Effective Antibacterial and Antihemolysin Activities of Ellipticine Hydrochloride against Streptococcus suis in a Mouse Model

In recent years, the inappropriate use of antibiotics has unnecessarily caused the continuous emergence of resistant bacteria. The antimicrobial resistance of Streptococcus suis has also become an increasingly serious problem.

ABSTRACT

Streptococcal toxic shock-like syndrome (STSLS) caused by the epidemic strain of Streptococcus suis leads to severe inflammation and high mortality. The life and health of humans and animals are also threatened by the increasingly severe antimicrobial resistance in Streptococcus suis. There is an urgent need to discover novel strategies for the treatment of S. suis infection. Suilysin (SLY) is considered to be an important virulence factor in the pathogenesis of S. suis. In this study, ellipticine hydrochloride (EH) was reported as a compound that antagonizes the hemolytic activity of SLY. In vitro, EH was found to effectively inhibit SLY-mediated hemolytic activity. Furthermore, EH had a strong affinity for SLY, thereby directly binding to SLY to interfere with the hemolytic activity. Meanwhile, it was worth noting that EH was also found to have a significant antibacterial activity. In vivo, compared with traditional ampicillin, EH not only significantly improved the survival rate of mice infected with S. suis 2 strain Sc19 but also relieved lung pathological damage. Furthermore, EH effectively decreased the levels of inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-α]) and blood biochemistry enzymes (alanine transaminase [ALT], aspartate transaminase [AST], creatine kinase [CK]) in Sc19-infected mice. Additionally, EH markedly reduced the bacterial load of tissues in Sc19-infected mice. In conclusion, our findings suggest that EH can be a potential compound for treating S. suis infection in view of its antibacterial and antihemolysin activity.

IMPORTANCE In recent years, the inappropriate use of antibiotics has unnecessarily caused the continuous emergence of resistant bacteria. The antimicrobial resistance of Streptococcus suis has also become an increasingly serious problem. Targeting virulence can reduce the selective pressure of bacteria on antibiotics, thereby alleviating the development of bacterial resistance to a certain extent. Meanwhile, the excessive inflammatory response caused by S. suis infection is considered the primary cause of acute death. Here, we found that ellipticine hydrochloride (EH) exhibited effective antibacterial and antihemolysin activities against S. suis in vitro. In vivo, compared with ampicillin, EH had a significant protective effect on S. suis serotype 2 strain Sc19-infected mice. Our results indicated that EH, with dual antibacterial and antivirulence effects, will contribute to treating S. suis infections and alleviating the antimicrobial resistance of S. suis to a certain extent. More importantly, EH may develop into a promising drug for the prevention of acute death caused by excessive inflammation.

INTRODUCTION

As one of the most important zoonotic pathogens, Streptococcus suis seriously threatens human health and the development of the swine industry and causes major economic losses worldwide. S. suis infection can cause a variety of diseases, including septicemia, meningitis, endocarditis, arthritis, and even streptococcal toxic shock-like syndrome (STSLS) (1–3). As an emerging infectious pathogen, S. suis has been classified into 29 (1 to 19, 21, 23 to 25, 27 to 31, and 1/2) serotypes based on the pathogen’s capsular polysaccharides. Streptococcus suis serotype 2 (S. suis 2) is the most frequent and pathogenic of all serotypes and is prone to infect humans and animals (4, 5). Since the first human infection with S. suis was discovered in 1968, over 1,600 human cases have been reported worldwide (6, 7). In 1998 and 2005, two large outbreaks of S. suis occurred in China, causing severe economic losses and many human deaths (8, 9). In addition, it has been reported that S. suis is also an important pathogen causing human meningitis in Vietnam, Thailand, and Hong Kong (10–12). So far, the treatment of S. suis infection mainly relies on the use of antibacterial drugs, including β-lactams, aminoglycosides, amphenicols, and fluoroquinolones, but these antibacterial drugs are ineffective against STSLS, which is considered the primary cause of acute death, with mortality higher than 80% even after adequate treatment (13). In addition, the unreasonable use of antibiotics has resulted in the increasing antimicrobial resistance of S. suis and multiple-drug resistance, which has brought difficulties to clinical treatment (14, 15). Therefore, developing a new strategy for suppressing excessive inflammation and alleviating antimicrobial resistance will undoubtedly contribute to treating S. suis infections and reducing mortality rates.

Previous studies demonstrated that suilysin (SLY), as one of the important virulence factors, plays a significant role in inducing S. suis 2 infection and inflammatory response (16, 17). SLY has been reported to lyse red blood cells to release hemoglobin in synergy with S. suis cell wall components, thus increasing the levels of proinflammatory mediators in vivo (18). In some previous studies, the virulence of S. suis 2 was reduced by flavonoids, including amentoflavone, fisetin, and myricetin, which can weaken S. suis 2’s pathogenicity by inhibiting the hemolytic activity of SLY (19–21). S. suis strains with high levels of SLY are more likely to cause high mortality in infected mice than nonvirulent strains, indicating that the pathogenicity and virulence of S. suis can be enhanced by increasing the production of SLY (22, 23). In addition, the NLRP3 inflammasome activation induced by the high expression of SLY in the S. suis 2 strain is the main reason for the excessive inflammatory response and multiorgan damage in STSLS (13). Therefore, a novel antivirulence compound which can inhibit the hemolytic activity of SLY remains to be developed to greatly relieve inflammation caused by S. suis with high levels of SLY.

Our recent study described a compound called ellipticine hydrochloride (EH), which can effectively inhibit the pathogenicity of multidrug-resistant Escherichia coli (ExPEC) with the mcr-1 gene (24). Ellipticine derived from the leaves of Apocynaceae plants was reported to display potent anticancer, anti-HIV, and anti-inflammatory activities (25–27). In addition, ellipticine was also reported to exhibit excellent antiplasmodial activity or antimalarial properties in a mouse model (28, 29). Our previous studies demonstrated that ellipticine can significantly reduce the bacterial load and tissue damage in colistin-resistant E. coli-infected mice (24). Nevertheless, no reports on the antibacterial and antihemolysin activities of ellipticine in S. suis are available.

In this study, we explored the antibacterial and antihemolysin activities of EH against S. suis. Our results demonstrated that in addition to good antibacterial activity, EH also exhibited strong antihemolysin activity. In particular, compared with traditional ampicillin, our animal experiments indicated that EH significantly relieved inflammation and organ damage and improved the survival rate of S. suis 2-infected mice. Overall, our findings suggest that EH can be a promising compound for treating S. suis infection due to its antibacterial and antihemolysin activities.

RESULTS

Inhibition of S. suis growth by EH.

The MICs of EH against the S. suis strains are summarized in Table 1. EH possessed potent antibacterial activity against clinical S. suis with a MIC ranging from 0.125 μg/ml to 0.5 μg/ml. The time-kill curves demonstrated that EH exerted effective killing effects on S. suis 2 strain Sc19. At the concentration of 4× MIC at 8 h after coincubation, the bacteria were thoroughly eliminated (Fig. 1), indicating a significant antibacterial effect of the compound against S. suis. These results suggested that EH effectively inhibited the growth of S. suis.

TABLE 1.

MICs of EH against S. suis^a

Strain ID	Phenotypic properties	Source	EH (mg/liter)
Sc19	Resistant to CLI, TET, and LEV	China (Sichuan)	0.25
S. suis 160413	Resistant to CLI, TET, AMP, and LEV	China (Hubei)	0.125
S. suis 16042	Resistant to CLI, TET, AMP, and LEV	China (Hunan)	0.25
S. suis 16091	Resistant to TET, AMP and LEV, and STX	China (Hubei)	0.5
S. suis 16095	Resistant to CLI, TET, AMP, and STX	China (Guangzhou)	0.25
S. suis 16072	Resistant to CLI, TET, AMP, LEV, and STX	China (Hubei)	0.125
S. suis 18051	Resistant to CLI, TET, AMP, and LEV	China (Hubei)	0.125
S. suis 180515	Resistant to TET, AMP, and LEV	China (Hubei)	0.25
S. suis 170612	Resistant to CLI, TET, AMP, and STX	China (Hubei)	0.25
S. suis 170601	Resistant to CLI, TET, AMP, and LEV	China (Hubei)	0.25
S. suis 170603	Resistant to TET, AMP and LEV, and STX	China (Zhejiang)	0.125

^aAMP, ampicillin; TET, tetracycline; LEV, levofloxacin; CLI, lincomycin; STX, trimethoprim and sulfamethoxazole.

FIG 1.

Survival of S. suis was determined after treatment with EH at the concentration of 4× MIC.

Effective antihemolysin activity of EH against S. suis.

The effect of EH against the activity of SLY was evaluated with hemolysis assays. The structure of EH is shown in Fig. 2A. We found that the supernatants from S. suis 2 Sc19 culture medium showed hemolytic activity, as shown in Fig. 2B. The supernatants of 125 μl Sc19 culture lysed more than 95% of defibrated sheep erythrocytes (Fig. 2B), but the supernatants from the Δsly strain’s culture medium had no hemolytic activity, as shown in Fig. 2C. We found that EH significantly decreased the hemolytic activity of Sc19 culture supernatants in a concentration-dependent manner in the coculture system of Sc19 supernatants and EH. The optimal inhibitory effect was achieved at a concentration of 32 μg/ml (Fig. 2D). Furthermore, the purified recombinant SLY was used to more directly evaluate the effect of EH on SLY. Consistent with the inhibitory effect of EH on supernatants as described above, the hemolytic activity of SLY protein was significantly inhibited by EH (Fig. 2E). Additionally, an experiment involving coincubation of defibrated sheep erythrocytes and EH was carried out to determine the effect of this compound on those cells. The results indicated that EH (ranging from 1 to 128 μg/ml) alone did not lyse erythrocytes (Fig. 2F). In summary, the above-described results indicated that EH decreased the hemolytic activity of SLY or directly neutralized SLY-mediated hemolysis.

FIG 2.

Antihemolysin activity of EH against S. suis. (A) Chemical structure of EH. (B) The hemolytic effect of Sc19 culture supernatant on defibrated sheep erythrocytes was determined by measuring the optical density at 543 nm. Positive control, 2.5% Triton X-100. Negative control, culture medium alone. (C) The hemolytic effect of Sc19 Δsly culture supernatant on defibrated sheep erythrocytes was determined as described above. (D) EH inhibited the hemolytic activity of Sc19 culture supernatant. The 125-μl culture’s supernatant was incubated with different concentrations of EH at 37°C for 30 min and then incubated with 875 μl PBS containing 2% defibrated sheep red blood at 37°C for 30 min. (E) EH reduced the hemolytic activity of the purified SLY protein. In the hemolytic assay, the hemolytic activity of the purified SLY protein was determined after coincubation with the different concentrations of EH. (F) Effect of EH at final concentrations of 1, 2, 4, 8, 16, 32, 64, and 128 μg/ml on defibrated sheep erythrocytes. EH at different concentrations was incubated with cells for 1 h at 37°C. Positive control, 2.5% Triton X-100. The two-tailed unpaired t test was used for statistical analysis. The data shown are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

EH binding site on SLY revealed by molecular docking and MST assay.

The EH binding site on SLY was identified by the molecular docking-based calculation. The blind docking results indicated that a total of 100 docking phases were generated, 90 of which were contained in Pose1 with the minimal (or optimal) energy of −6.11 kcal/mol at the 75th docking phase. Most small molecules were docked in Pose1, indicating that Pose1 was a small molecule binding pocket (Fig. 3A). As shown in Fig. 3B, the theoretical binding mode of EH was confirmed. The compact conformation of EH was shown in SLY. Amino acids I87, D86, L110, N112, D111, T195, K190, N50, T191, S374, and N82 of SLY formed the hydrophobic pocket cavity (Fig. 3B), which allowed good hydrophobic action with small molecules. These hydrophobic actions played a dominant role in the interaction between the EH and SLY. Importantly, two key hydrogen bonds were established between the EH and SLY residues S84 and L110. All these interactions helped EH to anchor at its binding site to SLY. To confirm the binding site in the SLY-EH complex, the microscale thermophoresis (MST) assay was performed for the complex systems involving the L110A/S84A-Sly mutant with EH, and the K_d values of the complex were then obtained using the monolith affinity analysis K_d fit. As predicted by molecular docking, the K_d values for the complexes revealed that the K_d of the L110A/S84A-Sly mutant showed a marked increase compared with the wild-type (WT) Sly with the EH complex, which indicated that the binding affinity between the mutant and EH was significantly weakened (Fig. 3C and D).

FIG 3.

Interaction between EH and SLY. (A) EH was bound into the pose1 of SLY. (B) EH is indicated with green sticks; the two hydrogen bonds are indicated with the black dotted line. (C) Interaction between RED-NHS second-generation dye-labeled purified Sly with EH. Data are presented as the mean ± standard error of the mean (s.e.m.) from three independent experiments. (D) Interaction between RED-NHS second-generation dye-labeled purified L110A/S84A-Sly with EH. Data are presented as the mean ± s.e.m. from three independent experiments. (E) The interaction of SLY protein and EH was analyzed using the ITC assay. EH (0.2 mmol/liter) was dripped into the purified SLY protein (0.02 mmol/liter) in PBS buffer (pH 7.4) at 25°C. The data were analyzed to obtain the equilibrium dissociation constant (K_d,  1.235 × 10⁻⁷ mol/liter), stoichiometry (n = 2.079), and changes of enthalpy (ΔH, −142.8 kJ/mol) and entropy (ΔS, –362.2J/mol/K). (F) An LSPR assay explored the kinetic parameters of the binding reaction between SLY and EH. The equilibrium dissociation constants (K_d) of SLY and EH were found to be 1.86 × 10⁻⁶ M using Trace Drawer software.

Direct binding reaction of EH with SLY.

To further verify the interaction between EH and SLY, isothermal titration calorimetry (ITC) and localized surface plasmon resonance (LSPR) assays were conducted to determine the binding affinity between EH and SLY. As shown in Fig. 3E, in addition to the dissociation constant (K_d), the thermodynamic parameters ΔH and ΔS were obtained in the ITC assay. Both the binding-induced enthalpy increase and ITC profile rising trend showed that the binding reaction of EH and SLY resulted in heat absorption. The equilibrium dissociation constant (K_d) between EH and SLY was identified to be 0.1235 μM (Fig. 3E), which indicated a strong binding interaction. In order to further validate our results, the LSPR assay was performed to characterize the interactions between EH and SLY. We observed strong surface plasmon resonance (SPR) signal responses when the increasing concentrations of EH flowed through SLY immobilized on the chip. The binding data processed by the kinetic model of the evaluation software showed that the K_d value of the affinity between EH and SLY was 1.86 μM (Fig. 3F). Our results suggested the EH and SLY had a strong direct interaction.

Therapeutic effects of EH on S. suis 2 Sc19-infected mice.

To determine the therapeutic effect of EH in vivo, a mouse model infected with S. suis 2 Sc19 was established. First, the protective effect of EH on infected mice was evaluated based on the survival rate. As shown in Fig. 4A, the survival rate of EH-treated mice increased to 60% compared with that of untreated infected mice, and that observed in the group treated with ampicillin was 40%. Afterward, the pathological changes in lung and brain and the levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were investigated to assess the anti-inflammation activity of EH and ampicillin in vivo. We found that EH significantly relieved inflammation and pathological damage, including infiltration of inflammatory cells, alveolar interstitial congestion, and edema in the lung and brain of infected mice (Fig. 4B). Moreover, compared with ampicillin, EH treatment resulted in a significant decrease in the expression levels of IL-6 and TNF-α (Fig. 5A). Finally, we assessed the effect of EH and ampicillin on the levels of alanine transaminase (ALT), aspartate transaminase (AST), and creatine kinase (CK) and the bacterial load of Sc19 in various tissues of infected mice. Our results indicated that EH obviously decreased the levels of blood biochemistry enzymes in infected mice (Fig. 5B). In addition, because the decrease of bacterial burden in the tissue in the ampicillin group was slightly greater than that in the EH group. (Fig. 6), the decreased inflammatory response was not due to a decreased bacterial load. Therefore, these data indicate that the anti-inflammatory effect of EH is essential to improve the survival of S. suis-infected mice. Taken together, these results showed that EH had a good therapeutic effect on S. suis 2 Sc19-infected mice.

FIG 4.

Survival rate and tissue pathological changes of Sc19-infected mice. The dose and interval of each treatment were 5 mg/kg and 12 h, respectively. (A) Survival rate of mice per day. Compared with ampicillin, EH has a higher protection rate for Sc19-infected mice (log-rank and chi-square tests, n = 10). (B) Pathological changes of lung and brain tissue after EH and ampicillin treatment. EH alleviated tissue damage of infected mice.

FIG 5.

Levels of inflammatory cytokines and blood biochemistry enzymes in Sc19-infected mice. The dose and interval of each treatment were 5 mg/kg and 12 h, respectively. (A) Expression levels of IL-6 and TNF-α in infected mice. Compared with ampicillin, EH reduced the production of inflammatory cytokines. (B) Levels of ALT, AST, and CK in the blood of infected mice. Compared with ampicillin, EH decreased the levels of blood biochemistry enzymes of Sc19-infected mice. The two-tailed unpaired t test was applied for statistical analysis. The data shown are representative of three independent experiments. *, P < 0.05; ***, P < 0.001.

FIG 6.

Tissue bacterial load in Sc19-infected mice. The dose and interval of each treatment were 5 mg/kg and 12 h, respectively. Both EH and ampicillin can reduce the bacterial titers in different tissues in mice infected with Sc19. The bacterial counts of Sc19 in the lung, spleen, kidney, and liver are shown. The two-tailed unpaired t test was applied for statistical analysis. The data shown are representative of three independent experiments. **, P < 0.01; ***, P < 0.001.

DISCUSSION

With the overuse of antibiotics and the slow development of new antibacterial agents, the antibiotic resistance of bacteria has become increasingly severe. Targeting virulence has become a promising approach to solving the problem of antibiotic resistance (30–32). As a prevalent pathogen, S. suis can cause human deaths and severe economic losses. Two large-scale outbreaks of S. suis in China with high mortality and STSLS have raised worldwide concern (33). Targeting virulence can reduce the selective pressure of bacteria on antibiotics, thereby alleviating the development of bacterial resistance to a certain extent (34). As a virulence factor, SLY has an important effect on the pathogenic process of S. suis (35). SLY was reported to activate high levels of the inflammasomes which played an important role in STSLS. In addition, SLY was also reported to play a significant role in meningitis caused by S. suis (23). Therefore, targeting inflammation-related SLY is a novel strategy for treating S. suis-induced diseases and relieving antibiotic resistance development.

Excessive inflammation can cause organ damage and accelerate disease progression, which are among the serious consequences of S. suis infection. SLY was reported to play an important role in inducing an excessive inflammatory response (13). In this study, EH significantly reduced the hemolytic activity of Sc19 culture supernatants and the purified recombinant SLY. Some previous studies suggested that flavonoids inhibited the hemolytic activity of SLY, thereby exerting an anti-inflammatory effect and weakening S. suis 2’s pathogenicity (19–21). EH and these compounds had the same function, which indicated that there may be similarities in the functional group of chemical construction. Moreover, it was also reported that ellipticine exhibited no hematological toxicity and had quite limited toxic side effects (36). Here, we also confirmed that EH had no hemolytic toxicity. In addition, the results of molecular docking suggested that one potential binding site existed in the protein SLY and that the EH interacted with SLY through hydrophobic interactions and hydrogen bonds.

Furthermore, we found that the binding K_d value of L110A/S84A-Sly mutant protein with EH increased significantly, indicating the weakened binding force between the protein and EH, which verified the molecular docking results. Subsequent K_d values of ITC and SPR assays also showed that EH interrupted the protein-receptor interaction by direct strong binding to the SLY in vitro, which was the main reason for antihemolysin activity of EH. Although traditional antibiotics can play an effective role in eliminating S. suis, these antibiotics cannot improve the survival rate of infected mice by inhibiting the excessive proinflammatory responses (37). EH showed favorable in vitro activity against multidrug-resistant clinical isolates of S. suis. Specifically, the mouse infection model showed a higher protection rate than commonly used drugs. Our results show that EH exerted an anti-inflammatory effect by inhibiting the hemolytic activity of SLY, thereby improving the survival rate of infected mice. This phenomenon reminds us that for some severely infected patients, EH alone or in combination with first-line drugs may be a better choice.

Currently, the treatment of S. suis infection often involves the combinational application of traditional anti-inflammatory and antimicrobial agents (37). Many previous studies reported that bacterial pathogenicity could be reduced by using the antivirulence activity of a single drug or the microbicidal and antivirulence activities of drugs combined with adjuvants (19, 38, 39). In this study, we found that EH also exhibited direct antibacterial activity in addition to antihemolysin activity. The MICs and the time-kill curves demonstrated that EH effectively inhibited the growth of S. suis. Our recent study reported that ellipticine inhibited topoisomerase IV activity to kill multidrug-resistant E. coli and exhibited broad-spectrum antimicrobial activities against the tested strains (24). The antibacterial activity of EH against S. suis might be related to targeting bacterial topoisomerase, which remains to be further investigated. In addition, EH treatment decreased the levels of blood biochemistry enzymes (ALT, AST, CK) and bacterial load in the tissues of the mice infected with S. suis 2 strain Sc19, which contributed to the higher survival rate of infected and untreated mice than that of infected mice treated with the single antihemolysin compound fisetin in our previous study (20). The results indicated that EH simultaneously exhibited direct antibacterial activity and antihemolysin activity. EH will have the potential to be used as a single agent or together with conventional antibiotics to treat S. suis infection since it has dual antibacterial and antivirulence effects. In summary, our findings indicate that EH can provide a novel therapeutic approach to S. suis infection due to its antibacterial and antihemolysin activities. This study lays a foundation for developing EH into a new drug against S. suis.

MATERIALS AND METHODS

Bacterial strains, growth conditions, and ellipticine hydrochloride (EH) preparation.

S. suis 2 strain Sc19, as a virulent strain, was isolated from the brain of a dead pig during an outbreak of S. suis in Sichuan Province, China, in 2005 (40). Ten clinical multidrug-resistant S. suis strains and the mutant Δsly were obtained from our laboratory strain library (20). S. suis strains were cultured in tryptic soy broth (TSB) or plated on tryptic soy agar (TSA) (Summus Ltd., China) with 5% (vol/vol) fetal bovine serum (Sijiqing Ltd., China) at 37°C. EH was purchased from Topscience and dissolved with dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA).

MIC determination.

The MIC of EH against S. suis was determined by referring to the Clinical and Laboratory Standards Institute (CLSI) guidelines (41). The microdilution broth method was applied to 96-well plates (Costar 3599, Corning, NY, USA). A 0.22-μm syringe filter (Millipore, USA) was used to filter EH dissolved in DMSO. The final concentration of the strain culture was 5 × 10⁵ CFU/ml. EH (0.03125 to 32 mg/liter) was tested in triplicate.

Time-kill curve.

To further evaluate the antibacterial activity of EH, a time-kill curve was plotted with an EH concentration of 4× MIC. The Sc19 culture was diluted with a broth medium containing 5% fetal bovine serum to a final concentration of 10⁶ cells/ml. EH with a concentration of 4× MIC was added, and the mixture was incubated at 37°C. At 2-h intervals, samples were taken out to be serially diluted and then plated on a TSA plate and incubated overnight at 37°C. The time-kill curve was determined by counting the colonies. The measurement was conducted in triplicate.

Effect of EH on hemolytic activity of Sc19 culture supernatant.

The S. suis Sc19 strain was cultured for 12 h at 37°C and centrifuged at 10,000 rpm for 10 min at 4°C. Subsequently, the supernatant was collected and incubated with different concentrations of EH (0, 2, 4, 8, 16, and 32 μg/ml) for 30 min at 37°C. Then, 2% defibrated sheep blood in phosphate-buffered saline (PBS; pH 7.4) solution was added and incubated at 37°C for 30 min. Finally, the mixture was centrifuged at 1,000 rpm for 5 min at 4°C. Subsequently, 200-μl aliquots of supernatant were collected, and their optical densities were measured at 543 nm using a BioSpectrometer instrument (Eppendorf). Additionally, to determine the effect of EH on sheep erythrocytes, a separate experiment was carried out. EH (ranging from 1 to 128 μg/ml) was incubated alone with cells for 1 h at 37°C. The sample was treated with 2.5% Triton X-100 and set as a 100% cleavage control. The effect of EH on the hemolytic activity of the Sc19 culture supernatant was evaluated based on the ratio of the optical density at 543 nm (OD₅₄₃) of each sample to that of the 100% cleavage control.

Experiment of EH resisting hemolytic activity of recombinant protein SLY.

The prokaryotic expression plasmid pET-28a(+)-SLY was constructed by subcloning SLY cDNA into the pET-28a(+) vector (Novagen, Madison, WI, USA) using BamHI and NdeI restriction enzyme cutting sites. E. coli BL21 transformed with the recombinant plasmid was cultured in the medium containing 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 16 h at 16°C. The resultant rSLY was purified by loading the supernatant of bacterial cell lysates onto a Ni-NTA column. The antihemolysin activity of EH was directly evaluated by coincubation of the purified protein (100 ng/ml) and EH at different concentrations (0, 2, 4, 8, 16, and 32 μg/ml) with the procedures described above.

Molecular docking.

AutoDock 4.2 was used in the docking procedure of SLY and EH to analyze the interaction mode between SLY and EH. The crystalline structure of SLY was obtained from the Protein Database (PDB). The two-dimensional (2D) structure and three-dimensional (3D) structure of EH were drawn using ChemBioDraw Ultra 14.0 and ChemBio3D Ultra 14.0, respectively (21).

Expression and purification of L110A/S84A-Sly.

The L110A/S84A-Sly gene was synthesized by Integrated DNA Technologies (Qingke Biological Co. Ltd., Beijing, China) by changing amino acids 84 and 110. The gene was then cloned into pET28a digested with BamHI and NdeI (MRKSSHLILSSIVSLALVGVTPLSVLADSKQDINQYFQSLTYEPQEILTNEGEYIDNPPATTGMLENGRFVVLRREKKNITNNAADIAVIDAKAANIYPGALLRADQNLADNNPTLISIARGDLTLSLNLPGLANGDSHTVVNSPTRSTVRTGVNNLLSKWNNTYAGEYGNTQAELQYDETMAYSMSQLKTKFGTSFEKIAVPLDINFDAVNSGEKQVQIVNFKQIYYTVSVDEPESPSKLFAEGTTVEDLKRNGITDEVPPVYVSSVSYGRSMFIKLETSSRSTQVQAAFKAAIKGVDISGNAEYQDILKNTSFSAYIFGGDAGSAATVVSGNIETLKKIIEEGARYGKLNPGVPISYSTNFVKDNRPAQILSNSEYIETTSTVHNSSALTLDHSGAYVAKYNITWEEVSYNEAGEEVWEPKAWDKNGVNLTSHWSETIQIPGNARNLHVNIQECTGLAWEWWRTVYDKDLPLVGQRKITIWGTTLYPQYADEVIE). E. coli BL21 transformed with the recombinant plasmid was cultured in the medium containing 0.2 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 16 h at 16°C. The resultant rL110A/S84A-Sly was purified by loading the supernatant of bacterial cell lysates onto a Ni-NTA column.

Microscale thermophoresis (MST) assay.

The K_d values of the binding of Sly or L110A/S84A-Sly to EH were measured using a Monolith NT.115 Pico instrument (Nanotemper Technologies GmbH, Munich, Germany). Protein was labeled with the RED-NHS second-generation dye for 30 min at room temperature in the dark according to the manufacturer’s instructions (Monolith Protein Labeling kit RED-NHS 2nd generation; Nanotemper Technologies GmbH). EH was serially diluted in the reaction buffer (50 mM HEPES buffer [pH = 7.4] containing 0.05% Tween 20). Then, 100 nM labeled protein was added to the serial dilution of the compound in a 1:1 volume ratio. After incubation for 30 min at room temperature, the samples were examined with Monolith NT.115 in capillaries (MO-K022) at medium MST power and 5% LED/excitation power. The K_d values were determined using the Monolith affinity analysis K_d fit from triplicate experiments.

Isothermal titration calorimetry (ITC) assay.

The interaction of SLY protein and EH was determined by calorimetry using affinity ITC (TA NANO) in vitro. The purified SLY protein (0.02 mmol/liter) and EH (0.2 mmol/liter) were dissolved in PBS (pH 7.4). EH was injected into the sample cell filled with the purified SLY protein, and the injection was repeated 20 times with an equilibrium interval of 200 s. The experiment was conducted at 25°C. The equilibrium dissociation constant (K_d), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) were obtained using nanoAnalyzer software.

Localized surface plasmon resonance (LSPR) assay.

The equilibrium dissociation constants of SLY and EH were determined by open SPR to further identify the interaction of SLY protein and EH (24). The SLY protein was fixed on the COOH-sensor chip (Nicoya, Canada). The chip was flushed with PBS (pH 7.4) to obtain a stable detection baseline. EH at increasing concentrations (1.272 mg/liter, 2.545 mg/liter, 5.090 mg/liter, 10.179 mg/liter) was injected into the chip. PBS was set as a negative control. The speed of each cycle was set to 20 μl/min. Trace Drawer software (Ridgeview Instruments AB, Sweden) was applied to analyze the data obtained from the assay. The interaction between SLY and EH was evaluated with the kinetic parameters of the binding reaction.

Establishment of the S. suis 2 Sc19-infected mouse model in vivo.

Seven-week-old female BALB/c mice were purchased from China Three Gorges University to establish a mouse model of S. suis 2 Sc19 infection. Animal experiments conformed to animal ethics, and all experiments were conducted under the guidance of the Protection, Supervision, and Control Committee of Animal Experiments of Huazhong Agricultural University (HZAUMO-2020-0009). S. suis 2 Sc19 was transferred into TSB medium at 1:100 and cultured at 37°C until the OD₆₀₀ reached 0.6. The bacteria were collected after centrifugation at 10,000 rpm for 10 min at 4°C and then suspended with PBS (pH 7.4). In the survival rate assay, the concentration of Sc19 in intraperitoneally infected mice was 1.25 × 10⁹ cells/ml (200 μl). After 2 h of infection, mice were treated with EH or ampicillin by intraperitoneal injection. The dose and interval of each treatment were 5 mg/kg and 12 h, respectively. The control group (10 per group) was injected with 200 μl PBS (pH 7.4). Based on assay data, the survival curve of mice was constructed.

In addition, mice (5 per group) were intraperitoneally infected with 200 μl Sc19 at a concentration of 2.5 × 10⁸ cells/ml, and then EH or ampicillin was injected, as described above. The control group was injected with PBS (pH 7.4). At 12 h postinjection, the cardiac blood of anesthetized mice was collected to analyze the effects of EH on the levels of blood biochemistry enzymes (ALT, AST, CK) in Sc19-infected mice. The lung, spleen, kidney, and liver were ground, diluted, and inoculated onto a TSA plate containing 5% fetal bovine serum. The samples were cultured overnight at 37°C, and then the bacteria were counted. For the electrochemiluminescence studies, the U-PLEX Biomarker mouse assay from Meso Scale Discover (MSD; UNIV, China), which measures IL-6 and TNF-α, was used. MSD plates were analyzed on the MSD QuickPlex SQ120 (42). Finally, the lung and brain tissue were immobilized in 4% paraformaldehyde to analyze the pathological changes. All the pathological images were obtained through the same microscope (Olympus, Japan), and any potential observed lines are likely the result of an artifact in the AI software (Adobe, USA) or microscope.

Statistical analysis.

All experimental data were expressed as the mean ± standard deviation (SD) and analyzed with GraphPad Prism 7.0. The two-tailed unpaired t test was used to reveal the significant difference.