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MOLECULAR BIOLOGY OF PATHOGENS

Structure and Biological Activities of Beta Toxin from Staphylococcus aureus

Medora Huseby, Ke Shi, C. Kent Brown, Jeff Digre, Fikre Mengistu, Keun Seok Seo, Gregory A. Bohach, Patrick M. Schlievert, Douglas H. Ohlendorf, Cathleen A. Earhart
Medora Huseby
1Department of Biochemistry, Molecular Biology and Biophysics
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Ke Shi
1Department of Biochemistry, Molecular Biology and Biophysics
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C. Kent Brown
1Department of Biochemistry, Molecular Biology and Biophysics
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Jeff Digre
1Department of Biochemistry, Molecular Biology and Biophysics
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Fikre Mengistu
1Department of Biochemistry, Molecular Biology and Biophysics
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Keun Seok Seo
3Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho
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Gregory A. Bohach
3Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho
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Patrick M. Schlievert
2Department of Microbiology, University of Minnesota, Minneapolis, Minnesota
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Douglas H. Ohlendorf
1Department of Biochemistry, Molecular Biology and Biophysics
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Cathleen A. Earhart
1Department of Biochemistry, Molecular Biology and Biophysics
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  • For correspondence: Earhart@umn.edu
DOI: 10.1128/JB.00741-07
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ABSTRACT

Beta toxin is a neutral sphingomyelinase secreted by certain strains of Staphylococcus aureus. This virulence factor lyses erythrocytes in order to evade the host immune system as well as scavenge nutrients. The structure of beta toxin was determined at 2.4-Å resolution using crystals that were merohedrally twinned. This structure is similar to that of the sphingomyelinases of Listeria ivanovii and Bacillus cereus. Beta toxin belongs to the DNase I folding superfamily; in addition to sphingomyelinases, the proteins most structurally related to beta toxin include human endonuclease HAP1, Escherichia coli endonuclease III, bovine pancreatic DNase I, and the endonuclease domain of TRAS1 from Bombyx mori. Our biological assays demonstrated for the first time that beta toxin kills proliferating human lymphocytes. Structure-directed active site mutations show that biological activities, including hemolysis and lymphotoxicity, are due to the sphingomyelinase activity of the enzyme.

Staphylococcus aureus produces a large number of cell surface and secreted virulence factors that allow this organism to cause a myriad of human illnesses, ranging from relatively mild boils and subcutaneous abscesses to highly severe toxic shock syndrome and necrotizing pneumonia (16, 20). The ability of this organism to cause human illnesses is thought to depend in part on three major activities: (i) colonization of mucosal and skin surfaces with concomitant surface immune evasion, (ii) production of cytolysins that target large numbers of host cells locally for additional immune evasion and also nutrient acquisition, and (iii) production of superantigens that become systemic and induce body-wide immune evasion (16), (20).

The majority of S. aureus infections begin with mucous membrane or skin colonization of the human host. Many of the cell surface virulence factors are thought to facilitate adherence of the organism to host tissue and, in addition, to interfere with host phagocytic cell function, such as protein A binding to the Fc of immunoglobulin G antibodies (16). S. aureus also secretes multiple toxins and leukocidins (cytolysins), many of which affect large numbers of epithelial, immune, and red blood cells, primarily acting in the immediate area of infection (16). In addition to damaging the local immune system, these factors likely also provide nutrients to the organism in its efforts to expand the colonization and infection site. Finally, the organism may secrete a variety of superantigens which have limited local tissue receptors but which cross-link major histocompatibility complex class II molecules with the variable part of certain β chains of T-cell receptors and in doing so interfere with host immune function throughout the body (17, 20).

Among the cytolysins are alpha, beta, gamma, and delta toxins (16). Of these, alpha toxin is a heptamer pore-forming exotoxin that lyses primarily rabbit erythrocytes but is toxic to human epithelial cells (12). Gamma toxin is a two-component exotoxin comprising at least six different combinations of proteins, one of which is also a leukocidin (10). Delta toxin is a low-molecular-weight exotoxin that forms multimeric structures with the ability to lyse many cell types. This toxin is also encoded by the gene that simultaneously makes RNA III which functions as a global regulator of both cell surface and secreted virulence factors by being part of the accessory gene regulator (22).

The exotoxin that we know the least about is beta toxin. This molecule has a molecular mass of 35 kDa and appears to function as a sphingomyelinase (SMase). Beta toxin is also known as the hot-cold toxin because of its unique activity on sheep blood agar plates. At 37°C, beta toxin interacts with sheep red blood cells but does not lyse them. If the red cells are then placed at 4°C, the cells lyse; this is observed as a lack of hemolysis on blood agar plates at 37°C and then complete hemolysis at 4°C. A survey by Aarestrup et al. (1) found that beta toxin was produced in 72% of bovine mastitis isolates, in 11% of healthy human nasal isolates, and in 13% of human septicemia isolates. Due to the likelihood of contamination from one or more cytolysins and the differential and species-dependent susceptibility to beta toxin, the literature has been rather variable regarding the effects of beta toxin on leukocytes. In addition, in most prior work workers studied beta toxin effects on neutrophils. However, Marshall et al. (18) demonstrated using biological assays and electron microscopy that both lymphocytes and neutrophils are susceptible to beta toxin and that the toxicity is enhanced by Mg2+. However, both of these cell types are considerably less sensitive than sheep erythrocytes, presumably due to the presence of relatively less sphingomyelin.

This study was undertaken to determine the three-dimensional structure of beta toxin as an SMase, to characterize its active site amino acid residues, and to obtain evidence that this toxin significantly affects human immune cell function.

MATERIALS AND METHODS

Protein production.Beta toxin was produced from S. aureus strain RN4220 and from the beta toxin gene cloned into a pET28b vector containing an N-terminal His6 tag and expressed in Escherichia coli (this toxin is referred to below as wild-type recombinant beta toxin). The resulting vector was chemically transformed, grown in BL21(DE3) competent cells to mid-log phase, and induced with 1 ml of 200 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were harvested after 18 h of induction at 25°C. The cells were cracked using lysis buffer consisting of 50 mM sodium dihydrogen phosphate and 0.5 M sodium chloride (pH 8.0) and ultrasonic treatment (Branson Sonifier 450), followed by centrifugation for 30 min at 75,000 × g. Nickel resin (Novagen His · Bind Resin 69670) was used to purify beta toxin. The protein was incubated with the resin, washed extensively with lysis buffer, and then eluted from the column by increasing the amount of imidazole from 25 to 250 mM over a gradient. Fractions were collected and assayed on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, and the pure beta toxin was dialyzed into 0.1 M imidazole-0.5 M sodium chloride-1 mM β-mercaptoethanol (MP Biomedicals) and 1 mM EDTA (pH 8.0) for 3 h. The protein was concentrated using Centricon tubes (Amicon), and the concentration was determined with a spectrophotometer (Beckman DU 640). Alternatively, non-His6-tagged beta toxin was produced in S. aureus RN4220 cultured in a dialyzable beef heart medium and was purified by ethanol precipitation and thin-layer isoelectric focusing (4).

Active site mutations (H289N and H150N) were made via QuikChange (Stratagene) site-directed mutagenesis, and the mutants were purified and crystallized by the methods used for the wild-type recombinant beta toxin.

Crystallization, data collection, and data processing.Crystals were grown in sitting drops by vapor diffusion using 96-well plates (Emerald BioSystems plate type EBS-XJR). The initial crystallization conditions were obtained from Hampton Salt Rx screen condition 2, and crystals were further improved by using 0.1 M bis-Tris propane (pH 6.8 to 7.4; Sigma), 3.0 to 3.2 M sodium acetate (Fisher), and 1 mM β-mercaptoethanol (MP Biomedicals) and stored at 18°C. Diffraction-quality crystals appeared in 2 to 3 days and were found to be perfectly merohedrally twinned. Because of the difficulty in solving and refining structures using perfectly twinned crystals, an extensive search of alternate crystallization conditions and of the crystals obtained failed to produce nonmerohedrally twinned crystals.

Diffraction-quality crystals were placed in a 3.2 M sodium acetate-0.1 M bis-Tris propane (pH 7.0) solution with 20% glucose and then frozen and stored, and diffraction data were collected at −170°C. Data sets for wild-type recombinant beta toxin were collected on Molecular Biology Consortium's beamline 4.2.2 at the Advanced Light Source synchrotron facility at the Lawrence Berkeley National Laboratory with a NOIR1 detector (E. M. Westbrook, J. Morse, R. E. Fischer, W. M. McGuigan, S. K. Onishi, P. Vu, I. Naday, C. Bauer, J. Phillips, T. A. Thorson, and R. D. Durst, presented at X-Ray and Gamma-Ray Detectors and Applications IV, Seattle, WA, 2003). The solution of the structure was found by molecular replacement using the coordinates of a previously solved SMase, Smase, from L. ivanovii (24), as the search model. SHELXL (27) was used for initial structural model refinement, followed by REFMAC (21).

Biological assays.The SMase activity was measured as described by Dziewonowsoka et al. (11). This involved spectrophotometrically assaying the amount of cleavage that occurred via a sphingomyelin analog. Hemolysis activity was assayed on agar plates containing sheep red blood cells kindly provided by the microbiology teaching lab of the University of Minnesota. Beta toxin was added at concentrations of 1, 0.1, 0.01, and 0.001 mg/ml and incubated at 37°C for 24 h. The plates were then shifted to 4°C for 24 h, and the zones of lysis were measured.

Human peripheral blood lymphocytes were obtained from heparinized venous blood samples after dilution in RPMI (catalog no. 12-167F; BioWhitaker) and separation on Histopaque-1077 (Sigma). The mononuclear cells were cultured with the superantigen toxic shock syndrome toxin 1 (TSST-1) [from S. aureus strain RN4220 (pCE107)] in the absence and presence of simultaneously added different concentrations of beta toxin or the H289N or H150N mutant and analyzed in quadruplicate using the procedure described by Barsumian et al. (3). Lymphocyte density was measured with a hemacytometer (Fisher Scientific), and the preparation was diluted to obtain 1 × 105 cells/experimental well. The plates were then incubated under a 5% CO2 atmosphere in a humidified incubator at 37°C for 3 days. The cells were labeled with 1 μCi of [methyl-3H]thymidine (catalog no. NET-027Z; PerkinElmer) and incubated for 24 h at 37°C. A multiwell semiautomated cell harvester (Cambridge Technology model 200A) was used to harvest the cells. Three milliliters of scintillation fluid (EcoLite from MP Biomedicals [formerly ICN]) was added, and the counts were determined with a scintillation counter. Beta toxin isolated from S. aureus strain RN4220 was used for comparison to the recombinant protein. Cell counting was done in triplicate with a hemocytometer, and the averages are reported below.

Modeling the beta toxin-spingomeylin complex.Starting with the beta toxin structure, metal ions were placed as found by Ago et al. (2). Next, the phosphorylcholine part of sphingomyelin was placed to optimally interact with His-150 and His-289, as suggested by Matsuo et al. (19) and Ago et al. (2). Ceramide was then manually placed in an extended conformation adjacent to beta toxin residues. The natural shape of the surface made the general location of the lipid tails clear (see Fig. S5 in the supplemental material). The model was minimized using simulated annealing with CNS (5), first holding beta toxin and the phosphorus and quaternary amine of sphingomyelin fixed. Next, beta toxin was released except for the Cαs, and the model was again minimized using simulated annealing. Finally, the Cαs and substrate phosphorus and quaternary amine were restrained using a harmonic potential and the model was minimized by simulated annealing.

RESULTS AND DISCUSSION

Structural overview.The 2.4-Å resolution structural model (Fig. 1) was determined and has the statistics shown in Table 1. The R work and R free values are somewhat higher than the typical values due to the crystal twinning. The final model contains density for residues 6 to 297. The extreme amino terminus containing the His6 tag is disordered and is not seen.

FIG. 1.
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FIG. 1.

(A) Stereo drawing of beta toxin secondary structure. Residue colors range from blue (N terminal) to red (C terminal). N and C termini, sheets A and B, and secondary structural elements are labeled. (B) Stereo drawing of active site. The view is roughly from the left of the view in panel A.

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TABLE 1.

Data collection and refinement statistics

Beta toxin folds into a four-layer sandwich with two β sheets at the center (see Fig. S1 in the supplemental material). Sheet A consists of strands β11, β14, β1, β2, β5, and β3; sheet B consists of strands β4, β6, β7, β8, β9, and β10. In the outer layer on top of sheet A are helices α1 and α2, a 25-Å β duplex formed by strands β12 and β13, and an Ω loop connecting helix α4 and strand β10. In the outer layer on top of sheet B are helices α3 and α4 and an Ω loop connecting strands β6 and β7.

A unique structural feature of the SMases relative to other members of the DNase I superfamily is the long β duplex (strands β12 and β13). As mentioned above, this duplex interacts with the N-acyl chain of sphingomyelin. The residues facing sphingomyelin are Trp-272, Val-274, Ala-276, Phe-277, Tyr-280, and Tyr-283. Most of these residues are absolutely conserved; the only exceptions are residues 276 and 277, which are serine and tryptophan in Staphylococcus epidermidis, and residue 280, which is threonine in S. epidermidis and Staphylococcus schleiferi. None of the other residues in the exposed end of the duplex is conserved in all three species.

The amino acid sequences encoded by the beta toxin gene (hlb) in the known genomic sequences of S. aureus are essentially identical. The exceptions are strains in which phages φ42 and φ13 recombine with the 5′ end of the beta toxin, producing hlb mutant phenotypes (6-8). In strains 8325, USA300, MSSA476, MRSA252, and MW2 this results in deletion of the first 24 residues of the amino terminus of the mature beta toxin sequence. The effects of this change include deletion of the export signal, as well as strand β1, which is an internal strand of sheet A, thereby explaining the negative phenotype. It should be noted that strain 8325-4 produces functional beta toxin because it has been cured of phages φ11, φ12, and φ13 (7). In strains N315 and Mu50 the insertion results in replacement of residues 1 to 24 with the sequence RDSKPNKYCCYKVS. This sequence is long enough to replace strand β1. However, this would result in replacement of the buried hydrophobic residues Leu-10 and Val-13 with arginine and lysine, respectively. Not only is there insufficient space for these larger residues, but these changes would result in burial of unpaired charges, which is extremely destabilizing (9), again producing the negative phenotype.

Two orthologs of S. aureus beta toxin that are 72 and 52% identical are found in S. schleiferi and S. epidermidis, respectively. These sequences are shown in Fig. 2. Examination of the positions of the sequence differences in light of the structure revealed that the significant changes are either on the surface or away from the active site. This suggests that these proteins should be active if they are expressed. In addition, there are four open reading frames in Leptospira interrogans which show significant (37 to 47%) sequence homology to beta toxin (26) that have beta-hemolytic and SMase activities (32).

FIG. 2.
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FIG. 2.

Alignment of beta toxin homologs. The first three sequences are the sequences of three species of Staphylococcus; the last 10 sequences are the sequences of structural homologs found by a DALI (13) search of the Protein Data Bank in order of decreasing similarity. 1ZWX is a neutral SMase from L. ivanovii, 2DDR is the SMase from B. cereus, 1BIX is human endonuclease HAP1, 1AKO is E. coli endonuclease III, 1VYB is the endonuclease domain of human Line-1 retrotransposon, 1I9Y is inositol polyphosphate 5-phosphatase from S. pombe, 3DNI is bovine pancreatic DNase I, 1WDU is the endonuclease domain of TRAS1 from B. mori, 1SR4 is H. ducreyi cytolethal distending toxin, and 1NTF is salivary nitrophorin from C. lectularius. The colors used to indicate amino acids are as follows: blue, R and H; red, D, E, and H; yellow, C; orange, Y, W, and F; green, T, N, Q, and S; violet, G and P; and pink, I, V, L, A, and M. β Strands and α helices of S. aureus beta toxin are indicated by red arrows and blue bars, respectively. The numbers are the positions in S. aureus beta toxin. Residues at the bottom of the active site are indicated by solid black diamonds, and residues along the sides of the active site are indicated by solid blue diamonds.

The primary sequence of beta toxin is 55.7% identical to the sequence of SmcL, a neutral SMase from L. ivanovii (24), and 59.2% identical to the sequence of SMase from Bacillus cereus (2). Its structure is also homologous to the structures of these proteins (PDB entries 1ZWX and 2DDR, respectively), with root mean square differences (RMSDs) of 1.21 and 1.07 Å, respectively, for all common Cαs and 1.37 and 1.39 Å, respectively, for all common atoms (see Fig. S2 in the supplemental material).

Beta toxin belongs to the DNase I folding superfamily (CATH [25] class 3.60). The proteins which are most structurally homologous to beta toxin, aside from the SMases, as revealed by a DALI (14) and SSM (15) search, are human endonuclease HAP1 (PDB entry 1bix; RMSD over 221 Cαs, 3.3 Å), E. coli endonuclease III (PDB entry 1ako; RMSD over 221 Cαs, 3.5 Å), the endonuclease domain of human Line-1 retrotransposon (PDB entry 1vyb; RMSD over 207 Cαs, 3.0 Å), inositol polyphosphate 5-phosphatase from Schizosaccharomyces pombe (PDB entry 1i9y; RMSD over 214 Cαs, 3.2 Å), bovine pancreatic DNase I (PDB entry 3dni; RMSD over 220 Cαs, 3.3 Å), the endonuclease domain of TRAS1 from Bombyx mori (PDB entry 1wdu; RMSD over 193 Cαs, 2.9 Å), Haemophilus ducreyi cytolethal distending toxin (PDB entry 1sr4; RMSD over 208 Cαs, 3.0 Å), and salivary nitrophorin from Cimex lectularius (PDB entry 1ntf; RMSD over 210 Cαs, 3.3 Å). Figure S3 in the supplemental material shows superposition of the Cα traces of human HAP1 endonuclease and bovine DNase I onto beta toxin. Despite the structural homology the levels of amino acid identity between beta toxin and these proteins are only 10 to 19%.

Active site.The active site of beta toxin is at the bottom central sheets in a deep cleft, as it is in all DNase I superfamily members. A comparison of the active sites of beta toxin and the two other SMase structures showed strong conservation of the 27 residues whose side chains line the sites (RMSD over all common atoms of these residues, 1.06 Å). The nine residues lining the bottom of the active site are 100% conserved (Fig. 2). For most of the other active site residues the changes are conservative; the only exceptions are Phe-124 (alanine in Listeria and proline in Bacillus enzymes) and Asn-236 (serine in Listeria enzyme) at the opening of the active site and Ser-153. Ser-153 is immediately above Asn-196; this residue is an alanine in both reported SMase structures and in the putative beta toxins from S. epidermidis and S. schleiferi.

Ago et al. (2) reported three metal ions in crystals of Co, Ca, and Mg complexes of Listeria SMase. All the reported metal ligands are conserved in beta toxin. In the Listeria SMase edge metal-binding site a metal is bound between Glu-98 and Asp-99 (beta toxin numbering). During purification of beta toxin, metal ions were removed through dialysis so as to not interfere with the metal affinity chromatography. In the absence of metal in beta toxin, the Glu-98 side chain points away from Asp-99. Ago et al. (2) reported that in the central metal-binding site the Co and Mg ions sit between Glu-53 and His-289 (beta toxin numbering). In beta toxin no metal is seen, and the Glu-53 side chain is rotated 66°, making an interaction with a metal untenable without side chain rotation.

As all proteins in the DNase I superfamily cleave phosphodiesterase bonds, it is not surprising that the six absolutely conserved residues shown in Fig. 2 either line the bottom of the active site (His-16, Asp-194, Asn-196, and His-289) or are critical in positioning residues there. Specifically, Asp-246 forms hydrogen bonds with the side chains of Thr-224 and His-289, and Gly-193's unusual main chain conformation (φ = 135°, ψ = −74°) allows Asp-194 N and O to form H bonds with Thr-159 O and Thr-151 N, respectively, positioning the Asp-194 side chain in the active site.

Models of SMases have been constructed using the structures of nucleases (19, 28). Matsuo et al. (19) predicted that Glu-53 would interact with the C-1 hydroxyl of glycerol and the carbonyl oxygen of the ceramide. They also suggested that His-150 and His-289 acted as the general acid and base in a catalytic mechanism derived from homology with DNase I. Obama et al. (23) predicted that Glu-53 interacts with the phosphate through a bound Mg2+ ion. Ago et al. (2) modeled a complex and proposed a similar role for Glu-53. Using the structure of beta toxin as a starting point, the complex with sphingomyelin was modeled and energy minimized (Fig. 3). The sphingosine chain lies in a cleft formed by the β1-α1, β2-α2, and β4-β5 loops. Its hydroxyl and a phosphate oxygen are proposed to be coordinated by a metal ion (M1 in Fig. 3) (2). The N-acyl chain of the substrate lies in a cleft between the β1-α1 and α4-β10 loops and against the β12-β13 duplex. The carbonyl of the amide is recognized by Tyr-237 (Fig. 3), which is conserved in Listeria SMase and S. epidermidis beta toxin. In B. cereus SMase and S. scheiferi beta toxin this residue is a phenylalanine (Fig. 2). In other structural homologs this residue is not conserved. The cationic choline projects between the β8-α3 and α4-β10 loops and is recognized by the side chains of Glu-154 and Glu-243 (Fig. 3). These residues are conserved in the beta toxins and SMases (the exceptions are Asp-154 in Listeria SMase and Gln-243 in S. epidermidis beta toxin) but not in other structural homologs (Fig. 2).

FIG. 3.
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FIG. 3.

(A) Stereo drawing of a model of beta toxin-sphingomyelin complex. SMase is shown in space filling with oxygen atoms red, carbon atoms light blue, nitrogen atoms navy, and sulfur atoms cyan. The two metals found by Ago et al. (2) are indicated by small magenta spheres. (B) LIGPLOT (31) drawing of a model of the complex of beta toxin with sphingomyelin (Spl) metal sites reported by Ago et al. (2). Dashes indicate hydrogen bond interactions. Red semicircles indicate hydrophobic interactions. The bond cleaved is indicated by a green arrow.

To verify the importance of His-150 and His-289 as suggested by Matsuo et al. (19), these residues were separately mutated to Asn for analysis. Both mutants crystallized isomorphously with the His-tagged recombinant beta toxin (see data collection and refinement statistics in Table 1). The RMSDs over Cαs between the two mutant structures and the wild type are 0.44 and 0.60 Å, and the F obs mutant-F obs wild type maps have no significant features other than the mutations. These data indicate that the structures of these mutants and the wild-type beta toxin are identical within experimental error.

SMase activity.The abilities of wild-type recombinant beta toxin and the H150N and H289N mutants to cleave sphingomyelin were assayed. As shown in Fig. 4, mutation of either His-150 or His-289 abolished SMase activity. In addition, the ability to perform hot-cold lysis of sheep erythrocytes was assayed using wild-type recombinant beta toxin and the H289N and H150N mutants (see Fig. S4 in the supplemental material). The activities of these mutants were reduced 60- and 200-fold, respectively.

FIG. 4.
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FIG. 4.

SMase and hemolytic activities of beta toxin and the H289N and H150N mutants. (A) SMase activity as described by Dziewanowska et al. (11). The positive control (SMase) was a neutral SMase purchased from Fisher and isolated from S. aureus. (B) Hemolytic assay performed with sheep erythrocytes (see Fig. S4 in the supplemental material).

Openshaw et al. (24) proposed that the duplex plays a significant role in penetrating the membrane in preparation for cleaving sphingomyelin. This appears to be unlikely because of conservation in the character of the residues in the duplex. In particular, in S. epidermidis residues 275, 279, and 282 are lysines. Ago et al. (2) reported binding of the buffer 2-morpholinoethanesulfonic acid adjacent to a twisted β duplex in the crystal form of B. cereus SMase containing bound Mg2+. The relevance of this structural change is uncertain as 2-morpholinoethanesulfonic acid is more than 13 Å from the active site metals and bears little resemblance to a substrate. Ago et al. also mutated Trp-279 and Trp-290 to alanine and reported reduced binding to sphingomyelin liposomes and loss of the ability to disrupt these liposomes or to lyse sheep erythrocytes. Verification of a regulatory role for the β duplex of beta toxin from S. aureus awaits further study.

Lymphocyte proliferation.The response of lymphocytes to superantigens, such as TSST-1, and the cytotoxicity of beta toxin for resting lymphocytes have been documented (17, 18). However, the effect of beta toxin on proliferating lymphocytes is unknown. When lymphocytes were incubated with TSST-1 and beta toxin, a decrease in the amount of tritium incorporation into the DNA was observed (Fig. 5). The experiment was repeated twice, and the same results were obtained.

FIG. 5.
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FIG. 5.

Beta toxin lysis of TSST-1-induced lymphocytes. (A) Tritium incorporation into induced lymphocytes in the presence of diffferent concentrations of beta toxin and the H289N and H150N mutants. The experiment was performed in quadruplicate, and averages are shown. The three control experiments were done on three different days. (B) Tritium incorporation into lymphocytes in the absence of beta toxin and in the absence (lymphocytes only) or presence (1 μg TSST-1) of TSST-1. (C) Photographs of representative experimental wells containing 10 mg of beta toxin or the H289N or H150N mutant. The brown clumps are colonies of proliferating lymphocytes. Some autostimulation of the lymphocytes was observed (lymphocytes only). (D) Cell counts obtained with a hemocytometer. Counting was done in quadruplicate, and the averages after 4 days of incubation are shown. The error bars indicate standard deviations.

To ensure that it was beta toxin that was inhibiting incorporation of [3H]thymidine, mutants with two active site mutations, H289N and H150N, were constructed. As stated above, both active site mutants showed no sphingomyelin cleavage in vitro (Fig. 4A). When the mutants were tested for hemolysis on sheep erythrocytes, very little lysis was observed after 48 h of incubation (Fig. 4B; see Fig. S4 in the supplemental material), although the structures of the H150N and H289N mutants are virtually identical to the structure of wild-type beta toxin. However, these two mutants failed to interfere with TSST-1-induced lymphocyte proliferation.

To differentiate between lymphocyte death induced by beta toxin and interference with the TSST-1 mitogenic signal, cells were counted and photos were taken of representative experimental wells (Fig. 5C). In these assays, there was significant autostimulation of lymphocytes in the absence of added superantigen or in the presence of beta toxin mutants, as shown by incorporation of [3H]thymidine into lymphocyte DNA (Fig. 5D). However, beta toxin significantly reduced incorporation of thymidine in proliferating cells (Fig. 5D). TSST-1 caused proliferation of lymphocytes, as shown in Fig. 5C (TSST-1 only well), in the form of large numbers of colonies, presumably due to proliferation of T lymphocytes containing Vβ-2-bearing T-cell receptors. Lymphocytes incubated without TSST-1 lacked these colonies. The proliferative (colony-forming) effect was negated in the presence of beta toxin, either isolated from S. aureus or expressed in E. coli (Fig. 5D). These results also show that the N-terminal His6 tag does not affect biological activity.

Conclusion.Iron is a limiting nutrient for all organisms and is necessary in many biological processes. S. aureus has a multicomponent heme-scavenging system, which imports heme from host heme proteins and extracts the iron (29). Potent sources of heme are hemoglobin and myoglobin, which can be found in erythrocytes. Erythrocytes may be early host defense cells encountered during infection. One role long proposed for bacterial hemolysins, such as beta toxin, is their potential to endow the organisms with the ability to acquire iron in the form of heme.

Our results suggest that beta toxin may also contribute to immune modulation of the host in the presence of accessory virulence factors, such as superantigens. Strains of S. aureus, such as RN6390 (= NCTC8325-4 = RN4220), have been known to secrete large amounts (up to 500 μg/ml) of beta toxin. These strains are important pathogens in animals but were thought not to be important in humans. This study shows conclusively that beta toxin affects human peripheral blood lymphocytes, specifically by killing proliferating T lymphocytes. Other human cells which are susceptible to beta toxin are monocytes (30), resting lymphocytes, and polymorphonuclear leukocytes (18). The action on human lymphocytes most likely occurs in order to evade the host immune machinery, as well as to scavenge nutrients.

The activities of beta toxin have been shown to be tied to its SMase activity. The cleavage of a phosphodiester bond uses the structural framework found in DNase I and an extended hydrophobic binding site for the fatty acid chains and specific recognition for the choline head group. Whether activities are due to changes in the physical properties of the membrane or to production of ceramide as a progenitor of apoptosis requires further study.

ACKNOWLEDGMENTS

Work on this project was supported by NIH grant R01 AI57585 to C.A.E., by NIH grant R01 HL36611 to P.M.S., and by NIH grants P20 RR15587 and P20 RR016454 to G.A.B. K.S. was partially supported by NIH training grant 5T32-DE007288-11.

Diffraction data were collected using the facilities of Molecular Biology Consortium at the Advanced Light Source (Beamline 4.2.2). Computational facilities were provided by the Basic Sciences Computer Laboratory of the Minnesota Supercomputing Institute. We also acknowledge the superb technical assistance of Zu-Yi Gu and Edward Hoeffner (University of Minnesota), as well as Claudia Deobald and Katarzyna Dziewanowska (University of Idaho).

FOOTNOTES

    • Received 10 May 2007.
    • Accepted 6 September 2007.
  • Copyright © 2007 American Society for Microbiology

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Structure and Biological Activities of Beta Toxin from Staphylococcus aureus
Medora Huseby, Ke Shi, C. Kent Brown, Jeff Digre, Fikre Mengistu, Keun Seok Seo, Gregory A. Bohach, Patrick M. Schlievert, Douglas H. Ohlendorf, Cathleen A. Earhart
Journal of Bacteriology Nov 2007, 189 (23) 8719-8726; DOI: 10.1128/JB.00741-07

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Structure and Biological Activities of Beta Toxin from Staphylococcus aureus
Medora Huseby, Ke Shi, C. Kent Brown, Jeff Digre, Fikre Mengistu, Keun Seok Seo, Gregory A. Bohach, Patrick M. Schlievert, Douglas H. Ohlendorf, Cathleen A. Earhart
Journal of Bacteriology Nov 2007, 189 (23) 8719-8726; DOI: 10.1128/JB.00741-07
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KEYWORDS

Bacterial Toxins
Hemolysin Proteins
Sphingomyelin Phosphodiesterase
Staphylococcus aureus

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