role in initial cell contact, thereby imparting cell and tissue tropism, is consistent with a subsequent interaction of the CDCs with a cholesterol-rich lipid raft that is immediately adjacent to these glycoconjugate receptors on the cell surface. We have confirmed the importance and relevance of these receptors in toxin function using the ex vivo human model system of hemolytic activity assays, which is a common and well-accepted assay for this entire class of toxins. The identification of these glycan receptors for each of the CDCs studied here opens the door to further detailed analysis of the role of these glycans in cell, tissue, and host specificity. We anticipate that, like the example of ILY where we have shown that glycans are a crucial component of the CD59 cellular receptor, the glycoproteins and glycolipids that carry the glycan receptors for other CDCs identified here will be discovered, which will help to explain toxin tropism. The identification of these receptors and the molecular mechanisms of their engagement by D4 of the CDCs, exemplified by SLY, will facilitate studies on small-molecule inhibitors and precisely targeted immunological strategies for prevention and treatment of diseases caused by this important group of human pathogens.

MATERIALS AND METHODS

Cloning, expression, and purification of CDCs, domains, and mutants

Ply and SLO were expressed and purified as previously described (9). PFO, LLY, and LLY without the lectin domain, VLY, and ILY were expressed and purified as previously described (47–49). Primers were designed to amplify sequences encoding the following: LLO without the signal sequence (residues 25 to 529) and D4 of LLO (residues 415 to 529) from L. monocytogenes American Type Culture Collection 9525, SLY without the signal sequence (residues 28 to 497) and D4 of SLY (residues 389 to 497) from S. suis strain P1/7, and D4 of ILY (residues 416 to 532) (accession no. AB029317.1). DNA used as template in polymerase chain reactions (PCRs) was purchased from Integrated DNA Technologies. Amplified sequences were cloned into the expression vector pET-15b (Novagen) and were confirmed by DNA sequencing. SLY site-directed mutants were created using inverse PCR to introduce mutations into the sly_pET-15b expression construct. The resultant His-tagged expression constructs were transformed into Escherichia coli BL21 (DE3) for protein expression. Bacterial cultures were grown in Luria-Bertani broth/Amp at 30°C with 200 rpm shaking until an optical density at 600 nm (OD₆₀₀) of ~0.4 was reached. Protein expression was induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) final concentration for 20 hours at 30°C or for 16 hours at 37°C for SLY and SLY D4. Cells were harvested, resuspended in a wash buffer [50 mM sodium phosphate and 300 mM NaCl (pH 7.0)] with deoxyribonuclease (10 μg/ml), lysozyme (1 mg/ml), and EDTA-free protease inhibitor cocktail (Roche). Cells were freeze/thawed and then lysed further with 0.1-mm glass beads using a Qiagen TissueLyser. Soluble His-tagged proteins were purified using TALON cobalt metal affinity resin (Takara Bio) according to the manufacturer’s protocol. Protein concentration was determined using a BCA (Bicinchoninic Acid) protein assay kit (Thermo Fisher Scientific). SDS–polyacrylamide gel electrophoresis (PAGE) analysis of purified recombinant CDCs is shown in fig. S1.

Glycan array

Glycan array slides were printed on SuperEpoxy 3–activated substrates as previously described (50) using the list of glycans previously described in (51) with the addition of glycan IDs 17A to 19P to the glycan library (also see data S1). Glycan array slides were blocked with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) with 2 mM MgCl₂/CaCl₂ (assay PBS) for 5 min. One microgram of His-tagged Ply, SLO, PFO, LLY, VLY, LLO, SLY, and ILY was diluted to 400 μl in assay PBS with precomplexed anti-His monoclonal antibody (Sigma-Aldrich), rabbit anti-mouse Alexa Fluor 555 (Life Technologies), and goat anti-rabbit Alexa Fluor 555 antibodies (Life Technologies) and bound to the glycan array for 15 min at room temperature. Slides were washed twice for 5 min in assay PBS to remove excess protein and were then fixed with 5% formaldehyde in PBS. The slides were lastly dried by centrifugation in a 50-ml tube for 5 min at 150g. The array slides were scanned with a ProScan Array scanner at 555/565 nm, and the results were analyzed using ScanArray Express software (PerkinElmer). Background relative fluorescence units (RFU) was defined as the average fluorescence of 12 sets of blank spots plus 3SDs of the mean. Binding was therefore defined as visible spots with an RFU value greater than 1 when average fluorescence of printed glycan spots was divided by background RFU (blank spot mean + 3SD). Statistical analysis of the data was performed by a Student’s t test with a confidence level of 99.99% (P ≤ 0.0001).

SPR analysis

SPR is a label-free method for testing interactions between two molecules. Using the Biacore T200 system used in these experiments, a minimum of two response units (RU) at the highest concentration tested is required for valid analysis. No data presented in this publication were determined from responses lower than 5 RU at the highest concentration tested. Interactions between the CDC proteins/domains and test glycans were analyzed using SPR as described by Shewell et al. (9), with the following modifications. Proteins were immobilized onto a CM5 chip at pH 4.5 with a flow rate of 5 μl/min for 600 s with an ethanolamine blank flow cell (FC) as a control. The CDCs were captured three at a time on FC2, FC3, and FC4, and due to this arrangement, data obtained from FC3 and FC4 contained timing spikes caused by the double reference subtraction with the data from FC3 and FC4, in particular, being out of phase with the data on FC1. This does not affect the analysis of the data as the region of the curves containing these spikes is not used for calculation of the affinity of the interactions. The range of CDC levels captured on CM5 sensor chips varies for each protein sample. Because of capture level differences between proteins and experiments, no direct RU comparison between tested glycans can be performed with the raw sensorgrams displayed in data S2. Glycans were initially tested between 160 nM and 100 μM, with 1:5 dilutions. Final concentrations of glycans were adjusted based on where saturation was observed, with 100 μM being the maximum concentration used. Representative sensograms from triplicate repeat experiments are shown in data S2. All glycans were obtained from Elicityl (France), except for cellobiose, which was obtained from Carbosynth (UK). All glycans were analyzed by NMR and high performance anion exchange chromatography for structure validation and purity by the supplier. sLeC (Neu5Acα2-3Galβ1-3GlcNAc), sTF (Neu5Acα2-3Galβ1-3GalNAc), and TF (Galβ1-3GalNAc) were synthesized as previously described (52). Cholesterol was sourced from Sigma-Aldrich, and PS (sodium salt; soluble up to 500 μM in aqueous buffers) was sourced from Steraloids Inc. (Newport, RI., USA). Cholesterol was tested at a range of concentrations (1.6 nM to 10 μM), which represents a range of physical states from soluble monomeric (<25 to 40 nM), to rod-like micelles (>25 to 40 nM) (53), to insoluble complexes (>5 μM). SPR studies with Mac-1 (R&D Systems) and recombinant I-domain were conducted as previously described (24). The presence of a N-linked sLeX glycosylation in the I-domain of the recombinant Mac-1 produced in Chinese hamster ovary cells (R&D Systems) was confirmed by NanoLC–tandem mass spectrometry (MS/MS) analysis (see below). Removal of N-linked sLeX from Mac-1 was achieved by incubation with peptide N-glycosidase F (PNGase F) (New England Biolabs) and confirmed by SPR analysis with anti-sLeX monoclonal antibody (clone 258-12767, Creative Diagnostics).

NanoLC-MS/MS analysis

Recombinant Mac-1 (R&D Systems) was analyzed by reverse-phase high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC-ESI-MS/MS) using a Dionex UltiMate 3000 RSLC nano-LC system (Thermo Fisher Scientific) connected to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific) with details described in the Supplementary Materials.

Cytotoxicity assays

Hemolytic assays were performed essentially as previously described (9), with the following modifications: CDCs were prereduced at a concentration of 100 μg/ml with 2 mM dithiothreitol, except for ILY; absorbance of released hemoglobin was measured at 417 nm. Human RBCs were provided by the Australian Red Cross under material supply agreement 17-02QLD-18.

Cytotoxicity assays against THP-1 cells were performed as previously described (24), except rather than intoxication with a purified toxin, a multiplicity of infection of 2.5 of S. pneumoniae D39 were added to the cells. A nontoxic Ply mutant was used to demonstrate that the cytotoxic effect observed was Ply dependent. shRNA knockdown of CD11b expression (26) was used to demonstrate that Ply cytotoxicity was CD11b dependent.

Inhibition of CDC hemolytic activity with candidate glycan receptors

The minimum concentration of toxin able to achieve approximately 100% lysis of 1% (v/v) human group O RBCs (HD₁₀₀) was determined for each CDC by titrating the toxin against the particular donor RBCs before performing inhibition of hemolytic activity assays (see fig. S8 for examples). CDCs at the HD₁₀₀ were preincubated with the appropriate concentration of exogenous free glycan in PBS for 15 min at room temperature before the addition of a final concentration of 1% (v/v) of human group O RBCs. Cellobiose was used in all assays as a nonbinding glycan control. For analysis of the combination of blood group B type IV pentasaccharide and PS against SLO (Fig. 2H), SLO at the HD₁₀₀ (fig. S7G) was preincubated with the indicated concentration of free glycan, PS (sodium salt), or a combination of free glycan and PS before the addition of 1% (v/v) of human group O RBCs. In cases of CDCs with multiple candidate glycan receptors (PFO and SLY), where the blocking effect of a single glycan may be masked by the presence of multiple distinct receptors, HD₅₀ was used in inhibition assays to increase sensitivity. It was observed during hemolytic assays that particular glycans (sTF and Neu5Acα2-6LacNAc) caused agglutination or aggregation of RBCs, resulting in a nonspecific protective effect against hemolysis. There are previous reports of carbohydrates in the form of polysaccharides causing hemagglutination (54). In the case of ILY, where this agglutination or aggregation was observed, whole RBC enzyme-linked immunosorbent assay (ELISA) was used to assess binding of toxin to the RBC surface (see the method below). Several CDCs recognize receptors terminating with sialic acid. An obvious strategy, in addition to inhibiting hemolytic activity with exogenous free glycan, described above, is to treat the cells with neuraminidase to remove the candidate sialylated receptor. Whole RBCs could not be treated with neuraminidase as this caused aggregation of the RBCs and interfered with hemolytic activity assays, presumably due to the reduction in net surface charge (55).

Analysis of RBC surface binding by flow cytometry

Flow cytometry analyses were performed essentially as previously described (9), with modifications and details described in the Supplementary Materials.

Whole RBC ELISA

Whole RBC ELISA was performed as described by Koganei et al. (56), with minor modifications and details described in the Supplementary Materials.

Biotin labeling of ILY

ILY was labeled with biotin using the EZ-link Micro Sulfo-NHS-Biotinylation Kit (Thermo Fisher Scientific) as per the manufacturer’s instructions using a fivefold molar excess of biotin reagent. The hemolytic activity of ILY following labeling was examined in a hemolytic assay and was found to be no different from unlabeled ILY.

Far Western/Western blot analyses

Solubilized RBC membrane proteins (described in the Supplementary Materials) were prepared by incubating 100 μg of RBC membranes with a final concentration of 3% (w/v) of 3-[(3-cholamidopropyl)di-methylammonio]-1-propanesulfonate in a 20 μl of reaction volume for 60 min at room temperature. Soluble and insoluble fractions were separated by centrifugation at 13,000g for 60 min at room temperature. Soluble RBC membrane proteins were treated with enzyme buffer only, α2-3 neuraminidase, both α2-3 neuraminidase and O-glycosidase or PNGase. The O-glycosidase used removes core 1 (Galβ1-3GalNAc) and core 3 (GlcNAcβ1-3GalNAc) O-linked disaccharides from glycoproteins. Sialic acid residues must be removed to allow this O-glycosidase to cleave O-linked glycans. All enzymes were from New England Biolabs and used as recommended by the manufacturer’s instructions. Untreated and treated soluble RBC membrane proteins were combined with LDS (lithium dodecyl sulfate) sample buffer without reducing agent and boiled for 10 min. Proteins were separated on a NuPAGE 4 to 12% bis-tris gel (Life Technologies) with NuPAGE MES SDS running buffer (Life Technologies) and then transferred to a nitrocellulose membrane. Membranes were blocked with 5% BSA in PBS for 60 min at room temperature. Equal loading of protein samples was confirmed by staining with Coomassie blue. hCD59 was detected using a CD59-purified MaxPab mouse polyclonal antibody (Abnova) at 1:1000 as primary antibody and anti-mouse immunoglobulin horseradish peroxidase antibody (Dako) at 1:10,000 as secondary antibody for 60 min at room temperature. For far-Western analyses, the membrane containing transferred solubilized RBC membrane proteins was incubated with ILY-biotin (500 ng/ml) for 2 hours at room temperature, washed three times for 5 min with PBS, incubated with streptavidin-peroxidase (Sigma-Aldrich) at 1:10,000 for 60 min at room temperature, and then washed three times for 5 min with PBS. Blots were developed by the addition of SuperSignal West Pico Chemiluminescence Substrate (Thermo Fisher Scientific) as per the manufacturer’s instructions.

Expression of double-labeled (¹³C-¹⁵N) SLY D4

Double-labeled ¹³C and ¹⁵N SLY D4 was produced by growing E. coli strain BL21 (DE3) transformed with the SLY D4 expression construct (slyD4_pET15b) in M9 minimal media containing N₁₅ NH₄Cl (1 g/liter) as a nitrogen source and C₁₃ glucose (4 g/liter) as a carbon source. Cells were grown to mid-log phase (OD₆₀₀ of ~0.35), induced with 0.5 mM IPTG final concentration, and then grown overnight at 37°C with 200 rpm shaking. SLY D4 containing a N-terminal 6xHis tag was purified using TALON cobalt metal affinity resin (Takara Bio) according to the manufacturer’s protocol with further details provided in the Supplementary Materials.

NMR spectroscopy

Uniformly ¹³C- and ¹⁵N-labeled purified SLY D4 was dissolved in 180 μl of 10 mM phosphate buffer (pH 7.4) containing 50 mM NaCl and 2.7 mM KCl in 10% D₂O, and 90% H₂O to a final concentration of 0.5 mM SLY D4. 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) was added as internal calibration standard. NMR experiments were carried out at 298 K (25°C) on a 600-MHz Bruker Avance spectrometer fitted with a 5-mm triple resonance cryoprobe, and a 600-MHz Agilent DD2 NMR spectrometer was also equipped with a cold probe (Creative Biostructures, NY, USA). ¹H NMR, 2D ¹H-¹⁵N HSQC, 3D HNCACB, and 3D CBCA(CO)NNH experiments were acquired. Proton chemical shifts were referenced against external DSS, whereas nitrogen chemical shifts were referenced indirectly to DSS using the absolute frequency ratio. One hundred twenty-eight data points in the t₁ domain and 1024 data points in the t₂ dimension were collected. The sweep width was 18.0 parts per million (ppm) in the ¹H dimension and 31.8 ppm in the ¹⁵N dimension. Triple resonance (¹H/¹³C/¹⁵N) NMR experiments were recorded with 128 increments in the t₁ domain, 40 increments in the t₂ domain, and 2048 increments in the t₃ domain. Spectra were processed using TopSpin and VnmrJ and analyzed using NMRPipe and CCPNmr. Secondary structure assignments were generated by the backbone chemical shift analysis program DANGLE incorporated in the CCPNmr software.

Backbone assignment of uniformly labeled ¹³C-¹⁵N SLY D4

Backbone ¹H_N, ¹⁵N, ¹³Cα, and ¹³Cβ chemical shifts were assigned for SLY D4 using standard triple resonance methodology, with details provided in the Supplementary Materials.

¹H-¹⁵N HSQC titration experiments of SLY D4 in complex with Galili and P₁ antigen

¹⁵N HSQC experiments were acquired using a phosphate buffer (pH 7.4) containing 50 mM NaCl and 2.7 mM KCl in 10% D₂O and 90% H₂O to a final concentration of 0.05 mM SLY D4 in 200 μl of total volume. Galili epitope was added at a protein:ligand ratio of 1:0.1, 1:0.5, 1:1, 1:5, and 1:10, whereas P₁ antigen was added resulting in protein:ligand ratios of 1:1, 1:2, and 1:10. Cellobiose was added at a protein:ligand ratio of 1:10 to serve as a nonbinding glycan control. All ¹H-¹⁵N HSQC titration experiments NMR were carried out at 298 K (25°C) on a 600-MHz Bruker Avance spectrometer fitted with a 5-mm triple resonance cryoprobe equipped with a cold probe. Data points in F2 were set to 256 with a total number of scans of 16.

Molecular modeling

To evaluate a potential binding site of the P₁ and Galili antigens, a docking experiment was performed using the AutoDock Vina protocol (57). It has been shown that AutoDock Vina has the highest scoring power among commercial and academic molecular docking programs (58) implemented in the YASARA molecular modeling package (ver. 16.46) (59).

An unbiased global docking experiment of α-galactose was conducted with a box size of 40 × 40 × 40 Å (Trp⁴³⁶), covering two-thirds of the protein surface. Results from these docking experiments revealed two frequently occupied ligand sites. These sites were used for a local docking experiment of the Galili and P₁ antigen. The preferred Galili epitope binding site was found close to Arg⁴⁶⁵, which showed significant intensity changes in the ¹H-¹⁵N HSQC NMR experiments. Docking results from the P₁ antigen resulted in a low-energy conformation at a site involving Ser³⁹⁶ that also is in excellent agreement with the ¹H-¹⁵N HSQC NMR results. Our unbiased NMR and molecular modeling approach has revealed two independent major glycan binding sites with distinct linkage specificity.

Drug library screening of Ply and SLO

Ply and SLO were immobilized to a CM5 sensor chip as described above. A combination of two drug libraries, Microsource-CPOZ (2400 drugs) and ML Drug (741 drugs), comprising U.S. Food and Drug Administration–approved drugs and other small-molecule compounds were purchased from Compounds Australia and binding screened by SPR analysis. The analysis was performed as previously described (60).

Data availability statement

The ¹H_N, ¹⁵N, ¹³Cα, and ¹³Cβ spectra for SLY D4 were deposited in the BioMagResBank (BMRB) (www.bmrb.wisc.edu/) under the BMRB accession code 27707. All other data generated or analyzed during this study are included in this manuscript and in the Supplementary Materials.

Statistical analysis

Data were analyzed for statistical significance with Prism 5 software (GraphPad Software Inc.) using a two-tailed unpaired Student’s t test. P < 0.05 was considered significant.

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/21/eaaz4926/DC1

View/request a protocol for this paper from Bio-protocol.