MAE4, an eLtaS monoclonal antibody, blocks Staphylococcus aureus virulence

Staphylococcus aureus causes a wide range of infectious diseases. Treatment of these infections has become increasingly difficult due to the widespread emergence of antibiotic-resistant strains; therefore, it is essential to explore effective alternatives to antibiotics. A secreted protein of S. aureus, known as eLtaS, is an extracellular protein released from the bacterial membrane protein, LtaS. However, the role of eLtaS in S. aureus pathogenesis remains largely unknown. Here we show eLtaS dramatically aggravates S. aureus infection by binding to C3b and then inhibiting the phagocytosis of C3b-deposited S. aureus. Furthermore, we developed a monoclonal antibody against eLtaS, MAE4, which neutralizes the activity of eLtaS and blocks staphylococcal evasion of phagocytosis. Consequently, MAE4 is capable of protecting mice from lethal S. aureus infection. Our findings reveal that targeting of eLtaS by MAE4 is a potential therapeutic strategy for the treatment of infectious diseases caused by S. aureus.

eLtaS binds to C3b. Given that eLtaS inhibits MAC formation and that C3 is the central component protein in all three complement pathways, we investigated the interaction between eLtaS and C3. Binding of eLtaS to C3 from both human and mouse serum was detected by capture ELISA (Fig. 2a,b), indicating a species-independent interaction. This interaction was not observed with serum from C3-deficient (C3 −/− ) C57BL/6 mice (Fig. 2b).
Two forms of C3 convertase (C4b2a and C3bBb) cleave C3 into C3a and C3b. C3b is further cleaved into C3c and C3d by Factor I 20,23,24 . To identify the fragment responsible for eLtaS binding, a capture ELISA was performed with C3 and its proteolytic fragments, C3b, C3c, and C3d, as coating antigens. eLtaS was found to bind the C3d domain of C3b, but not C3c (Fig. 2c). The relative affinity of eLtaS for human C3 and C3b was determined respectively (Table S1).
C3b binds covalently to bacterial surfaces through a reactive intramolecular thioester in the C3d domain of the α -chain, either to hydroxyl groups via an ester linkage or to primary amino groups via an amide bond 25,26 . Three strains of S. aureus (8325-4, Newman, and 04018) were incubated with increasing concentrations of human serum for 15 min and the deposition of C3b was detected by flow cytometry (FCM) using an anti-C3b-FITC antibody. The percentage of C3b-deposited S. aureus increased in a serum concentration-dependent manner ( Fig. 2d and S4). Using an eLtaS-specific antibody, we further demonstrated that, following incubation, eLtaS bound to C3b deposited on the bacterial surface (Fig. 2d). Sera from C3 +/− and C3 −/− C57BL/6 mice were incubated with S. aureus 8325-4 prior to addition of FITC-labeled eLtaS. Analysis by FCM showed that eLtaS only bound to S. aureus incubated with serum from C3 +/− C57BL/6 mice and not from C3 −/− C57BL/6 mice (Fig. 2e), confirming that eLtaS is able to bind C3b deposited on the bacterial surface. eLtaS attenuates phagocytosis of C3b-deposited S. aureus. Immune effector cells, such as neutrophils, target C3b-deposited S. aureus for destruction 27 . We considered whether eLtaS blocked the process of C3b deposition in the three complement activation pathways. S. aureus Efb (extracellular fibrinogen binding protein), which is known to inhibit C3b deposition in the AP, was used as a positive control 28,29 . As expected, Efb inhibited C3b deposition in AP. However, deposition of C3b was not Scientific RepoRts | 5:17215 | DOI: 10.1038/srep17215 blocked by eLtaS in any of the pathways (Fig. S5a-c), suggesting that the interaction site of eLtaS on C3d was different from that of Efb. We further demonstrated that eLtaS could not inhibit binding between C3d and Efb. Similarly, the interaction between C3d and eLtaS was not interrupted by Efb (Fig. S6a,b). The ability of eLtaS to block C3b deposition was tested by incubating human serum with S. aureus 8325-4 in the presence or absence of eLtaS. As shown in Fig. S7, the level of C3b deposited on the S. aureus surface was unaffected by the presence of eLtaS. were incubated with 25% pre-cleared normal human serum in the presence of eLtaS at the concentrations indicated for 30 min at 37 °C. The samples were centrifuged, and the absorbance of the supernatants was measured at 405 nm. (b-d) eLtaS inhibits C5b-9 formation via the classic (2% serum; (b)), lectin (10% serum; (c)), and alternative (20% serum; (d)) pathways in a concentration-dependent manner. Serum samples were pre-incubated with eLtaS at the concentrations indicated. The serum and eLtaS mixture was added to plates coated with fibrinogen immune complex (classic pathway), immobilized mannan (lectin pathway), or LPS (alternative pathway). The formation of C5b-9 was detected using an anti-C5b-9 antibody. Data are presented as the mean ± SD. e-g. Survival rate of mice challenged with different S. aureus strains in the acute peritoneal infection model. S. aureus 8325-4 (5 × 10 8 cfu/mouse), Δ ltaS (2 × 10 9 cfu/mouse) or Δ ltaSR (5 × 10 8 cfu/mouse) was injected into the peritoneal cavity of CD-1 mice (e). CD-1 mice were intraperitoneally injected with Δ ltaS (2 × 10 9 cfu/mouse) in the presence of eLtaS (f). S. aureus 8325-4 (2 × 10 8 cfu/mouse) was injected in the presence of eLtaS (g). The survival rate was calculated at different time points post challenge. Data are presented as percentages of surviving mice (n = 8). Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test. To determine whether eLtaS attenuates phagocytosis of C3b-deposited S. aureus by neutrophils, we incubated SYTO9-labeled S. aureus 8325-4 with fresh human or murine serum and then added human or murine leukocytes. As neutrophils constitute more than 90% of granulocytes, we detected the proportion of SYTO9-positive granulocytes that had engulfed S. aureus by FCM. We found an increased percentage of SYTO9-positive granulocytes in the presence of serum, and this increase was reduced in a concentration-dependent manner in the presence of eLtaS (Fig. 3a,b). Furthermore, human neutrophils were isolated from peripheral blood 30 and incubated with C3b-deposited S. aureus 8325-4 in the presence of eLtaS. The number of S. aureus cells engulfed by neutrophils was found to decrease with increasing amounts of eLtaS (Fig. 3c).
Murine peritoneal cavity cells were isolated and incubated with SYTO9-labeled S. aureus, and the percentage of phagocytes, including neutrophils and macrophages, that engulfed S. aureus was determined by FCM. We found that eLtaS attenuated the engulfment of C3b-deposited S. aureus by peritoneal phagocytes (Fig. 3d). (a) eLtaS binds to C3 from human serum in a concentration-dependent manner. eLtaS was coated onto a 96-well plate and human serum was added at various concentrations. Bound C3 was detected using an anti-C3 antibody. Data are representative of three independent experiments and shown as the mean ± SD. (b) eLtaS binds to murine C3. Serum (3% and 1.5%) from C3 + /− or C3 −/− mice was added to an eLtaS coated 96-well plate. C3 was detected using an anti-C3 antibody. Data are representative of three independent experiments and shown as the mean ± SD. Significant differences between groups were evaluated using a two-tailed Student's t test. (c) eLtaS binds to C3, C3b, and C3d, but not C3c. Human complement C3 and its fragments, C3b, C3c, and C3d, were coated (1 μ g/well) onto a 96well plate. Bound eLtaS was detected using an anti-eLtaS antibody. Data are presented as the mean ± SD of three independent experiments. (d) eLtaS binds to C3b-deposited S. aureus. S. aureus 8325-4 was incubated with the indicated concentrations of human serum for 15 min. After washing with PBS, S. aureus was incubated with eLtaS for a further 30 min. Deposited C3b and bound eLtaS were detected by FCM with anti-C3b and anti-His antibodies. (e) eLtaS binds to C3b. Serum was isolated from C3 +/− and C3 −/− C57BL/6 mice and incubated with S. aureus 8325-4 FITC-labeled eLtaS protein was added and specific binding was detected by FCM.  We then studied the effect of eLtaS on phagocytosis of S. aureus in vivo using C3 +/− C57BL/6 mice. Mouse peritoneal cavity cells were isolated after intraperitoneal injection of SYTO9-labeled S. aureus in the presence or absence of eLtaS. We found that the percentage of phagocytes that had engulfed S. aureus was decreased in the presence of eLtaS (Fig. 3e). However, eLtaS had no significant inhibitory effect on the phagocytosis of S. aureus by peritoneal phagocytes of C3 −/− C57BL/6 mice (Fig. 3f). These results further demonstrated that eLtaS attenuated the engulfment of S. aureus by phagocytes through interaction with C3.

Monoclonal antibody 4 against eLtaS (MAE4) prevents S. aureus infection. Because eLtaS was
found to aggravate S. aureus infection, we considered whether an antibody directed against eLtaS could be protective. Mouse monoclonal antibodies to eLtaS were generated according to standard protocols. Seven monoclonal antibodies (MAE1-7) were obtained (Fig. S8a) and one of these, MAE4, was found to block the interaction between eLtaS and C3b (Fig. S8b). MAE4 had an EC 50 of 80.89 ng/ml (Fig. 4a) and was of an IgG2a isotype (Fig. S8c).
We assessed the protective effect of MAE4 in the murine acute peritoneal infection model. It was found that MAE4 was able to completely rescue mice from lethal S. aureus 8325-4 infection (Fig. 4b). As expected, MAE4 had no effect when used in combination with a lethal dose of S. aureus Δ ltaS (Fig. 4c). These results suggest that MAE4 protected mice from S. aureus infection by directly targeting eLtaS. This effect was further studied in a murine model of staphylococcal pneumonia. MAE4 was injected intramuscularly at 30 min, 24 h and 48 h post challenge with S. aureus 8325-4. Histopathological examination of the lungs of the mice showed that MAE4 decreased S. aureus-induced tissue damage (Fig. 4d). We also assessed the effect of MAE4 in a murine pneumonia infection model with two clinical S. aureus strains (S. aureus Newman and 04018) 31,32 . MAE4 also inhibited infection caused by these two S. aureus strains (Fig. S9).
In addition, the protective effect of MAE4 was assessed using a mouse intravenous challenge model. MAE4 was injected (100 μ g/mouse) into the peritoneal cavity of BALB/c mice 2 h before intravenous challenge with a sublethal dose of S. aureus Newman (1 × 10 7 cfu/mouse). Five days post infection, the kidneys were examined by histopathology for internal abscesses (Fig. 4e) and the number of bacterial colonies was determined (Fig. 4f). MAE4 was also tested in a lethal challenge model with S. aureus Newman (1 × 10 8 cfu/mouse). Survival rates were monitored for ten days (Fig. 4g). These results further confirm that MAE4 protects mice from S. aureus infection.

MAE4 blocks evasion of phagocytosis mediated by eLtaS.
We further determined whether MAE4 could neutralize the effect of eLtaS. Using a competitive ELISA, we demonstrated that MAE4 blocked the interaction between eLtaS and C3b in a concentration-dependent manner (Fig. 5a). The effect of MAE4 on eLtaS-mediated evasion of phagocytosis was detected in vitro by FCM. We found that eLtaS did not attenuate engulfment of S. aureus by human or murine neutrophils in the presence of MAE4 (Fig. 5b,c). We further tested the neutralization effect of MAE4 in vivo and demonstrated that treatment with MAE4 increased the percentage of neutrophils and macrophages that engulfed S. aureus in the peritoneal cavity (Fig. 5d).
Given that LtaS is located on the cell membrane, we tested whether MAE4 could directly bind bacterial cells and affect the growth of S. aureus in vitro. Analysis by FCM showed that MAE4 did not bind S. aureus cells (Fig. 6a). We further demonstrated that synthesis of LTA and growth of S. aureus were unaffected by MAE4 (10 μ g/ml) (Fig. 6b,c). The metal-binding domain and substrate-binding domain of LtaS are considered important for LTA synthesis, and mutation of two amino acids (T300 and H347) in these domains decreases the production of LTA 15 . These two amino acids were substituted with alanine to generate two mutant proteins (T300A and H347A). As shown in Fig. S10a,b, MAE4 binding to these two mutant proteins was maintained, indicating that MAE4 only targeted secreted eLtaS protein and had no effect on the activity of membrane-bound LtaS.
(e,f) Evaluation of the engulfment of S. aureus by murine peritoneal phagocytes in vivo in C3 +/− C57BL/6 (e) and C3 −/− C57BL/6 (f) mice. Peritoneal cavity cells were isolated 30 min after intraperitoneal injection of SYTO9-labeled S. aureus 8325-4 (5 × 10 8 cfu/mouse) in the presence of eLtaS. The percentage of neutrophils and macrophages that had engulfed S. aureus was determined by FCM. Data are representative of three independent experiments and shown as the mean ± SD. Significant differences between groups were evaluated using a two-tailed Student's t test. (g,h) The role of eLtaS in the mortality of C3-deficient mice. C3 −/− (g) and C3 +/− C57BL/6 (h) mice were injected intraperitoneally with S. aureus 8325-4 cells (5 × 10 8 cfu/mouse for C3 +/− C57BL/6 mice, 1 × 10 8 cfu/mouse for C3 −/− C57BL/6 mice) in the presence of eLtaS. The survival rate was calculated at different time points post challenge. Data are presented as the percentage of mice surviving. Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test (n = 8) (NS, non-significant). For the lethal challenge model, S. aureus Newman cells (1 × 10 8 cfu/mouse) were administered and survival rates were monitored for 10 days (g). Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test (n = 8) (NS, non-significant).

Discussion
The spread of multi-drug-resistant S. aureus is an increasingly serious threat to global public health. Global efforts to develop new antibiotics have not been fast enough to combat the evolution of antimicrobial resistance [33][34][35] . Targeting of virulence factors is regarded as a promising strategy that would apply less selective pressure for the development of bacterial resistance than traditional strategies 36 .
In vivo, S. aureus bacteria are mainly cleared by phagocytes (neutrophils and macrophages). Phagocytosis is strongly enhanced by the opsonization of bacteria with antibodies and the deposition of complement activation products on the surface of the bacteria 37,38 . Peritoneal cavity cells were isolated, and the percentage of SYTO9-labeled neutrophils and macrophages, indicating engulfment of S. aureus was determined by FCM. Data are representative of three independent experiments and shown as the mean ± SD. Significant differences between groups were evaluated using a two-tailed Student's t test.
Scientific RepoRts | 5:17215 | DOI: 10.1038/srep17215 In this study, we developed a monoclonal antibody against eLtaS, MAE4, which is capable of blocking eLtaS-mediated evasion of phagocytosis and dramatically reduces S. aureus infection. eLtaS, the C-terminal extracellular domain of LtaS, is released from the cell membrane by peptidase SpsB as a soluble peptide whose function was unknown until now 15 . Here, we demonstrate that eLtaS aggravates S. aureus infection through binding to C3b.
Complement activation leads to rapid deposition of C3b on the surface of bacteria, promoting phagocytosis of the opsonized bacteria 27 . We found that eLtaS had no effect on the deposition of C3b, but the interaction between eLtaS and C3b resulted in attenuated engulfment of C3b-deposited S. aureus by phagocytes, demonstrating that eLtaS aggravates infection by mediating evasion of phagocytosis by C3b-deposited S. aureus.
Furthermore, we demonstrated that the mouse monoclonal antibody MAE4 inhibited the interaction between eLtaS and C3b and consequently blocked evasion of phagocytosis of C3b-deposited S. aureus. In vivo studies also showed that administration of MAE4 promoted engulfment of S. aureus by phagocytes and protected mice against challenge with drug-sensitive (8325-4, Newman) and drug-resistant (04018) strains of S. aureus.
Our results in vitro showed that MAE4 did not target LtaS located on the surface of S. aureus; moreover, it had no effect on LTA synthesis and growth of S. aureus, suggesting that MAE4 could not inhibit the activity of membrane-bound LtaS. No observed interaction between MAE4 and LtaS on S. aureus may be because the epitope on LtaS recognized by MAE4 was covered. Whether MAE4 could directly target LtaS and has the inhibitory effect on S. aureus growth and LTA synthesis in vivo will be addressed in the future.
Of note, S. aureus expresses several small secreted proteins (~10-15 kDa) that bind to C3b, such as staphylococcal complement inhibitors (SCINs) and Efb 39 . These proteins share a similar structure for interaction with C3b 39 . Compared with these proteins, eLtaS is larger (~50 kDa) and a competitive ELISA also indicated that the C3b-binding site of eLtaS was different. Cell-associated LTA from S. aureus 8325-4 (2.5 × 10 9 cfu) cultured with MAE4 or isotype control IgG (50 μ g/ml) was extracted, and the level of LTA was analyzed by western blotting using a monoclonal LTA-specific antibody.
In conclusion, we have shown that the secreted protein eLtaS is an important virulence factor of S. aureus that mediates immune evasion by interfering with phagocytosis of complement-deposited bacterial cells. Targeting of eLtaS using the neutralizing antibody MAE4 that we have generated is a potential therapeutic strategy for treatment of infectious diseases caused by S. aureus.

Methods
Ethics Statement. All animal experimental protocols of the study are in accordance with the national guidelines for the use of animals in scientific research "Regulations for the Administration of Affairs Concerning Experimental Animals" and were approved by the Animal Care and Use Committee of Beijing Institute of Basic Medical Sciences, with the approval number BMS-111248.
Bacterial strains and growth conditions. S. aureus strains were grown in 5 ml of brain heart infusion (BHI) (BD) at 37 °C for 12 h with shaking at 200 rpm.
Hemolysis assay. For preparation of bacteria-free culture supernatant, a single colony was used to inoculate 5 ml of BHI in a 50-ml conical tube. The cultures were incubated at 37 °C with shaking at 200 rpm for 6 h and 12 h, at which time the samples were placed on ice. The cultures were diluted with BHI to equalize the OD 600 to a value at 10, pelleted by centrifugation at 4 °C, and sterile filtered through a 0.2-μ m filter. The classical pathway-mediated hemolytic assay was performed as previously described with some modifications 40 . Briefly, normal human sera were first incubated with sheep erythrocytes to pre-clear serum of antibodies directed against erythrocytes. Concurrently, sheep erythrocytes were opsonized with anti-erythrocyte IgM. The opsonized sheep erythrocytes (2 × 10 7 ) were then incubated with 25% precleared normal human serum, in the presence of eLtaS or the supernatants of S. aureus 8325-4 or Δ rnc in HBS ++ buffer (20 mM HEPES, 140 mM NaCl plus 5 mM CaCl 2 , 2.5 mM MgCl 2 and 0.1% Tween-20, pH 7.4). After 30 min at 37 °C, the samples were centrifuged, and the absorbance of the supernatants at 405 nm was measured. eLtaS protein expression and purification. The eltaS gene was amplified by PCR from the genomic DNA of S. aureus 8325-4 using the primer sequences (forward-EcoRI) ccggaattctctgaagatgacttaacaaa and (reverse-XhoI) ccgctcgagttattttttagagtttgctt. The PCR fragment was subcloned into the expression vector pET-28(a) and expressed in Escherichia coli (BL21) as an N-terminal his-tag fusion protein. The fusion protein was purified by Ni-NTA agarose (Qiagen) . Purified eLtaS protein was passed through the pierce high-capacity endotoxin removal resin to remove residual E. coli endotoxins (Thermo Scientific). Protein concentrations were determined by use of the bicinchoninic acid (BCA) protein assay, and proteins were stored at − 70 °C until use.
Enzyme-linked immunosorbent assay. Capture ELISA was performed as previously described with some modifications 29 . Plates were coated with eLtaS (5 μ g/well). The human or mouse sera were diluted in GVB-EDTA buffer (VBS containing 0.1% gelatin and 40 mM EDTA) and then added for 1 h at 37 °C. Detection of C3 was performed using anti-C3 (Cerdalane, 1:1000), followed by HRP-conjugated goat anti-rabbit IgG.
The binding ELISA was performed as previously described with some modifications 22 . Human complement C3 and its fragments C3b and C3d (Merck Millipore) were coated onto ELISA plates (1 μ g/well). eLtaS protein was diluted in PBS and then added for 1 h at 37 °C. Bound eLtaS was detected using anti-eLtaS antibody (1:1000) followed by HRP-conjugated goat anti-rabbit IgG.
In the eLtaS competitive ELISA, CR1 protein (USCN) was coated at 1 μ g per well; then 1 μ g C3b protein was added to each well with a serial dilution of eLtaS protein. Bound C3b proteins were detected using anti-C3 (Santa Cruz Biotechnology) followed by HRP-conjugated goat anti-rabbit IgG (1:20,000, Jackson ImmunoResearch Laboratories). In the MAE4 competitive ELISA assay, eLtaS was coated at 1 μ g per well; then 2 μ g C3b was added to each well with a serial dilution of MAE4. Bound C3b proteins were detected using anti-C3 (Santa Cruz Biotechnology) followed by HRP-conjugated goat anti-rabbit IgG (1:20,000, Jackson ImmunoResearch Laboratories).
Acute peritoneal infection murine model. Male mice (CD-1, C3 −/− C57BL/6 and C3 +/− C57BL/6; 6-to 8-week-old) were intraperitoneally injected with S. aureus 8325-4 or Δ ltaS cells. Each type of mice was randomized into treatment groups of eight mice each. In the eLtaS or MAE4 group, 100 μ g protein/per mouse carried in 100 μ l PBS was injected into the peritoneal cavity. Mouse survival was recorded at the different time points post challenge.
Sublethal murine pneumonia infection model. Male CD-1 mice, 6-to 8-week-old, were challenged with S. aureus 8325-4 (1 × 10 7 cfu/mouse) via the tracheal route and were randomized into two groups of eight mice each. eLtaS or MAE4 protein (100 μ g per mouse in 300 μ l PBS) was intraperitoneally injected. After 72 h of challenge, the animals were sacrificed and the lung tissues were fixed and stained by H&E.
Renal abscess model and lethal challenge via intravenous injection. BALB/c mice (8-week-old, female) were intraperitoneally injected with 100 μ g antibody/per mouse in 100 μ l PBS. Mice were injected retro-orbitally with clinical S. aureus strain Newman (1 × 10 7 cfu) under anesthesia. On the 5th day post infection, mice were killed and the right kidneys were examined by histopathology for internal abscesses. Left kidneys were removed and homogenized in 0.1% Triton X-100. Aliquots were diluted and plated on agar medium for triplicate determination of cfu. For the lethal challenge model, all experimental conditions remained the same except that 1 × 10 8 cfu were administered and animals were monitored for survival for ten days post infection. SYTO 9 labeling S. aureus. S. aureus 8325-4 was labeled by SYTO9 as previously described with some modifications 41 . Briefly, S. aureus 8325-4 was grown on BHI for 12 h at 37 °C. The bacterial cells were collected by centrifugation (5,000 g, 5 min), washed with PBS, suspended in PBS to an optical density of 2 × 10 9 cfu/ml, and incubated at room temperature for 30 min in the dark with 5 μ M SYTO 9 (Molecular Probes). The cells were washed twice to remove excess dye and resuspended in PBS. A flow cytometer (BD) with a 530/30 band pass filter was used to detect SYTO 9 fluorescence. S. aureus with C3b deposition. C3b-deposited S. aureus 8325-4 was prepared as described with some modifications 42 . Human sera were obtained from normal donors after informed consent, according to human study protocols reviewed and approved by the institutional review boards at Beijing Institute of Basic Medical Sciences. S. aureus cells (1 × 10 7 cfu) were washed twice in PBS. A mixture of S. aureus cells and human serum was incubated in 100 μ l (final volume) of PBS, with or without eLtaS protein, for 10 min at 37 °C. Controls for C3b deposition experiments included S. aureus cells incubated with buffer alone and eLtaS alone.
Deposition of C3b was confirmed by direct immunofluorescence analysis using flow cytometry. For flow cytometric analysis, S. aureus cells (1 × 10 7 cfu) with C3b deposits were resuspended in PBS, incubated with FITC-conjugated mouse anti-human C3b antibody (1 μ g/ml, Cedarlane) for 30 min at room temperature, washed twice in PBS and resuspended in the same buffer at 1 × 10 6 cells/ml.

Assessment of phagocytosis in vitro.
The C3b-deposited S. aureus 8325-4 (5 × 10 6 cfu) was incubated with different concentrations of eLtaS for 30 min at room temperature. For colony-forming unites assay, human neutrophils were purified on a Ficoll (Amersham)/Histopaque (density 1.119; Sigma) gradient using heparinized whole blood from a single donor 30 .
Then the C3b-deposited S. aureus 8325-4 with or without eLtaS were incubated with human neutrophils for 10 min at 37 °C. The reaction was stopped by adding gentamicin (0.1 mg/ml) for 10 min at 37 °C. The bacteria were resuspended in 0.02% Triton X-100. Cfu were calculated by serial dilution plated on Todd-Hewitt agar (THA; 1.5% agar in Todd-Hewitt broth) plates.
For flow cytometry assays, leukocytes were isolated from whole blood by red blood cell lysis. The C3b-deposited S. aureus 8325-4 (labeled with SYTO 9) with eLtaS were incubated with leukocytes for 10 min. The cells were washed twice by PBS and the percentage of granulocyte cells that engulfed the SYTO 9-labeled S. aureus was detected by flow cytometry. SYTO9-labeled S. aureus 8325-4 (5 × 10 8 cfu/mouse) were intraperitoneally injected into individual mice together with eLtaS (100 μ g/mouse) in 500 μ l PBS. Peritoneal cavity cells were isolated 30 min after injection. The reaction between peritoneal cavity cells and S. aureus was stopped by adding gentamicin (0.1 mg/ml) for 10 min at 37 °C. After washing two times with PBS, cell suspensions were then preincubated with anti-CD16/CD32 mAb to block Fcγ RII/III receptors and stained in 4 °C for 15 min. Cells were then stained with APC-conjugated anti-CD11b (Mac-1) (M1/70), PE-conjugated anti-F4/80 (BM8) and PercP-Cy5.5-conjugated Gr-1(Ly-6 G) (1A8) (Biolegend) in 4 °C for 30 min. For dead cell exclusion, stained cells were resuspended in 5 μ g/ mL 7-AAD to exclude dead cells. Data were collected from 1 to 5 × 10 5 cells and analyzed with FlowJo software. To distinguish autofluorescent cells from cells expressing low levels of individual surface markers, we established upper thresholds for autofluorescence by running an unstained sample. Single-stain controls are then analysed to determine the correct voltage and gain settings.

Production and characterization of monoclonal antibodies. Monoclonal antibodies of eLtaS
were produced by fusing antibody-secreting spleen cells from eLtaS-immunized mice with immortal myeloma cells to create monoclonal hybridoma cell lines that express the eLtaS specific antibody in cell culture supernatant. Antibody samples from cell culture supernatants were screened to identify eLtaS-binding specificity. The titer and isotype of positive antibody samples were determined. To produce large quantities of antibody MAE4, the MAE4 hybridoma cells were injected into the abdomen of mice where the cells multiplied and produced antibody-filled fluid (ascites). The antibodies were purified using protein G agarose (GE Healthcare) according to the product instructions.
Statistical analysis. All experiments were repeated at least three times and the data were presented as the mean ± SD unless noted other wise. Significant differences between groups were evaluated using a two-tailed Student's t test. Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test. A P-value of less than 0.05 was considered statistically significant. GraphPad Prism software was used for statistical analyses.