Functional analysis of the type II toxin-antitoxin system ParDE in Streptococcus suis serotype 2

Abstract

Streptococcus suis (S. suis) is a major pathogen in swine and poses a potential zoonotic threat, which may cause serious diseases. Many toxin-antitoxin (TA) systems have been discovered in S. suis, but their functions have not yet been fully elucidated. In this study, an auto-regulating type II TA system, ParDE, was identified in S. suis serotype 2 strain ZY05719. We constructed a mutant strain, ΔparDE, to explore its functions in bacterial virulence, various stress responses, and biofilm formation capabilities. The toxicity exerted by the toxin ParE can be neutralized by the antitoxin ParD. The β-galactosidase activity analysis indicated that ParDE has an autoregulatory function. An electrophoretic mobility shift assay (EMSA) confirmed that the antitoxin ParD bound to the promoter of ParDE as dimers. In the mouse infection model, the deletion of ParDE in ZY05719 significantly attenuated virulence. ΔparDE also exhibited a reduced anti-oxidative stress ability, and ΔparDE was more susceptible to phagocytosis and killing by macrophages. Moreover, the biofilm formation ability of the ΔparDE strain was significantly enhanced compared to ZY05719. Taken together, these findings indicate that the type II TA system ParDE plays a significant role in the pathogenesis of S. suis, providing new insights into its pathogenic mechanisms.

Introduction

Streptococcus suis is a significant zoonotic pathogen that poses a huge threat to public health security [1]. It can cause serious diseases, including septicemia, arthritis, endocarditis, and meningitis, both in swine and humans [1, 2]. Based on the difference in capsular polysaccharide antigens, S. suis is classified into 29 traditional serotypes [3] and 28 novel capsular polysaccharide loci strains [4,5,6,7]. Among the various serotypes, S. suis serotype 2 (SS2) is the most virulent and has a global distribution [8, 9], making it a significant challenge. Two outbreaks of SS2 infections in humans occurred in China in 1998 and 2005, resulting in 14 and 39 fatalities, respectively [10, 11]. Capsular polysaccharide (CPS), suilysin, and muramidase-released protein (MRP) are considered important virulence factors in S. suis [12]. Additionally, several enzymes and transcriptional regulatory factors have also been implicated in the virulence of S. suis [13]. Despite the identification of numerous virulence factors and virulence-associated proteins, the precise pathogenic mechanism of S. suis remains elusive.

Toxin-antitoxin systems are small genetic elements composed of a toxin, which exerts inhibitory effects on cells under stress conditions, and a homologous antitoxin, which neutralizes the toxin [14]. Originally discovered in E. coli as mechanisms to ensure stable inheritance of plasmids through post-segregational killing (PSK) [15], TA systems have been subsequently found to be widely distributed in bacterial chromosomes. Based on the nature of antitoxin and its mode of action with toxins, TA systems have been classified into eight types [14], with type II TA systems being the most widely distributed in bacterial genomes and possessing critical biological functions. In the type II TA system, the labile antitoxin and the stable toxin are encoded by two genes in one operon. The antitoxin typically consists of two structural domains: the DNA-binding domain, which can bind to the promoter of the TA system to achieve auto-regulation, and the toxin-binding domain, which counteracts the toxin [16]. Numerous type II TA systems have been shown to be involved in bacterial virulence [17,18,19]. Several type II TA systems are also encoded in S. suis [17, 20,21,22], but many have not been characterized, and the role of these TA systems in S. suis virulence is unclear.

In our study, an auto-regulating type II ParDE system in SS2 strain ZY05719 was investigated. We demonstrated that ParDE modulates the oxidative stress response in strains. Meanwhile, the deletion of ParDE showed reduced pathogenicity in mice. These results confirm the importance of ParDE in regulating the pathogenesis of S. suis.

Materials and methods

Bacterial strains, plasmids, cell line and growth conditions

The bacterial strains and plasmids utilized in this study are detailed in Table S1 of the supplementary materials. All the primers used in this study are listed in Table S2. All cell lines were obtained from the National Biomedical Experimental Cell Resource Bank in Beijing, China. S. suis was cultivated at 37 °C in Todd-Hewitt broth (THB, Oxoid Cheshire, United Kingdom) or on THB agar (THA). E. coli was cultured in Luria-Bertani (LB) broth or on LB agar plates at 37 °C. When necessary, spectinomycin (100 µg/mL) was added to THB or THA for mutant selection. Antibiotics (kanamycin at 50 µg/mL, or chloramphenicol at 10 µg/mL) were added to the LB medium to maintain plasmids as required. The RAW264.7 cell line was maintained at 37 °C, 5% CO₂ in DMEM culture medium (Gibco, Invitrogen) supplemented with 10% fetal bovine serum (Gibco, Invitrogen).

Construction of mutant strain

Mutants were created by employing a modified natural DNA transformation approach [23]. The upstream and downstream sequences of the target gene parDE were amplified utilizing the genomic DNA of strain ZY05719. Subsequently, the amplified upstream and downstream fragments were fused with the sacB-Spc cassette via overlap PCR. The resulting fusion fragment, along with a synthetic peptide, was subsequently introduced into 100 µL of bacteria (OD₆₀₀, 0.042), and incubated at 37 °C for 2 h in THA supplemented with spectinomycin. A negative control, utilizing the sacB gene, which is sensitive to sucrose, was included. The positive mutants were subjected to another transformation using the homologous fusion fragment without the cassette, and the transformed cells were cultured on a THB plate containing 10% (w/v) sucrose. Finally, PCR screening was performed to identify the positive mutations.

Growth curve determination

BL21 (DE3) cells were cultured in LB medium supplemented with 10 µg/mL of chloramphenicol and 50 µg/mL of kanamycin. Upon reaching an OD₆₀₀ of 0.3 ∼ 0.5, the medium was supplemented with 0.2% L-arabinose and 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The S. suis overnight culture was diluted 1:100, and samples were collected at specified time points to measure the OD₆₀₀. OD₆₀₀ measurements were taken every 2 h and recorded. For the colony-forming units (CFU) assay, BL21 (DE3) cells were cultured in LB until the OD₆₀₀ reached 0.6. The cells were diluted with phosphate-buffered saline (PBS) and plated on LB agar plates supplemented with 0.2% L-arabinose and 1 mM IPTG. The plates were incubated at 37 °C overnight, and photographs were taken the following day.

Bioinformatics analysis

The putative type II TA system in SS2 ZY05719 was predicted by TAfinder [24]. BLAST from NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to analyze amino acid sequences. Phylogenetic analyses of the antitoxin and toxin were performed using the neighbor-joining (NJ) method with 1,000 bootstrap replicates in MEGA11. The three-dimensional (3D) structure of the antitoxin was predicted using the SWISS-MODEL server [25].

Promoter activity assay

A 200 bp fragment of the ParDE promoter region was amplified from the genomic DNA of strain ZY05719 using the pTCV-Lac-200-F/R primer pair. Subsequently, this fragment was inserted into the pTCV-Lac plasmid, generating the recombinant plasmid pTCV-Lac-200. This recombinant plasmid was then introduced into both the wild-type and ΔparDE strains. The method for the assessment of β-galactosidase activity was adapted from Miller et al. with appropriate modifications [26]. The strains were cultured overnight in THB and subsequently diluted at a ratio of 1:100 into fresh THB for continued growth at 37 °C in a CO₂ incubator until reaching the logarithmic phase. A 2 mL aliquot of the bacterial culture was collected, and the cells were harvested via centrifugation and washed twice with sterile PBS. The cells were then resuspended in 200 µL of pre-chilled Z-buffer supplemented with 50 mM β-mercaptoethanol. Next, 0.1% SDS and chloroform were added, thoroughly mixed, and incubated at 30 °C in a water bath for 5 min. Following this, 100 µL of O-nitrophenyl-β-D-galactopyranoside (ONPG, 4 mg/mL) was added, mixed thoroughly, and then incubated at 30 °C until a yellow color fully developed. The reaction was halted by the addition of 250 µL of sodium carbonate, and the duration of the reaction (t) was documented. Following centrifugation of the solution, 250 µL of the resulting supernatant was transferred to a 96-well plate, and the absorbance at wavelengths of 420 nm (A₄₂₀) and 550 nm (A₅₅₀) was quantified using a microplate reader. The β-galactosidase activity was calculated using the following formula:

Activity [MU] = [1, 000 × (A₄₂₀ − 1.75 × A₅₅₀)] / [t(min) × V(ml) × OD₆₀₀]

MU is Miller units; t is reaction time; and V is volume of culture assayed in milliliters. At least three independent cultures of each strain were assayed in each experiment.

Expression and purification of antitoxin

The ParD coding sequence was amplified, digested with restriction enzymes, and ligated to the plasmid pET28a in order to create the prokaryotic expression vector pET28a-ParD. Two ParD coding sequences were connected using the linker GGGGSGGGGSGGGGS according to reference [27], and the construct was ligated to pET28a to generate pET28a-ParD^dimer. This flexible linker ensures that enough dimers are harvested and helps maintain their activity during the experiment. The N-termini of the protein was tagged with His-tag. The pET28a-ParD and pET28a-ParD^dimer plasmids were introduced into BL21 (DE3) cells, which were grown until reaching an optical density at 600 nm of 0.4–0.6. Subsequently, 0.5 mM IPTG was added, and the cells were further cultured for 16 h at 16 °C. The cells were then harvested and sonicated in lysis buffer (20 mM Na₃PO₄·12H₂O, 0.5 mM NaCl, 30 mM imidazole, pH 7.4). Purification of ParD and ParD^dimer was achieved using a His-tag Ni-NTA affinity chromatography column. Elution was performed using a step-wise gradient of imidazole concentrations ranging from 50 to 500 mM. Protein concentrations were determined using BCA protein assays (Thermo Fisher Scientific, USA).

Electrophoretic mobility shift assay

The purified protein was subjected to incubation with a probe composed of the ParDE promoter fragment for analysis via native polyacrylamide gel electrophoresis. The 200 bp probe was amplified through PCR and purified using a kit (TaKaRa). A negative-control probe was produced by amplifying the 16 S rRNA. Subsequently, the purified protein and DNA probe were mixed with a binding buffer (10 mM Tris-base, 50 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol, 0.05% Nonidet P-40, 2.5% glycerol, pH 7.5) and incubated at 37 °C for a duration of 30 min. Following this, the samples underwent electrophoresis on a 6% native polyacrylamide gel in 0.5 × TBE buffer (44.5 mM Tris-base, 44.5 mM boric acid, 1 mM EDTA, pH 7.5) at 200 V for a duration of 45 min. Subsequently, the gel was stained with ethidium bromide in 0.5 × TBE for 20 min, and images were then captured.

Mouse infection assay

BALB/c mice were divided into three groups, with each group comprising 10 mice. These mice were then challenged intraperitoneally with a dosage of 5 × 10⁸ colony-forming units (CFU) per mouse. The control group was challenged with phosphate-buffered saline (PBS). Survival of the mice was monitored over a period of seven days, and a bacterial load assay was conducted to assess in vivo proliferation capacity. Each group, comprising six mice, was administered an intraperitoneal injection of 3 × 10⁸ CFU per mouse. At 6 h post-infection, the mice were anesthetized with isoflurane and euthanized via exposure to CO₂. Blood, brains, livers, and spleens were harvested, weighed, and homogenized in PBS. CFU enumeration was conducted by plating serial 10-fold dilutions of these homogenates and blood on THB-agar medium.

Swine blood survival assay

Late logarithmic phase bacteria were harvested and washed twice with PBS. The bacterial cells were then adjusted to a concentration of 1 × 10⁸ CFU/mL using sterile PBS. Following this, 100 µL of the prepared bacterial cells were added to a blood mixture consisting of 900 µL of fresh swine blood. The viable cells were quantified at specified time intervals by plating serial dilutions onto THB agar plates and incubating them at 37 °C overnight. The experiment was repeated at least three times.

Oxidative stress assay

Bacteria were cultured until reaching the mid-logarithmic phase, followed by treatment with THB medium supplemented with either 20 or 40 mM H₂O₂, while control cells were treated with PBS. After incubating at 37 °C for 20 min, the number of viable bacteria was determined by spreading serial dilutions on THB agar plates. The survival rate was calculated as the percentage of CFU at the 20-minute time point, with the CFU at the initial time point (0 min) serving as the reference. Data were obtained from at least three independent experiments.

Phagocytosis and intracellular survival assay

The method utilized for assessing the phagocytosis and intracellular survival capacity of S. suis in RAW264.7 macrophages was adapted from a previous study [28], with appropriate modifications. RAW264.7 cells were stored in our laboratory. RAW264.7 macrophages were cultured in 24-well cell culture plates until a monolayer was formed, with approximately 7 × 10⁵ cells per well. The bacteria were grown to the logarithmic phase and then used to infect the macrophages at a multiplicity of infection (MOI) of 1. Concurrently, the remaining bacteria underwent serial dilution and were plated on THB agar plates for overnight incubation to quantify the colonies. Following an incubation period of 1 h at 37 °C in a CO₂ incubator, the supernatant was discarded, and the cells were washed three times with PBS. Next, 500 µL of Dulbecco’s Modified Eagle Medium (DMEM) containing penicillin (final concentration 5 µg/mL) and streptomycin (final concentration 100 µg/mL) was added to the wells, and the plates were then incubated at 37 °C in a CO₂ incubator for 2, 4, or 6 h. Following seven washes with PBS, 1 mL of double-distilled water (ddH₂O) was added to the wells and incubated at room temperature for 10 min. The cells were then lysed by vigorous pipetting, and an appropriate dilution of the lysate was plated on THB agar plates. The plates were then placed in a CO₂ incubator at 37 °C overnight for the purpose of colony counting. The phagocytosis rate was calculated as CFUs at 2 h / CFUs in the original bacterial suspension × 100%. The intracellular survival rate was determined as CFUs at 4–6 h / CFUs at 2 h × 100%. 2 h after the addition of antibiotics was used as the starting point for survival analysis (0 h). Data were obtained from at least three independent experiments.

Antimicrobial susceptibility assay

The minimum inhibitory concentrations (MICs) of antibiotics against bacteria were determined in accordance with the guidelines set forth by the Clinical and Laboratory Standards Institute [29]. Bacterial strains were diluted 1,000-fold in THB medium, and 180 µL of the diluted culture was aliquoted into the initial well of a 96-well microtiter plate, with each subsequent well receiving 100 µL. Subsequently, 20 µL of the respective antibiotic was added to the first well, mixed thoroughly, and 100 µL of the resulting mixture was transferred to the following well. This process was repeated until the 10th well, with the 11th well designated as the positive control and the 12th well as the negative control. The 96-well plate was incubated at 37 °C for 20 h, and the results were recorded. Each experimental procedure was independently repeated three times.

Biofilm formation assay

The bacterial cultures were cultivated until achieving an optical density at OD₆₀₀ of 0.6, followed by a 1:100 dilution and inoculation into separate wells of a 96-well plate. The plate was incubated at 37 °C for 24, 48, and 72 h, with THB medium used as the control. After incubation, the medium was carefully removed, and the cells were gently washed twice with PBS to eliminate any unattached bacteria. The samples were subsequently fixed with methanol for 30 min, after which the fixation solution was discarded. The samples were air-dried at room temperature, and the biofilm was stained using 0.1% crystal violet for 30 min. After staining, the samples were washed with tap water, air-dried, and treated with 33% acetic acid. The mixture was placed on a shaker operating at 80 rpm for 30 min to facilitate the release of crystal violet. Following this, the absorbance was measured at 595 nm. Each strain was subjected to ten separate cultures for analysis.

Ethics statement

Female SPF (specific pathogen-free) BALB/c mice were purchased from the Comparative Medicine Centre of Yangzhou University (Yangzhou, China). Animal experiments were conducted at the Animal Center Laboratory of Nanjing Agricultural University and were approved by the Jiangsu Provincial Laboratory Animal Monitoring Committee (license no. SYXK(SU) 2021-0086), and were approved by the Animal Ethics Committee of Nanjing Agricultural University.

Statistical analyses

All experiments were carried out with a minimum of three repetitions to ensure reproducibility. GraphPad Prism version 8 was utilized for data analysis and creating graphical representations. The statistical significance threshold was set at P < 0.05. The data were subjected to analysis using either an unpaired two-tailed t-test or a log-rank (Mantel-Cox) test.

Results

Identification and phylogenetic analysis of ParDE in S. suis

Previous research has identified nine type II TA systems present in the genome of S. suis serotype 2 strain ZY05719 [22]. Subsequent alignment analysis revealed that ZY05719_09530 encodes the toxin protein ParE, while ZY05719_09535 encodes the antitoxin protein ParD, forming the type II ParDE system. To determine the toxicity of the toxin and whether the antitoxin can alleviate it, the two genes, parD and parE, were cloned into the plasmids pET28a and pBAD33, respectively. Recombinant plasmids or blank vectors were co-transformed into E. coli BL21 (DE3), and the cell’s growth was monitored. The results showed that cells harboring pET28 and pBAD33-parE plasmids exhibited growth inhibition after the addition of inducers L-arabinose and IPTG, while cells harboring pET28-parD and pBAD33-parE did not show significant differences in growth compared to the blank vector group (Fig. 1A). Furthermore, co-transformed cells were diluted and plated on agar plates supplemented with inducers, revealing that the pET28/pBAD33-parE group displayed no bacterial colony growth, whereas the pET28-parD/pBAD33-parE group exhibited normal growth (Fig. 1B). These findings indicate the toxicity of ParE and the neutralization effect of ParD. To understand the distribution of ParDE in Gram-positive bacteria, we conducted blast searches in the NCBI database and performed phylogenetic analyses based on the amino acid sequences of ParE and ParD from strain ZY05719. The ParD homologs were divided into five groups, and the predominant bacteria in groups II, III, IV, and V were all streptococci (Fig. 1C). Meanwhile, the ParE homologs were divided into six groups, and the predominant bacteria in the largest groups, VI and V, were both streptococci (Fig. 1D). These results indicate that ParDE is widely distributed in streptococci, suggesting that ParDE may play an important biological function in streptococci.

Fig. 1

Identification and phylogenetic analyses of type II TA system ParDE in S. suis. (A) The growth curve analyses of BL21 (DE3) harboring distinct plasmids. 0.2% L-arabinose and 1 mM IPTG were added when cells OD₆₀₀ reached 0.3 ∼ 0.5. The OD₆₀₀ was determined every 2 h. (B) BL21 (DE3) cells were cultured until the OD₆₀₀ reached 0.6, serially diluted, and plated on LB agar plates containing 0.2% L-arabinose and 1 mM IPTG. The original blot is presented in Supplementary material 3. The phylogenetic analyses of antitoxin ParD (C) or toxin ParE (D) were conducted by neighbor-joining, with 1, 000 bootstrap replicates using MEGA11

Identification of auto-regulation in ParDE

Given that type II toxin-antitoxin systems typically possess auto-regulatory capabilities [14], we verified whether ParDE has an auto-regulatory effect. A modified natural DNA transformation approach was employed to construct ΔparDE (Supplementary Fig. 1). The original gel is presented in Supplementary material 3. Analysis of β-galactosidase activity revealed a marked increase in the deletion strains as compared to the wild-type, indicating the inhibitory role of ParDE on its promoter (Fig. 2A). The SWISS-MODEL protein homology-modeling server was utilized to predict the tertiary structure of ParD, revealing that its N-terminus adopts a ribbon-helix-helix (RHH) structural domain. The structural comparison revealed that this domain overlaps almost completely with the N-terminal DNA-binding domain of the E. coli antitoxin DinJ, which has been resolved in the crystal structure [30], suggesting that the N-terminal RHH of ParD may have a similar DNA-binding function (Fig. 2B). In order to confirm the direct interaction of ParD and the ParDE promoter, ParD was expressed and purified. SDS-PAGE analysis showed that the ParD protein was successfully expressed and purified with the expected size of 10.9 kDa (Fig. 2C). The electrophoretic mobility shift assay (EMSA) was conducted with purified ParD and the ParDE promoter sequence probe. However, it displayed that ParD failed to bind to the ParD promoter (Fig. 2D). Previous studies have indicated that ParD forms dimers in both E. coli and M. opportunistum [30, 31]. We therefore tested whether ParD^dimer can bind directly to the ParDE promoter. Using the peptide linker sequence to ligate between the two same amino acid sequences of ParD to obtain ParD homodimer, we expressed and purified ParD^dimer (Fig. 2E). As expected, the EMSA result indicated that ParD^dimer specifically bound to ParDE promoter (Fig. 2F). These findings suggest that ParD binds to the promoter of ParDE as dimers, thereby exerting auto-regulatory functions.

Fig. 2

Characterization of auto-regulation of ParDE. (A) The ZY05719 and ΔparDE strains were transformed with the pTCV-Lac reporter plasmid (pTCV-lac-200) harboring the ParDE promoter sequence to analyze β-galactosidase activity. An unpaired two-tailed t-test was used to analyze the data: ∗∗∗ P < 0.001. (B) The tertiary structure of antitoxin ParD was predicted by the SWISS-MODEL protein homology-modeling server and aligned with E. coli DinJ (4q2u. 1. A). ParD (C) or ParD^dimer (E) were purified using HisTrap affinity columns. The EMSA showed that ParD did not exhibit binding affinity towards the ParDE promoter (D), whereas ParD^dimer bound to the ParDE promoter (F). DNA probe containing the ParDE promoter region was used at 60 ng per reaction mixture. Fragment amplified from 16 S rRNA served as a negative control. The original gels are presented in Supplementary material 3

ParDE system is required for the virulence of ZY05719

Multiple TA systems have been demonstrated to play a role in bacterial virulence [19, 21, 22]. To determine the role of ParDE in S. suis virulence, BALB/c mice were divided into three groups, infected with the wild-type and ΔparDE strains, respectively, injected with PBS as control, and their survival was monitored for seven days. All mice challenged by the wild-type strains died within one day post-infection, while those challenged by ΔparDE conferred a significant survival advantage with a survival rate of 100% at seven days post-infection (Fig. 3A). To exclude the effect of different growth rates of the wild-type and ΔparDE strains, we determined the growth curves and the result showed no difference between the wild-type and ΔparDE strains (Fig. 3B). To further confirm the decreased virulence of ΔparDE, we compared the in vivo bacterial load of the wild-type and ΔparDE strains in mice 6 h post-infection. As the results showed in Fig. 3C-F, the bacterial abundances of ΔparDE in blood, spleen, liver, and brain were significantly reduced compared to the wild-type strains, suggesting that ParDE contributes to the colonization of ZY05719 in blood and organs. These results demonstrate that the deletion of ParDE in ZY05719 largely attenuates its pathogenicity.

Fig. 3

ParDE facilitates to S. suis virulence. (A) BALB/c mice were divided into three groups, infected with the wild-type and ΔparDE strains at a dosage of 5 × 10⁸ CFU/mouse intraperitoneally, respectively, and challenged with PBS as a control. Their survival was continuously monitored for 7 days. The survival data were analyzed by the log-rank (Mantel-Cox) test. (B) The growth curves of the wild-type and ΔparDE strains were determined to ensure their same growth rate. After 6 h of infection, the in vivo bacterial abundance of the wild-type and ΔparDE strains in mice blood (C), spleen (D), liver (E), and brain (F) was enumerated (n = 6 mice per group). The y-axis is in Log10 scale. An unpaired two-tailed t-test was used to analyze the data: ∗∗∗ P < 0.001; ∗∗∗∗ P < 0.0001

ParDE facilitates ZY05719 resistance to oxidative stress

The oxidative stress response serves as a host defense mechanism against pathogen invasion, while bacteria counteract this stress through strategies such as reduction systems and scavenging enzymes [32, 33]. As the ParDE TA system has been reported to participate in oxidative stress alleviation in Mycobacterium tuberculosis [34], we wondered if it has a similar role in S. suis. In the bloodstream, substances such as reactive oxygen species (ROS), complement, and antimicrobial peptides possess bactericidal properties [35]. To determine the role of S. suis ParDE in escaping the bactericidal effects of blood, the prepared late log-phase wild-type and ΔparDE strains were added to swine blood and incubated, and the viable cells were counted at the indicated times to calculate the survival rate. The result displayed that after incubation for 1.5–3 h, the survival of ΔparDE in swine blood was 10.17% and 2.8%, respectively, which was significantly reduced compared to the wild-type strains (Fig. 4A). To further ascertain the sensitivity of wild-type and ΔparDE strains to oxidative stress, survival assays were conducted at different H₂O₂ concentrations, and it was observed that the survival rates of ΔparDE were 53.91% and 7.45% at concentrations of 20 mM and 40 mM H₂O₂, respectively, the viability of ΔparDE was substantially reduced compared to the wild-type strains (Fig. 4B). Furthermore, macrophages can eradicate invading bacteria by phagocytosis along with the generation of antimicrobial agents, such as ROS and lactoferrin [36]. We, therefore, tested the ability of the wild-type and ΔparDE strains to resist macrophage phagocytosis and their survival in macrophages. As indicated in Fig. 4C and D, ΔparDE was more easily phagocytosed and killed by RAW264.7, suggesting the facilitation of S. suis anti-phagocytosis and intracellular survival in macrophages by ParDE. These findings imply the crucial role of ParDE in S. suis tolerance to oxidative stress.

Fig. 4

ParDE contributes to tolerance to oxidative stress. (A) Survival rates of the wild-type and ΔparDE strains were measured after incubation in swine blood for 1.5 h and 3 h. (B) Survival assays of the wild-type and ΔparDE strains were conducted after 20 min at 20 or 40 mM H₂O₂ concentrations. The control group was treated with PBS. The effect of ParDE deletion on the ability of ZY05719 to resist phagocytosis (C) and its intracellular survival capacity (D) in RAW264.7 macrophages was investigated. After 1 h of incubation, the cells were washed and incubated in DMEM containing antibiotics for another 2 h, 4 h, or 6 h. The cells were then washed again and lysed to determine CFU numbers. An unpaired two-tailed t-test was used to analyze the data: ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗∗ P < 0.0001

The type II ParDE system is involved in biofilm formation

As the ParDE system has been identified to be involved in bacterial biofilm formation and antibiotic resistance [37, 38], we investigate these characteristics in S. suis ParDE. The results revealed that deletion of ParDE led to increased biofilm formation (Fig. 5A and B), confirming a role for ParDE similar to that of ParDE in Caulobacter crescentus [38]. For the comparison of the differences in the architecture and composition of the biofilm between ZY05719 and ΔparDE, further observation and analysis by confocal microscopy and scanning electron microscopy are required. In addition, we determined the sensitivity of the wild-type and ΔparDE strains to different types of antibiotics. The determination of MIC values indicated that ParDE does not affect the susceptibility of strains to antibiotics (Table 1). These results suggest that ParDE in S. suis plays an important role in biofilm formation.

Fig. 5

ParDE affects S. suis biofilm formation. Bacterial cultures of ZY05719 and ΔparDE were diluted 1:100 and inoculated into individual wells of a 96-well plate for 24, 48, and 72 h. The biofilm was stained using crystal violet (A) and the absorbance at 595 nm was determined (B). An unpaired two-tailed t-test was used to analyze the data: ∗∗∗∗ P < 0.0001

Table 1 The MICs of ZY05719 and ΔparDE to antibiotics

Discussion

As an important genetic element, TA systems are widely distributed in bacterial genomes, displaying various functions. In recent years, novel TA systems have been continuously discovered in pathogenic bacteria. In Pseudomonas species, the PacTA system exhibits a broad distribution and enhances bacterial pathogenicity through the regulation of iron acquisition [19]. In E. coli DarTG, the resistance against phage infection is achieved by the toxin DarT, which blocks the production of mature virions through the modification of viral DNA [39]. Mycobacterium abscessus VapC5 toxin activates multiple resistance pathways and contributes to persisters formation [40]. Investigating the structural and functional characteristics of TA systems provides novel insights into the pathogenicity and antibiotic resistance traits of bacteria. Several TA systems have been characterized in S. suis, a pathogen that poses a significant threat to public health security. Although the activity of ParDE in S. suis SC84 has been identified [41], little is known about its biological roles. In this study, we identified an auto-regulating ParDE system in SS2 strain ZY05719 and demonstrated its important role in S. suis virulence, oxidative stress response, and biofilm formation.

The tight regulation of TA systems enables the maintenance of a neutralized state and facilitates the rapid release of a substantial amount of toxin when bacteria are under stress. The regulation of type II TA systems is predominantly achieved at the transcriptional level [14]. Antitoxin of type II TA system consists of a structured DNA-binding domain that specifically recognizes and binds to the TA system promoter, thus achieving auto-regulation of the TA system [42]. In Staphylococcus aureus, the antitoxin SavR can bind to the palindrome sequence within the promoter region, leading to transcriptional repression, and the formation of toxin-antitoxin complex enhances this binding interaction [18]. Both the antitoxin HicB and the toxin-antitoxin complex HicBA in Streptococcus pneumoniae can bind to promoter DNA, and the ribbon-helix-helix motif at the C-terminus of HicB is responsible for the DNA binding interaction [43]. Our result demonstrated the negative regulatory effects of ParDE on its promoter, which is consistent with previous research in SS2 strain SC84 [41]. In the ParDE system, the N-terminus of ParD typically exhibits a ribbon-helix-helix (RHH) DNA binding domain, while the intrinsically disordered C-terminus domain interacts with ParE [31], in accordance with the predicted tertiary structure of S. suis ParD. Transcription of the Vibrio cholerae parDE2 operon is contingent upon the binding ratio of antitoxin ParD2 and toxin ParE2 [44]. The complex with three interacting ParD2 dimers bridged by two ParE2 can block the operator, while when a ParD2 dimer forms a heterotetramer with two ParE2, it fails to bind to the operator due to steric hindrance. The coexistence of heterotetrameric and heterohexameric complexes of ParDE1 in a dynamic equilibrium in solution in M. tuberculosis has been demonstrated [45]. These findings suggest that the various conformations of ParDE may impact its ability to bind to the promoter. In our research, the EMSA results confirmed that ParD binds directly to the promoter as dimers in S. suis. However, the precise structure of ParD and its specific binding site on the promoter DNA sequence remains elusive. Furthermore, a comprehensive understanding of the structure of the ParDE complex and its role in transcriptional regulation in this TA system necessitates further investigation.

As a zoonotic pathogen, the pathogenesis of S. suis has been extensively investigated. A serine-rich repeat (SRR) glycoprotein, SssP1, enhances adhesion to and penetration of the blood-brain barrier, contributing to S. suis meningitis [46]. Ornithine carbamoyltransferase (OTC) has been reported to be involved in S. suis virulence, which can promote adhesion to human laryngeal epidermoid carcinoma (HEp-2) cells of strains [47]. Previous studies have established a correlation between TA systems and virulence in S. suis [17, 22]. Specifically, in the type II TA system, the antitoxin functions as a transcriptional regulatory factor, facilitating both self-regulation and the ability to influence other co-transcribed genes, thereby controlling bacterial characteristics. In the construction of C∆parDE, we found that the complementary strain exhibited significant growth defects compared to ZY05719, which may be due to an imbalance in the expression of ParDE and its regulatory network. Type II TA system Xress-MNTss modulates the downstream streptomycin resistance gene via auto-regulation [22]. In this study, we observed that ParDE contributes to S. suis virulence in mice, promoting bacterial colonization in blood and organs, while ParDE negatively regulates biofilm formation. Similarly, deletion of gdpP in S. suis type 2 strains also resulted in a significant decrease in virulence and an increase in biofilm formation, and the decrease in virulence in gdpP mutant strains was associated with a significant down-regulation of the expression of several virulence genes [48]. The presence of self-regulation of ParDE suggests that ParDE may affect virulence by regulating virulence-related genes while regulating other genes to affect biofilm formation. However, the specific targets of ParDE action require further validation.

ParE toxin has been identified as a DNA gyrase inhibitor in various bacterial species, including M. tuberculosis, Pseudomonas aeruginosa, and Enterobacteriaceae [37, 45, 49]. DNA gyrase, a significant drug target in bacteria, is involved in maintaining the topological structure of DNA and altering supercoiling [50]. When ParE is expressed in a ParD-free background, it exhibits a bactericidal effect similar to that of ciprofloxacin [51]. However, ParE mediated a protective effect when bacteria were exposed to gyrase inhibitors such as quinolones, possibly as a result of the interaction of both ParE and antibiotics with DNA gyrase [49]. These results show that ParE toxin can exhibit either protective or toxic effects, contingent upon its concentration. ParE has been demonstrated to participate in bacterial tolerance to oxidative stress [34]. In this study, deletion of ParDE in S. suis resulted in reduced bacterial tolerance to H₂O₂ and more sensitivity to phagocytosis and killing by macrophages. Nevertheless, additional research is required to determine whether this effect is mediated by ParD or ParE and to elucidate its underlying mechanism. The mechanism of S. suis anti-phagocytosis has been extensively studied, in which capsular polysaccharide [52], HP0487 [53], two-component signaling systems [54, 55], sRNA [56], enolase [57], and the orphan response regulator CovR [58] play crucial roles in the anti-phagocytosis of S. suis. However, the role of the toxin-antitoxin system in the anti-phagocytosis of S. suis has not been reported. The self-regulatory ParDE system may affect the antiphagocytic ability of S. suis by regulating some important biological processes, and its specific mechanism of action needs to be further investigated. The enhanced biofilm formation was determined in S. suis ΔparDE, which is in contrast to the findings in E. coli [37], suggesting a new mechanism by which ParDE affects bacterial biofilm formation.

Based on these findings, we characterized the role of the ParDE TA system in S. suis. Toxin ParE exerts a toxic effect on E. coli BL21 (DE3), which can be alleviated by the antitoxin ParD. Moreover, ParDE is identified to be auto-regulating, with antitoxin ParD directly binding to ParDE promoter as dimers. ParDE enhances pathogenicity and resistance to oxidative stress in S. suis. In addition, the deletion of ParDE results in enhanced biofilm formation capability. These findings contribute to a deeper understanding of the mechanisms underlying the pathogenesis of S. suis, providing new insights into the prevention and control of S. suis disease.