A novel aquaporin Aagp contributes to Streptococcus suis H₂O₂ efflux and virulence

ABSTRACT

Streptococcus suis is a bacterium that can cause infections in pigs and humans. Although oxidative stress is common occurrence during bacterial growth and infection, the regulation networks of S. suis under oxidative stress remain poorly understood. To address this, we utilized RNA-Seq to reveal the transcriptional landscape of S. suis in response to H₂O₂ stress. We identified novel genes responsible for S. suis resistance to oxidative stress, including those involved in DNA repair or protection, and essential for the biosynthesis of amino acids and nucleic acids. In addition, we found that a novel aquaporin, Aagp, belonging to atypical aquaglyceroporins and widely distributed in diverse S. suis serotypes, plays a crucial role during H₂O₂ stress. By performing oxidative stress assays and measuring the intracellular H₂O₂ concentrations of the wild-type strain and Aagp mutants during H₂O₂ stress, we found that Aagp facilitated H₂O₂ efflux. Additionally, we found that Aagp might be involved in glycerol transport, as shown by the growth inhibition and H₂O₂ production in the presence of glycerol. Mice infection experiments indicated that Aagp contributed to S. suis virulence. This study contributes to understanding the mechanism of S. suis oxidative stress response, S. suis pathogenesis, and the function of aquaporins in prokaryotes.

KEYWORDS:

• Streptococcus suis
• oxidative stress
• aquaporin
• virulence
• glycerol transport

Introduction

Streptococcus suis can cause systemic diseases such as septicaemia and meningitis in pigs. It is also considered as a zoonotic pathogen for humans in close contact with infected pigs or contaminated by-products [1–3]. During the growth and infection process, S. suis may encounter oxidative stress. Reactive oxygen species (ROS) like hydroxyl radicals (HO•), hydrogen peroxide (H₂O₂), and superoxide anion (O₂⁻) are generated during many cellular activities [4]. O₂⁻ and H₂O₂ result from one-electron and two-electron reduction of O₂, respectively [5,6]. H₂O₂ can oxidize Fe²⁺ in ferritin, leading to inactivation of the enzymes and releasing Fe³⁺, and then Fe³⁺ is reduced to Fe²⁺ in the intracellular environment [5]. When Fe²⁺ reacts with H₂O₂, Fenton chemistry is occurred and generates highly-reactive HO• [7]. O₂⁻, H₂O₂, and HO• can cause cell damage by oxidizing amino acids, DNA, and lipids [5,8]. When bacteria invade the host, innate immune cells, including neutrophils, monocytes, and macrophages, are responsible for ROS production, which is essential to eliminate bacteria [9]. In addition, ROS participates as a signal molecular in activating various immune mechanisms [10]. In order to defend against oxidative stress, S. suis has developed various mechanisms that are regulated by transcriptional regulators, including PerR, Rex, SpxA1, FlpS, and SrtR, which target genes encoding superoxide dismutase, catalase, thioredoxin, glutathione reductase, iron uptake (Fur), and ferritin [11–15]. However, since antioxidant defence systems involve comprehensive and complex processes, studies focusing solely on individual regulators cannot fully reveal regulation networks under oxidative stress.

In this study, we performed RNA-sequencing (RNA-Seq) analysis to explore the global oxidative stress response of S. suis. Our findings revealed a comprehensive defence network in response to H₂O₂ stress, and we identified a novel aquaporin, Aagp, that plays a crucial role in facilitating S. suis H₂O₂ efflux and contributes to virulence.

Materials and methods

Bacterial strains and culture conditions

Supplementary Table S1 lists S. suis strains and plasmids. S. suis serotype 9 virulent strain GZ0565 was isolated from a meningitis pig [16]. S. suis strains were cultured in Todd-Hewitt broth (THB, Hopebio, Qingdao, China) and plated on an agar (THA) medium containing 6% (vol/vol) sheep blood at 37 °C with 5% CO₂. Escherichia coli strains were grown in Luria-Bertani (LB, Becton Dickinson, USA) broth at 37 °C. Antibiotics were added when in need as follows: spectinomycin (Spc, Macklin, China), 50 μg/mL for E. coli, and 100 μg/mL for S. suis, 10% (w/v) sucrose (sucrose, Macklin, China) for S. suis.

For studying glycerol utilization, S. suis strains were grown in THB to mid-exponential phase (OD₆₀₀ = 0.6), washed twice in PBS, and then transferred (with a dilution of 1:100) to a 50 mL flask with 10 mL of 1/3 diluted THB or 1/3 diluted THB containing glycerol with a final concentration of 10 mM or 100 mM. The growth rates were measured at OD₆₀₀ at 37 °C in a shaking incubator.

RNA-Seq analysis

To serve as a control group, S. suis strain GZ0565 was grown in THB to reach the mid-exponential phase. For H₂O₂ treatment group, when the bacteria had reached the mid-exponential phase, a final concentration of 25 mM H₂O₂ was added to THB medium and incubated for 25 min. After incubation, the cultures were centrifuged at 5000×g for 10 min at 4 °C, and the bacterial pellets were collected. The total RNA was extracted using the FastRNA Pro Blue Kit (MP Biomedicals) according to our previous report [17]. The extracted RNA samples were submitted to Realbio Technology (Shanghai, China) for RNA-seq analysis. Each experimental group was performed in duplicate. The library construction for RNA-seq was described in our previous study [18]. Sequencing was carried out using Illumina HiSeq 2500 (Illumina, USA) following the manufacturer’s protocol. The sequencing reads were aligned by Bowtie2 [19]. The gene expression was calculated by FPKM (Fragments Per Kilobase of transcript per Million mapped reads), and differential expression analysis was performed using edgeR [20]. The threshold of p < 0.01 and the absolute value of Fold change ≥ 2 were used to identify the differentially expressed genes.

Construction mutant strains

The detailed protocol for deleting Aagp (BFP66_RS01250) in strain GZ0565 background using S. suis-E. coli shuttle plasmid pSET4s was described in our previous study [21]. In addition, we created a mutation strain of Aagp in strain GZ0565 background (Aagp^mut) by inserting single “G” base into its coding sequence (CDS), causing a frameshift that rendered Aagp non-functional. This mutation strain was constructed through a two-step natural transformation [22]. Step I was performed to replace the upstream sequence of Aagp with SacB-Spc cassette and resulted in sucrose sensitive and spectinomycin resistant; step II was the process of cassette replacement for single base insertion via the negative selection on sucrose THA plate (Supplementary Figure S1). We also employed the same methodology to construct deletions of exodeoxyribonuclease III (exo III, BFP66_RS05940), AguB (BFP66_RS06520), metQ (BFP66_RS08225) in strain GZ0565 background, as well as Aagp in serotype 2 strain P1/7 background (Aagp_P1/7). Supplementary Table S2 contains a list of primers used for constructing all these strains.

Oxidative stress assays

To assess the role of the Aagp in oxidative stress response, S. suis strains were challenged with H₂O₂. Wild-type strain GZ0565 (WT), ΔAagp, and Aagp^mut were cultured to the mid-exponential phase. Then the final concentration of 25 mM H₂O₂ was added to THB. After incubation at 37 °C for 25 min, the number of bacteria was determined by spreading serial dilutions on THA plates. The survival rate at each time point was calculated as CFU at time point 25 min/CFU at time point 0. The functions of exo III, AguB, metQ and Aagp_P1/7 in response to oxidative stress were evaluated using the same methodology. Experiments were performed with three biological replicates. The statistical analysis was performed with a two-tailed unpaired t test.

To further assess the susceptibility of WT, ΔAagp, and Aagp^mut to H₂O₂, the Oxford cup supplemented with 10 µL 1 M H₂O₂ was conducted on the THA plates. After incubation at 37 °C with 5% CO₂ for 24 h, a transparent circle formed, indicating an inhibition zone.

Measurement of H₂O₂ in cells

After treated with 25 mM H₂O₂ for 25 min, one mL bacterial cultures were centrifuged at 5000 × g at 4 °C for 10 min and subsequently washed once with PBS and resuspended in 1 mL PBS. According to the manufacturer’s protocol, the concentration of H₂O₂ in cells was measured using the Pierce Quantitative Peroxide Assay Kits (Thermo Fisher Scientific, Shanghai, China). Experiments were performed with two biological replicates and repeated two times. A two-tailed unpaired t test was used for statistical analysis.

Determination of viable bacteria in organs

The virulence of WT and ΔAagp was assessed in mice according to our previous report [23]. Mice infection was carried out in the Laboratory Animal Center of Nanjing Agricultural University with the approval of the institution’s ethics committee (Permit number SYXK (Su) 2021–0086). Bacteria were cultured to the mid-exponential phase and washed twice with PBS. Six-week SPF CD1 female mice (SiPeiFu Biotechnology Co., Ltd, China) were used for infection (five mice per group). The mice were intraperitoneally injected with a dose of 1.5 × 10⁸ CFU of the WT or ΔAagp. All mice were euthanized at 24 h post-infection. Blood samples were collected from the heart, and liver, kidney, and brain samples were taken, weighed, suspended in PBS, and homogenized. Plating serial dilutions on THA determined the number of viable bacteria in organs and blood. Experimental results were shown as mean ± standard error of the mean (SEM). The statistical analysis was performed with a two-tailed unpaired t test.

RNA extraction and quantitative real-time PCR (RT-qPCR)

H₂O₂ treatment and RNA extraction were performed as described above for RNA-Seq analysis. The detailed protocol for RT-qPCR was described previously [18]. Supplementary Table 2 lists primers for RT-qPCR analysis. The gene BFP66_RS5620 (ParC) was used as the internal control. Three biological replicates were used. The 2^−ΔΔCT method was used to calculate the relative fold change. Data were shown as mean ±SEM.

Results

The transcriptional landscape of S. suis oxidative response

The RNA-Seq was used to explore the transcriptional landscape of S. suis in response to H₂O₂. Compared with the control condition in the THB medium, 371 differently expressed genes (DEGs) were observed in the H₂O₂ treatment condition, consisting of 112 upregulated and 259 downregulated genes (Supplementary Table 3). The putative functions of these genes were classified into different functional categories according to the gene ontology (GO) (Supplementary Figure S2A). The DEGs are involved in various biological processes, such as biosynthesis of amid acids, carbohydrate transport and metabolism, metal ion transport, and transcriptional regulation.

As shown in Figure 1a, we found that genes involved in DNA repair or protection were upregulated under H₂O₂ stress, including BFP66_RS05940 encoding an exodeoxyribonuclease III (exo III) and BFP66_RS06520 encoding an N-carbamoylputrescine amidase (AguB). The gene expression of enzymes encoded by BFP66_RS01580, BFP66_RS05715, BFP66_RS06935, BFP66_RS07745, BFP66_RS04160 (LDH) was upregulated under H₂O₂ condition, and these enzymes contribute to producing oxidized dinucleotide cofactors (NAD⁺, FAD⁺, and NADP⁺) which are essential for the biosynthesis of amino acids and nucleic acids. As shown in Table 1 and Figure 1, six genes involved in glycolysis were upregulated under H₂O₂ condition, contributing to producing more ATP. In contrast, several genes related to energy-consuming pathways were downregulated, including six genes related to sugar ABC transporters, 13 related to the PTS system, and seven related to glycerol metabolism. RT-qPCR was performed to verify the transcriptional data. Eight upregulated and four downregulated genes were selected, and their expressions were consistent with those obtained from the RNA-seq (Supplementary Figure S2B).

Figure 1. The transcriptional landscape of S. suis oxidative response. (a) RNA-Seq analysis was performed to explore the global H₂O₂ stress response of S. suis. The upregulated genes are shown in green, and the downregulated genes are shown in red. The detailed information and fold change of genes shown in this figure are summarized in table 1. (b) The survival rate of WT, ΔAagp, Δexo III, ΔAguB, and ΔmetQ was determined after 25 min treated with 25 mM H₂O₂. The statistical analyses were performed with a two-tailed unpaired t test. “****” indicate p < 0.001.

Table 1. The key differentially expressed genes under H₂O₂ stress^a.

ID	Fold Change (H2O2/THB)	Product	Function
BFP66_RS01250	3.66	aquaporin Aagp;	H2O2 efflux
BFP66_RS01545	−3.62	transcriptional regulator PerR;	oxidative stress regulation
BFP66_RS09925	−1.93	transcriptional regulator mntR;	oxidative stress regulation
BFP66_RS08225	1.35	methionine ABC transporter, MetQ	methionine transporter, converting H2O2 to H2O
BFP66_RS06520	2.01	N-carbamoylputrescine amidase;	converting agmatine to putrescine, which protects DNA from damage
BFP66_RS05940	2.89	exodeoxyribonuclease III;	DNA repair
BFP66_RS06160	2.19	N-acetyl-beta-hexosaminidase;	producing sugar by cell wall hydrolyzation
BFP66_RS04160	2.61	L-lactate dehydrogenase LDH;	generating NAD^+/FAD^+/NADP^+ for biosynthesis of amino acids and nucleic acids
BFP66_RS01580	2.02	NADP-dependent oxidoreductase;
BFP66_RS05715	2.30	NADH oxidase;
BFP66_RS07745	2.09	nicotinate phosphoribosyltransferase;
BFP66_RS06935	2.21	flavodoxin;
BFP66_RS01665	3.07	fructose-bisphosphate aldolase;	glycolysis
BFP66_RS07495	2.61	2,3-bisphosphoglycerate-dependent phosphoglycerate mutase;
BFP66_RS06470	2.19	pyruvate kinase;
BFP66_RS06865	2.36	enolase;
BFP66_RS02590	2.86	triose-phosphate isomerase;
BFP66_RS00790	2.16	type I glyceraldehyde-3-phosphatedehydrogenase;
BFP66_RS00845	−2.35	sugar ABC transporter substrate-binding protein;	sugar ABC transporters
BFP66_RS00850	−2.22	sugar ABC transporter permease;
BFP66_RS02995	−8.65	sugar ABC transporter permease;
BFP66_RS07040	−15.41	sugar ABC transporter permease;
BFP66_RS07045	−10.83	sugar ABC transporter substrate-binding protein;
BFP66_RS08960	−2.20	sugar ABC transporter substrate-binding protein;
BFP66_RS00910	−7.00	PTS sugar transporter subunit IIC;	PTS system
BFP66_RS02210	−3.46	PTS system mannose/fructose/N-acetylgalactosamine-transporter subunit IIB;
BFP66_RS02215	−1.90	PTS mannose/fructose/sorbose/N-acetylgalactosamine transporter subunit IIC;
BFP66_RS02695	−6.00	PTS glucose transporter subunit IIBC;
BFP66_RS03575	−14.07	PTS N-acetylgalactosamine transporter subunit IIA;
BFP66_RS04315	−3.47	PTS lactose/cellobiose transporter subunit IIA;
BFP66_RS04320	−3.65	PTS lactose transporter subunits IICB;
BFP66_RS08510	−10.66	PTS cellobiose transporter subunit IIC;
BFP66_RS08530	−53.00	PTS cellbiose transporter subunit IIC;
BFP66_RS08570	−5.96	PTS beta-glucoside transporter subunit EIIBCA;
BFP66_RS09840	−2.24	PTS sugar transporter subunit IIC;
BFP66_RS09850	−4.61	PTS lactose/cellobiose transporter subunit IIA;
BFP66_RS09855	−3.09	PTS sugar transporter subunit IIB;
BFP66_RS05750	−3.95	aquaporin family protein GlpF;	glycerol metabolism
BFP66_RS05760	−1.66	glycerol kinase GlpK;
BFP66_RS05755	−3.12	type 1 glycerol-3-phosphate oxidase GlpO;
BFP66_RS05785	−12.96	glycerol dehydrogenase GldA;
BFP66_RS05790	−51.82	acetaldehyde/alcohol dehydrogenase;
BFP66_RS05800	−2.54	Formate C-acetyltransferase/glycerol dehydratase family glycyl radical enzyme;
BFP66_RS09160	−2.28	dihydroxyacetone kinase transcriptional activator DhaS;

Note: ^a The threshold of p < 0.01 was used to identify the differentially expressed genes.

To further investigate the response of S. suis to H₂O₂ at the transcriptional level, we selected four DEGs, namely exo III, AguB, metQ (encoding a methionine transporter) and Aagp (encoding an aquaporin), to examine their function under oxidative stress conditions. After subjecting the deletion mutations to H₂O₂ treatment, the survival rates of mutants ΔAagp, Δexo III, ΔAguB, and ΔmetQ were 0.7075%, 0.0063%, 0.69%, and 9.1867%, respectively, which were significantly lower than that of wild-type (WT) strain (59.3133%) (Figure 1b). These findings suggest that these genes are involved in oxidative stress responses and support the transcriptional landscape data of S. suis in response to H₂O₂.

Aagp, a novel aquaporin, is widely distributed in S. suis

Out of the four genes associated with oxidative stress responses, Aagp showed the highest fold change (Table 1), making it the chosen candidate for further investigation. Aquaporins contain an aromatic/arginine (ar/R) substrate selectivity motif that comprises one arginine and three other amino acids, and these three amino acids are conserved in every aquaporin subfamily [24]. In S. pneumoniae, a new aquaporin subfamily called atypical aquaglyceroporin has YVPR as the substrate selectivity motif, which is different from glycerol-transporting aquaporins with WG(F/Y)R as the motif and water-transporting aquaporins with F(H/I)XR as the motif (Figure 2). S. suis aquaporin Aagp shares 68.17% amino acid identity with S. pneumoniae atypical aquaglyceroporin Pn-AqpC [25], belonging to a novel aquaporin subfamily. Aagp was predicted to be a transmembrane protein with five transmembrane loops, and it possesses substrate-selective residues YVPR as Pn-AqpC (Figure 2).

Figure 2. S. suis Aagp belongs to atypical aquaglyceroporins. The S. suis Aagp transmembrane helices were predicted (top panel). Asterisks specify the ar/R region residues, and black boxes mean the atypical aquaglyceroporins’ substrate-selective residues (YVPR).

A search for Aagp in the NCBI database revealed that 126 S. suis strains contained Aagp, distributed across numerous serotypes, including serotypes 1–9, 12, 16, 19, 24, 28, and 31 (Figure 3a). To confirm the function of Aagp further, a deletion strain of Aagp_P1/7 was constructed in S. suis serotype 2 strain P1/7. When challenged with H₂O₂, the survival rate of ΔAagp_P1/7 was reduced to 15.1%, compared to 37.0775% for strain P1/7 (Figure 3b). These findings provide further evidence supporting the role of S. suis Aagp in oxidative stress responses.

Figure 3. Aagp, a novel aquaporin, is widely distributed in S. suis. (a) The distribution of Aagp was assessed in 126 S. suis strains by examining Aagp sequences obtained from the NCBI database. (b) The survival rates of P1/7 and ΔAagp_P1/7 were determined after 25 min treated with 25 mM H₂O₂. The statistical analyses were performed with a two-tailed unpaired t test. “**” indicate p < 0.01.

Aagp facilitates H₂O₂ efflux and alleviates oxidative stress

Tong et al. reported that an aquaporin, So-AqpA, of Streptococcus oligofermentans contributes to the diffusion of H₂O₂ across the membrane [26]. To investigate the involvement of Aagp in H₂O₂ diffusion, we measured the concentration of H₂O₂ in bacterial cells treated with 25 mM H₂O₂. The concentration of H₂O₂ treated for 25 min in WT (59.22% survival rate) was 8.4067 pmol per 10⁶ cells, which was dramatically lower than 430.731 pmol per 10⁶ cells in ΔAagp (0.96% survival rate) (Figure 4a,b). Despite several attempts, we failed to construct an Aagp complementary vector based on plasmids pSET1, pSET2, and pSET3. Therefore, we created a frameshift mutation strain, Aagp^mut, by inserting one base into its CDS, as we reported in a previous study [27]. Similar to the results obtained from ΔAagp, we found that the survival rate was lower in Aagp^mut compared with WT, while the concentration of H₂O₂ in Aagp^mut was significantly higher than in WT (Figure 4c,d). Additionally, we compared the response to H₂O₂ between the WT and Aagp mutations via the Oxford cup test. We found that both ΔAagp and Aagp^mut strains exhibited larger inhibition zones than the WT strain (Figure 4e). These results collectively indicate that Aagp facilitates H₂O₂ efflux, thereby alleviating oxidative stress.

Figure 4. Aagp facilitates H₂O₂ efflux. (a) The survival rates of WT and ΔAagp were determined after 25 min treated with 25 mM H₂O₂. (b) The H₂O₂ concentrations of WT and ΔAagp were measured after 25 min treated with 25 mM H₂O₂. (c) The survival rates of WT and Aagp^mut were determined after 25 min treated with 25 mM H₂O₂. (d) The H₂O₂ concentrations of WT and Aagp^mut were measured after 25 min treated with 25 mM H₂O₂. (e) The response to H₂O₂ between the wild type and Aagp mutations via the Oxford cup test. The statistical analyses were performed with a two-tailed unpaired t test. “*” indicates p < 0.05 and “**” indicates p < 0.01.

Aagp may be involved in glycerol transport

Previous studies have shown that some aquaporins facilitate glycerol diffusion [28]. To determine the function of Aagp in relation to glycerol, the growth curves of ΔAagp and WT were performed in diluted THB medium (1/3 THB as a poor medium condition) containing 10 mM glycerol or 100 mM glycerol under aerobic condition. As shown in Figure 5a, b, the growth of WT was inhibited as the concentration of glycerol increased, whereas the growth of ΔAagp was not affected in the absence or presence of glycerol. As shown in Figure 5c, S. suis strain GZ0565 possesses genes involved in the phosphorylation pathway for glycerol metabolism, which is an energy-consuming pathway. Under the aerobic condition, glycerol may be phosphorylated by glycerol kinase (GlpK) and then oxidized by glycerol-3-phosphate oxidase (GlpO), which is accompanied by H₂O₂ production that can be toxic to bacteria [29]. To test this hypothesis, the concentrations of H₂O₂ in WT and ΔAagp were measured in 1/3 THB containing 10 mM glycerol or 100 mM glycerol. The concentration of H₂O₂ in WT cultured in 1/3 THB was 0.0178 pmol per 10⁶ cells, but in the presence of additional glycerol, the concentration of H₂O₂ increased and reached to 0.1007 pmol per 10⁶ cells when cultured in 1/3 THB containing 100 mM glycerol. Conversely, in ΔAagp, the concentration of H₂O₂ was similar in the absence or presence of glycerol (Figure 5d). These results suggest that Aagp may be involved in glycerol transport.

Figure 5. Aagp is involved in glycerol transport. The growth curves of WT (a) and ΔAagp (b) cultured in 1/3 THB, 1/3 THB containing 10 mM glycerol or 100 mM glycerol were shown. (c) S. suis strain GZ0565 possesses a phosphorylation pathway for glycerol metabolism. The downregulated genes are shown in red in H₂O₂ treatment compared with THB condition by RNA-Seq analysis. (d) The H₂O₂ concentrations of WT and ΔAagp cultured in 1/3 THB, 1/3 THB containing 10 mM glycerol or 100 mM glycerol were determined. The statistical analyses were performed with a two-tailed unpaired t test. “*” indicates p < 0.05.

Aagp contributes to S. suis virulence in a mouse infection model

Given that Aagp alleviates S. suis oxidative stress, the contribution of Aagp to S. suis virulence was assessed in mice. Compared with WT infection groups, the number of ΔAagp in blood, liver, spleen, kidney, and brain tissues was significantly reduced, indicating that Aagp contributes to S. suis virulence (Figure 6). These results indicate that Aagp contributes to S. suis virulence in a mouse infection model.

Figure 6. Aagp contributes to S. suis virulence in a mouse infection model. Five mice per group were injected intraperitoneally with 1.5 × 10⁸ CFU of WT and ΔAagp. All mice were euthanized at 24 h post-infection. Bacteria from blood, liver, spleen, kidney and brains were plated onto THA, and colonies were expressed as Log₁₀ CFU/mg or Log₁₀ CFU/mL. The statistical analyses were performed with a two-tailed unpaired t test. “***” indicate p < 0.001.

Discussion

Oxidative stress is a common challenge for S. suis to overcome in response to infection or environmental conditions. In this study, we employed RNA-seq to investigate the global transcriptional landscape of S. suis under H₂O₂ treatment and to reveal the adaptation mechanisms of S. suis response to oxidative stress. Our findings showed that multiple genes involved in oxidative stress were differentially expressed. Some of them have been identified in previous studies. We found that BFP66_RS05715 encoding NADH oxidase, known to contribute to oxidative stress tolerance [30], was upregulated 2.30-fold by H₂O₂ treatment. We also observed that BFP66_RS01615 encoding trigger factor, which contributes to stress tolerance [31], was upregulated 3.12-fold by H₂O₂ treatment. Additionally, we noted a downregulation of BFP66_RS01545 encoding Fur-like protein PerR by 3.62-fold in response to H₂O₂ stress, consistent with previous research showing that the PerR deletion mutant is less sensitive to H₂O₂ stress [12]. PerR negatively regulates metQ, and its derepression increases methionine utilization, converting H₂O₂ to H₂O [12]. Although RNA-Seq analysis indicated a modest 1.35-fold up-regulation of metQ (BFP66_RS08225) (p < 0.01), RT-qPCR analysis revealed a 10.65-fold upregulation (Supplementary Figure S2B). Moreover, compared to the WT, ΔmetQ was more sensitive to H₂O₂, confirming the role of metQ under H₂O₂ conditions (Figure 1b). These findings indicate that the identified genes are crucial in S. suis response to oxidative stress.

Our study has uncovered novel mechanisms that may be responsible for S. suis resistance against oxidative stress caused by ROS. ROS can cause damage to DNA, proteins, and lipids, and repairing these molecules is crucial to combating oxidative stress. In E. coli, exonuclease III is a major apurinic/apyrimidinic endonuclease that recognizes base lesions and cleaves the glycosylase bond to repair DNA [32]. We found that the gene BFP66_RS05940, which codes for exodeoxyribonuclease III was upregulated in response to H₂O₂ stress, indicating that it may be involved in DNA repair through cleaving lesions. Additionally, BFP66_RS06520 encoding an N-carbamoylputrescine amidase (AguB) was upregulated upon H₂O₂ stress. The AguAB pathway converts agmatine to putrescine, protecting E. coli from DNA damage via ROS scavenging [33]. Our deletion studies showed that exo III and AguB are crucial for H₂O₂ resistance in S. suis (Figure 1b). Furthermore, we observed upregulated genes contributing to the biosynthesis of amino acids and nucleic acid or transport, including five genes involved in producing oxidized dinucleotide cofactors (Figure 1a), BFP66_RS01165 encoding a component of an amino acid ABC transporter, and BFP66_RS04785 encoding a glutamate dehydrogenase. These findings indicated that S. suis enhances its biosynthesis and transport of amino acids and nucleic acid to respond to H₂O₂ stress. Interestingly, under ROS stress caused by 2,4-dichlorophenoxyacetic acid, E. coli reduces energy-consuming pathways to conserve energy for vital metabolism [33]. In our study, several genes related to energy-consuming pathways were downregulated under H₂O₂ stress (Figure 1a). In contrast, genes involved in glycolysis were upregulated, indicating that S. suis generates more ATP through enhanced glycolysis and reduces energy-consuming pathways for H₂O₂ detoxification.

The aquaporins are divided into three subfamilies: supergene channel superaquaporins, water-transporting aquaporin, and glycerol-transporting aquaglyceroporins [34–36]. Recent studies have demonstrated that aquaporins can transport H₂O₂ and regulate its levels in animals and plants, preventing cells from oxidative stress [37–40]. However, little is known about the function of aquaporins in H₂O₂ regulation in bacteria. S. oligofermentans aquaporin So-AqpA, sharing only 21.90% amino acid identity with S. suis Aagp, has been reported to facilitate the efflux of endogenous H₂O₂ and protect the bacterium from oxidant damage [26]. Both S. pneumoniae Pn-AqpC and S. suis Aagp are atypical aquaglyceroporins and contribute to bacterial resistance to H₂O₂ stress, but their mechanisms are different. Pn-AqpC can facilitate oxygen permeation into pneumococcal, which promotes the generation of endogenous H₂O₂ and helps S. pneumoniae adapt to higher exogenous H₂O₂ [25]. In contrast, S. suis Aagp contributes to resistance to oxidative stress by facilitating H₂O₂ efflux, similar to So-AqpA. Previous studies have shown that in S. oligofermentans, the expression of So-aqpA is negatively regulated by MntR and PerR [26,41]. In our RNA-seq data, we observed a significant reduction in the expressions of MntR (encoded by BFP66_RS09925) and PerR (encoded by BFP66_RS01545), accompanied by a significant upregulation of Aagp during H₂O₂ stress in S. suis. These findings suggest that MntR and PerR in S. suis may play similar roles in regulating Aagp under H₂O₂ stress conditions. Additionally, we propose that Aagp may possess dual functions of both H₂O₂ efflux and glycerol transport. In Lactobacillus plantarum, GlpF2, GlpF3, and GlpF4, sharing 30.37%, 36.63%, and 32.73% amino acid identity with Aagp, respectively, have been shown to be permeable to both H₂O₂ and glycerol [42]. In our study, we have confirmed that Aagp facilitates H₂O₂ efflux by oxidative stress assays and measurement of intracellular H₂O₂ level. However, further investigation is required to determine the glycerol transport ability of Aagp.

Through in-depth analysis, we have identified homologs of Aagp that play a role in virulence in other pathogenic bacteria. In Group B Streptococcus, GlpF, sharing 81% amino acid identity with Aagp, enhances the invasion capacity of Group B Streptococcus into eukaryotic cells [43]. In S. pneumococci, Pn-AqpC contributes to pneumococcal pathogenicity by modulating H₂O₂ production and pneumolysin release [25]. In L. monocytogenes, the expression of GlpF, sharing 37% amino acid identity with Aagp, is upregulated during intracellular growth [44]. When bacteria invade, host innate immune cells generate ROS such as H₂O₂. Neutrophils, monocytes, and macrophages, abundant in blood and tissues, play crucial roles in producing ROS to eliminate bacteria. The deletion of Aagp would impair the defence capability of S. suis survival in blood and tissues. Finally, we assume that Aagp contributes to S. suis oxidative stress resistance and promotes its survival in the host by facilitating H₂O₂ efflux.

In summary, we depicted S. suis defence network response to H₂O₂ stress and identified a novel aquaporin, Aagp, that alleviated S. suis oxidative stress via facilitating H₂O₂ efflux, which contributes to virulence. In addition, Aagp might be involved in glycerol transport.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data Availability statement

The data supporting this study’s findings are available from the corresponding author upon reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21505594.2023.2249789

Additional information

Funding

This work was supported by the National Key Research and Development Program of China (No.2021YFD1800402), National Natural Science Foundation of China (No.32172859), Open Project Program of Jiangsu Key Laboratory of Zoonosis (No. R2103), Open Project Program of Engineering Research Center for the Prevention and Control of Animal Original Zoonosis, Fujian Province University (No.2021ZW001).