Thr-phosphorylation of FadR protein is specifically mediated by STK

The SS2 genome encodes a stk gene, which includes the typical N-terminal kinase domain in the cytoplasm and the C-terminal PASTA domain in the cytoplasm, separated by a transmembrane region [25]. STK has been proven to be involved in many crucial physiological processes of bacteria [15,19,28]. The global phosphorylation profiles of wild-type (WT) SS2 and Δstk strains were compared through high-throughput phosphoproteomics and protein quantitative omics. According to the phosphoproteomic results, a phospho-peptide signal of FadR (ZY05719_05575) in Δstk strain was significantly attenuated (S1 Table) compared with that of WT SS2 strain, and the mass spectrometry results revealed that Thr230 was a potential phosphorylation site (S1 Fig). Next, we further investigated whether STK can phosphorylate FadR in vivo. Immunoprecipitation (IP) analysis revealed that compared with WT strain, the phosphorylation of FadR disappeared in Δstk strain, while there was no difference in the phosphorylation level of FadR in CΔstk strain (Fig 1A), which is consistent with the results obtained from mass spectrometry analysis. We subsequently attempted to determine the phosphorylation sites mediated by STK in FadR. The recombinant FadR protein and the non-phosphorylatable FadR-T230A mutant protein were expressed, purified, and incubated with nSTK in vitro. Phos-tag SDS‒PAGE assay was used to determine the phosphorylation level of FadR, and the results revealed that only FadR protein presented a shifted band (Fig 1B), which also indicated that only T230 at the C-terminal of FadR was phosphorylated by STK. In addition, the IP analysis reveals that the phosphorylation level of FadR in FadR-T230A strain was significantly lower than that in WT SS2 (Fig 1C). Altogether, these results indicated that Thr230 in FadR is the only target site for STK.

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Fig 1. FadR is phosphorylated by STK.

(A) FadR polyclonal antibody was used to perform immunoprecipitation (IP) experiments with whole proteins of WT SS2, Δstk, and CΔstk strains. The pulled-down protein was separated by SDS‒PAGE, and Western blot analysis was performed using anti-phosphorylated Thr antibody and FadR polyclonal antibody at a 1:1000 dilution. The band intensity was analyzed relative to the FadR protein phosphorylation level in WT SS2 strain. (B) The purified GST nSTK (15 μg) and substrate protein (5 μg) were mixed in phosphorylation buffer and incubated at 37°C for 2 h, and a phosphorylation reaction was performed. The samples were subjected to 12% conventional SDS‒PAGE (left) and Phosbind acrylamide SDS‒PAGE (right). The separated proteins were observed by Coomassie Brilliant Blue staining. (C) The IP experiments of WT SS2 and FadR-T230A whole proteins were performed separately with FadR polyclonal antibodies. The pull-down protein was detected by the method described in (A). The band intensity was analyzed relative to the FadR protein phosphorylation level in WT SS2 strain. All data are representative of at least three independent experiments with similar results. The data were statistically analyzed by One-way ANOVA (A) and an unpaired t-test (C). ^ns, P > 0.05; ^****, P < 0.0001.

https://doi.org/10.1371/journal.ppat.1013534.g001

FadR phosphorylation in SS2 is regulated by extracellular stimuli

The level of STK-mediated phosphorylation of substrate proteins in bacteria may be influenced by the external environment [25]. To explore the important physiological significance of FadR phosphorylation in the life process of SS2, we subjected WT SS2 strains to various environmental stresses and evaluated the in vivo phosphorylation level of FadR. To avoid the possibility that the observed differences in FadR phosphorylation states might be due to the degree of bacterial death rather than the nature of the applied stress, we conducted experiments using weak acid (THY, pH 5.5), low concentration hydrogen peroxide (THY, 10 mM H₂O₂), and low-salt (THY, 0.2 M NaCl) stress for a 30 min experiment. Additionally, we also tested the bacterial viability before and after the stress, and results showed that these environmental stresses had no significant effect on bacterial survival (S2 Fig). The results revealed that FadR phosphorylation levels significantly increased in acidic environments and hydrogen peroxide, while osmotic pressure had no effect on FadR phosphorylation (Fig 2A–2C). Therefore, we speculated that the STK-FadR axis may play an important role in the acid stress response of SS2.

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Fig 2. Increased phosphorylation level of FadR in acidic environments.

(A) Exponentially growing cells were treated with pH 5.5 THY adjusted with hydrochloric acid for 30 min. (B) Exponentially growing cells were treated with THY supplemented with 10 mM H₂O₂ for 30 min. (C) Exponentially growing cells were treated with THY supplemented with 0.2 M NaCl for 30 min. (A-C) Immunoprecipitation (IP) experiments of all WT SS2 whole proteins were performed separately with FadR polyclonal antibodies. The pull-down protein was detected by the method described in Fig 1A. Bar graphs showed the percentage of phosphorylated and total FadR in different treatment groups. All experiments were repeated three times. ^ns, P > 0.05; ^**, P < 0.01; ^****, P < 0.0001 (an unpaired t-test).

https://doi.org/10.1371/journal.ppat.1013534.g002

Phosphorylation of FadR enhances SS2 virulence

STK-mediated phosphorylation of substrates had a significant impact on the pathogenicity and virulence of bacteria [24,25]. To investigate the effect of STK-mediated FadR phosphorylation on the virulence of SS2, the growth curves of WT SS2 strain, fadR-deleted strain ΔfadR (S3A Fig), Complementation strain CΔfadR (S3B Fig), phospho-ablative strain FadR-T230A (mimics non-phosphorylated Thr residues, which alanine replaces threonine), and phosphomimetic strain FadR-T230E (mimics phosphorylated Thr residues) (S3C Fig) were first measured under the same conditions. The results showed that these mutant strains had no difference in growth rate compared to WT SS2 strain (S3D Fig). Furthermore, compared with WT SS2 strain, there were no significant differences in the transcription and expression levels of FadR in CΔfadR strains (S4A and S4B Fig).

To explore the effect of FadR and its phosphorylation on the virulence of SS2, an animal intraperitoneal infection challenge test was performed. Animal experimental model revealed that, compared with that of the mice injected with WT SS2 strain (30%), the survival rate of the mice injected with ΔfadR (80%) and FadR-T230A (60%) strains were significantly increased, the survival rate of the mice injected with FadR-T230E (0%) strain was significantly decreased, and the survival rate of the mice injected with CΔfadR (30%) strain showed no significant change (Fig 3A). In addition, to investigate the role of FadR in in vivo infection of SS2, we compared the colonization efficiency of WT SS2 and mutant strains in BALB/c mice. The results indicated that, compared with WT SS2 strain, the CFUs of blood and various organs in the mice infected with ΔfadR and FadR-T230A strains were significantly decreased, the CFUs of blood and various organs in the mice infected with FadR-T230E strain were significantly increased, and the CFUs of blood and various organs in the mice infected with CΔfadR strain showed no significant change (Fig 3B–3F). Meanwhile, compared with ΔfadR strain, the CFUs of blood and various organs in the mice infected with FadR-T230A strains were significantly increased (Fig 3B–3F). Notably, the results revealed that FadR and its phosphorylation significantly weakened the ability of tissues/organs in infected mice to clear pathogenic microorganisms.

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Fig 3. Phosphorylation of FadR enhances bacterial virulence.

(A) BALB/c mice were intraperitoneally injected with WT SS2, ΔfadR, FadR-T230A, FadR-T230E, and CΔfadR strains at a dose of 2 × 10⁸ CFUs/mice (n = 10 mice/group), and survival time was closely monitored. (B-F) After the intraperitoneal injection of 2 × 10⁷ CFUs of WT SS2, ΔfadR, FadR-T230A, FadR-T230E, and CΔfadR strains into BALB/c mice, the bacterial loads in the blood (B), liver (C), spleen (D), lungs (E), and brain (F) were measured at 24 hpi (n = 8 mice/group). (G) H&E staining of lung and spleen tissue sections at 24 hpi from mice inoculated with PBS, WT SS2, and FadR-T230E strains by intraperitoneal injection. In the enlarged area of the images (black dashed frame), the black arrow indicates hepatic steatosis, with varying numbers of small circular vacuoles in the cytoplasm, and many congested blood vessels and hepatic sinusoids. The red arrow indicates a decreased number of lymphocytes. The yellow arrow indicates alveolar congestion, thickening of alveolar walls, and alveolar collapse. The blue arrow indicates glial cell nodules, and some bruising had appeared. Scale bar, 100 μm. The data were statistically analyzed by the Log-rank test (A) and One-way ANOVA (B-F). ^ns, P > 0.05; ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001; ^****, P < 0.0001.

https://doi.org/10.1371/journal.ppat.1013534.g003

Streptococcus suis can usually cause meningitis, septicemia, endocarditis, and arthritis in pigs, with a high mortality rate [29]. Next, the histopathological analysis of the liver, spleen, lung, and brain tissues from mice challenged with SS2 revealed hepatic steatosis, with varying numbers of small circular vacuoles in the cytoplasm, and many blood vessels and hepatic sinusoids were congested; the number of lymphocytes in the spleen decreased; alveolar congestion, thickening of alveolar walls, and alveolar collapse were observed; glial cell nodules and some bruising appeared in the brain (Fig 3G). Compared with WT SS2 strain, the liver, spleen, lung and brain tissues of the mice infected with FadR-T230E presented more severe pathological manifestations (S5A–5D Fig). Altogether, these animal experiments indicated that FadR is indispensable for whole virulence and pathogenicity of SS2, and phosphorylation plays an important role in FadR functional process.

FadR binds to promoter regions of the adi

The above studies indicate that FadR and its phosphorylation regulate the acid resistance and virulence of SS2. However, as a transcription factor, it is bound to indirectly exert its regulatory function by regulating downstream target genes. Therefore, to identify the genes regulated by FadR, we used RNA-seq technology to measure the gene expression profiles of ΔfadR and WT SS2 strains. The RT‒qPCR results revealed that when WT SS2 strain was cultured to an OD₆₀₀ of 0.8, the transcription level of fadR was the highest (S6 Fig). The total bacterial RNA of WT SS2 and ΔfadR strains during this growth period was extracted and subjected to RNA-seq analysis. The raw data for comparative transcriptomics has been submitted to the SRA database (The BioProject accession number: PRJNA1283375). All comparative transcriptomic results are listed in the S2 Table. Genes with transcription level differences ≥ 2.0 and P values ≤ 0.05 were considered to have significant expression differences. Compared with WT SS2 strain, ΔfadR strain contained 88 up-regulated genes and 51 down-regulated genes (Fig 4A). The up-regulated genes were related mainly to carbohydrate metabolism, including hydrolytic enzymes, synthetases, transporter protein permeases, and dehydrogenases (S3 Table). However, other genes involved in transcription, transport, and kinase and deaminase activity were down-regulated in ΔfadR strain (S3 Table). Through KEGG pathway enrichment analysis, it was found that most of these genes were related to glucose metabolism (Fig 4B).

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Fig 4. Phosphorylation of FadR increases the transcription of adi.

(A) Volcano plot showing the differential gene expression of the RNA-seq results between WT SS2 and ΔfadR strains. The genes that were significantly upregulated or downregulated by at least 2-fold are represented by red and green dots, respectively. (B) The statistical analysis of KEGG pathway enrichment is represented by scatter plots of the differentially expressed genes. (C) FadR, FadR-T230A, and FadR-T230E proteins were incubated with DNA fragments (80 ng) ~300 bp in length from the adi promoter region. The negative control was a DNA fragments (80 ng) ~197 bp in length from the enolase promoter region. The reaction was carried out at 37°C for 30 min, separated with a 6% polyacrylamide gel in 0.5 × TBE buffer, and stained with ethidium bromide for imaging. (D) The adi transcript levels in WT SS2, ΔfadR, FadR-T230A, and FadR-T230E strains were determined by RT‒qPCR. (E) The pTCV-lacZ recombinant plasmid containing the adi promoter sequence was electroporated into the WT SS2, ΔfadR, FadR-T230A, and FadR-T230E strains, and adi promoter activity was measured and normalized to the internal control β‐galactosidase activity. (F) ChIP assay was used to detect the binding of FadR and adi promoter regions. CΔfadR-flag was grown to the logarithmic stage, and DNA fragments were sonicated after washing with PBS. The DNA fragments that interacted with FadR were precipitated with anti-Flag antibodies, and healthy mouse IgG was used as a negative control. The purified DNA fragments were used as PCR templates to amplify the target region of the adi promoter. The PCR product of the 16S rRNA gene was used as a negative control. (G) The expression of ADI in WT SS2, ΔfadR, FadR-T230A, and FadR-T230E strains were detected by Western blots (left). The band intensity relative to that of WT SS2 group was analyzed (right). The data represent three independent experiments, and are presented as the means ± standard deviations. Statistical analysis was conducted by using One-way ANOVA (D, E, and G). ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001; ^****, P < 0.0001.

https://doi.org/10.1371/journal.ppat.1013534.g004

Among all the differentially expressed genes, the gene adi, which breaks down arginine, attracted our attention, as it is involved in the acid resistance and virulence of Streptococcus suis [30,31]. We first conducted in vitro validation experiments using the FadR protein at different concentrations (ranging from 0 ng/μL to 40 ng/μL) and 80 ng of ~300 bp promoter DNA fragments for electrophoretic mobility shift assays (EMSA). The results indicated that the FadR protein could bind to the promoter of adi, and the higher the protein concentration was, the more DNA was bound (Fig 4C). To examine whether our in vitro observations reflect in vivo phenomena, the Flag-tag was fused to the C-terminus of FadR to construct the CΔfadR-flag strain, and the expression of FadR-Flag was detected by Western blotting (S7A Fig). The results of the mouse macrophage phagocytosis experiments revealed that the presence of the Flag-tag did not affect the biological function of SS2 (S7B Fig). In addition, the ChIP results also revealed that FadR can directly bind to the adi promoter region in vivo (Fig 4F). These findings suggested that FadR may play a role in acid resistance by regulating the transcription of adi.

Phosphorylation of FadR promotes adi transcription

To further investigate the effect of phosphorylation on FadR regulation, we used an EMSA to detect the ability of the phosphomimetic protein FadR-T230E to bind to the adi promoter. The results showed that compared to the FadR protein, the binding ability of the FadR-T230E protein to the adi promoter was significantly enhanced, while the binding ability of the FadR-T230A protein to the adi promoter showed no significant difference (Fig 4C). In addition, our study shows that the transcription level of adi decreased significantly when SS2 against acid stress, which is similar to the previous report (S8 Fig) [30]. Compared with that of WT SS2 strain, the RT‒qPCR results revealed that the adi transcription level of ΔfadR and FadR-T230A strains were significantly decreased, while the adi transcription level of FadR-T230E strain was up-regulated approximately 1.5-fold (Fig 4D). Compared with that of WT SS2 strain, Western blot results revealed that ADI protein expression level of ΔfadR and FadR-T230A strains were significantly decreased, while ADI protein expression level of FadR-T230E strain was significantly increased (Fig 4G). To analyze the activity of the adi promoter in vivo, the pTCV-lacZ plasmid was electroporated into WT SS2 and fadR variants. As expected, compared with that of WT SS2 strain, the promoter activity of adi were significantly decreased in both ΔfadR and FadR-T230A strains, while the promoter activity of adi was significantly increased in FadR-T230E strain (Fig 4E). These discoveries indicated that FadR exert positive regulatory effects for adi, and phosphorylation further enhances its binding ability to adi promoter and also increases the transcription level of adi.

The increased virulence of FadR-T230E strain is due to transcriptional activation of adi

At present, it has been shown that adi was an important virulence factor of bacteria [32–34]. Therefore, we suspected that the increased virulence of the FadR-T230E strain was related to the increased transcription of adi. The promoter of impdh [35] was fused to the adi gene sequence, after which the pSET2-imp-adi plasmid was constructed to avoid the regulatory effect of FadR on adi [24]. The complemented plasmid (pSET2-imp-adi) was electroporated into FadR-T230E competent cells lacking adi gene to obtain the FadR-T230E-Cadi strain. In addition, we tested the growth curves of the strains and confirmed that carrying the plasmid did not affect its growth rates (S9 Fig). The RT‒qPCR results revealed that the transcription level of adi in the FadR-T230E-Cadi strain was not significantly different from that in WT SS2 strain (Fig 5A). In addition, animal experimental model revealed that, compared with that of the mice injected with WT SS2 strain (30%), the survival rate of the mice injected with Δadi (90%) strain was significantly increased, while the survival rate of the mice injected with the FadR-T230E-Cadi (30%) strain showed no significant difference (Fig 5B). Meanwhile, the CFUs of the blood and various organs in the mice infected with FadR-T230E-Cadi strain in mice decreased to WT SS2 strain level (Fig 5C). Overall, these above results indicated that the increased virulence of SS2 caused by STK/FadR phosphorylation pathway is due to the increased transcription of adi.

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Fig 5. The increased virulence and acid resistance capacity resulting from the phosphorylation of FadR are associated with the promotion of adi transcription.

(A) The adi transcript levels in WT SS2 and FadR-T230E-Cadi strains were determined by RT‒qPCR. (B) BALB/c mice were challenged by intraperitoneal injection of WT SS2, Δadi, and FadR-T230E-Cadi strains at a dose of 2 × 10⁸ CFUs/mouse (n = 10 mice/group), and the survival time was continuously monitored. The control group is PBS. (C) Bacterial loads in the blood, liver, spleen, lung, and brain of BALB/c mice were challenged with 2 × 10⁷ CFUs of WT SS2 and FadR-T230E-Cadi strains by intraperitoneal injection at 24 hpi (n = 5 mice/group). (D) WT SS2, ΔfadR, CΔfadR, FadR-T230A, FadR-T230E, Δadi, CΔadi, and FadR-T230E-Cadi strains were treated with PBS at pH 5.0 for 1 h, and the number of viable bacteria was subsequently determined by CFU plate counts. The bacterial survival rate was expressed as the ratio of the number of viable bacteria at 1 h to that at 0 h. (E) RAW264.7 cells were infected at an MOI of 10:1 with WT SS2, ΔfadR, CΔfadR, FadR-T230A, FadR-T230E, Δadi, CΔadi, and FadR-T230E-Cadi strains for 1 h. After 1 h of antibiotic sterilization, the amount of internalized bacteria was regarded as 100% to calculate percent survival. After 3 h of antibiotic sterilization, sterile water was used to lyse the cells to release bacteria. Then bacteria were serial-diluted in PBS buffer and spread onto THY plates, incubated at 37°C for 16 h. Black bars correspond to the control group, and red bars correspond to the CQ group. The data were representative of three independent experiments and are presented as the means ± standard deviations. Statistical analysis was performed by using an unpaired t-test (A and C), Long-rank test (B), One-way ANOVA (D), and Two-way ANOVA (E). ^ns, P > 0.05; ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001; ^****, P < 0.0001.

https://doi.org/10.1371/journal.ppat.1013534.g005

FadR phosphorylation affects acid resistance capacity and survival in host macrophages

The adi is involved in bacterial acid resistance [14,30,36], whether FadR phosphorylation affects the acid resistance of SS2 by regulating adi needs further exploration. Here, to demonstrate the correlation between FadR phosphorylation and the acid resistance of SS2, we compared the survival ability of WT SS2, fadR variants, and adi variants under acid stress conditions. The results showed that after 1 h of stimulation in a PBS environment at pH 5.0, compared with that of WT SS2 strain, the survival rate of ΔfadR, FadR-T230A, and Δadi strains were significantly decreased, the survival rate of the FadR-T230E strain was significantly increased, while the survival rates of CΔfadR, CΔadi, and FadR-T230E-Cadi strains showed no significant difference (Fig 5D). In addition to its acid resistance, bacteria can also produce the acid tolerance response (ATR) [16,37]. To investigate whether the STK/FadR signaling pathway affects SS2’s ATR, we first measured the sub-lethal pH and lethal pH in WT strain (S10A Fig). Subsequently, we compared ATR levels among WT SS2, Δstk, ΔfadR, FadR-T230A, and FadR-T230E strains. The results showed that after sub-lethal pH treatment, the survival rates of WT SS2 significantly increased, while those of Δstk, ΔfadR, FadR-T230A, and FadR-T230E strains remained unchanged (S10B Fig). Therefore, the above results indicate that STK enhances the acid resistance and ATR of SS2 by phosphorylation of FadR, and the effects of FadR phosphorylation on acid resistance of SS2 were closely related to the increase in adi transcription.

Many pathogens have the ability to survive for a period of time in a membrane-bound acidic chamber within macrophages [38]. Therefore, we further investigated whether FadR phosphorylation enhances the intracellular survival ability of SS2 in RAW264.7 macrophages. After lysosome acidification, an acidic pH environment (typically ranging between pH 4.5 and pH 5.0) is formed, which subsequently kills and degrades pathogens [39]. Intracellular survival was measured in the absence or presence of Chloroquine (CQ), an effective inhibitor of lysosomal acidification, to investigate the effect of the macrophage lysosomal acidification on WT SS2 and mutant strains. In the absence of CQ, compared with that of WT SS2 strain, the intracellular survival rate of ΔfadR, FadR-T230A, and Δadi strains were significantly decreased, the intracellular survival rate of FadR-T230E strain was significantly increased, while the intracellular survival rates of CΔfadR, CΔadi, and FadR-T230E-Cadi strains showed no significant difference (Fig 5E). In the presence of CQ, the intracellular survival rates of all strains increased, with no significant difference observed between WT SS2, CΔfadR, FadR-T230A, FadR-T230E, CΔadi, and FadR-T230E-Cadi strains, while the intracellular survival rates of ΔfadR and Δadi strains did not fully recover to WT SS2 level (Fig 5E). The treatment with CQ eliminated the survival defect of SS2 within macrophages, revealing that the bactericidal effect of macrophage lysosomal acidification was effectively neutralized or weakened by the FadR phosphorylation pathway. In summary, the above results indicated that the STK/FadR pathway not only enhances the acid resistance of the bacteria but also significantly improves their ability to survive in RAW264.7 macrophages.

The mechanism of SS2 utilizing the STK/FadR axis to resist acid and promote macrophage survival

The arginine deaminase system can decompose arginine, ultimately producing substances such as ornithine and ammonia, thereby increasing the acid resistance of bacteria [13]. Meanwhile, we used the promoters of impdh (weak promoter) [35] and enolase (strong promoter) [40] to control the expression of ADI in SS2, and then constructed knockdown strain ADI^Imp (replace the promoter of adi with weak promoter impdh) and overexpression strain ADI^Eno (replace the promoter of adi with strong promoter enolase) of ADI to more accurately investigate the effect of ADI content on SS2 virulence. In addition, we tested the growth curves of the strains and confirmed that carrying the plasmid did not affect their growth rates (S9 Fig). We measured the arginine content in bacteria and found that compared with WT SS2 strain, the arginine content in ΔfadR, FadR-T230A, and Δadi strains were significantly increased, the arginine content in FadR-T230E and ADI^Eno strains were significantly decreased, while the arginine content in FadR-T230E-Cadi and ADI^Imp strains showed no significant difference (Fig 6A). In vitro enzymatic reaction assays were performed, and the results showed that recombinant arginine deaminase could decompose arginine, which further confirmed our hypothesis (S11 Fig) [30]. To further determine the ammonia production of bacteria, we first established a standard curve with an ammonia content detection kit, and the results revealed an almost linear relationship between the ammonia concentration and fluorescence intensity (S12A Fig). We measured the ammonia content inside and outside the bacteria. The results revealed that the ammonia contents of FadR-T230E and ADI^Eno significantly increased compared with those of WT SS2, the ammonia contents of ΔfadR, FadR-T230A, and Δadi strains significantly decreased compared with those of WT SS2, whereas the ammonia levels in FadR-T230E-Cadi and ADI^Imp were the same as those in WT SS2 (Fig 6B and 6C). Altogether, these results indicated that the increase of ADI accelerates the conversion of arginine to ammonia, which in turn improves the survival ability of SS2 in acidic environments.

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Fig 6. Molecular mechanism of acid resistance in SS2.

(A) WT SS2, ΔfadR, FadR-T230A, FadR-T230E, Δadi, FadR-T230E-Cadi, ADI^Imp, and ADI^Eno strains were placed in pH 5.5 PBS and incubated for 1 h before the levels of intracellular arginine were measured. (B) Intracellular ammonia contents of WT SS2, ΔfadR, FadR-T230A, FadR-T230E, Δadi, FadR-T230E-Cadi, ADI^Imp, and ADI^Eno strains were determined according to the method used in (A). (C) The ammonia released by WT SS2, ΔfadR, FadR-T230A, FadR-T230E, Δadi, FadR-T230E-Cadi, ADI^Imp, and ADI^Eno strains were measured using the method described in (A). (D) WT SS2, ΔfadR, FadR-T230A, FadR-T230E, Δadi, FadR-T230E-Cadi, ADI^Imp, and ADI^Eno strains were placed in pH 7.6 PBS to measure the intracellular pH, then quickly transferred to pH 5.5 PBS and incubated for 1 h before the intracellular pH was measured again. (E) WT SS2, ΔfadR, FadR-T230A, FadR-T230E, Δadi, FadR-T230E-Cadi, ADI^Imp, and ADI^Eno strains were added into the RAW264.7 cell holes at an MOI of 100:1 incubating for 1 h. The hole without strain was used as the control. The culture medium in these wells was replaced with DMEM containing gentamicin (100 µg/mL) and penicillin (10 µg/mL). After 3 h of antibiotic sterilization, the arginine content produced by RAW264.7 was determined using an arginine assay kit. (F) WT SS2, ΔfadR, FadR-T230A, FadR-T230E, Δadi, FadR-T230E-Cadi, ADI^Imp, and ADI^Eno strains were added into the RAW264.7 cell holes at an MOI of 100:1 incubating for 1 h. The culture medium in these wells was replaced with DMEM containing gentamicin (100 µg/mL) and penicillin (10 µg/mL). The NO content produced by RAW264.7 cells was determined using a micro NO content assay kit after 24 h. (G) WT SS2, ADI^Imp, and ADI^Eno strains were treated with PBS at pH 5.0 for 1 h, and the number of viable bacteria was subsequently determined by CFU plate counts. The bacterial survival rate was expressed as the ratio of the number of viable bacteria at 1 h to that at 0 h. (H) RAW264.7 cells were infected at an MOI of 10:1 with WT SS2, ADI^Imp, and ADI^Eno for 1 h. After 1 h of antibiotic sterilization, the internalized bacterial count was regarded as 100% to calculate percent survival. After 3 h of antibiotic sterilization, sterile water was used to lyse the cells to release bacteria. Then bacteria were serial-diluted in PBS buffer and spread onto THY plates, incubated at 37°C for 16 h. The data shown represent three independent experiments and are presented as the means ± standard deviations. Statistical analysis was performed by One-way ANOVA (A-C and E-H) and Two-way ANOVA followed by Bonferroni’s multiple comparisons test (D). ^ns, P > 0.05; ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001; ^****, P < 0.0001.

https://doi.org/10.1371/journal.ppat.1013534.g006

To resist acidic environments and improve their intracellular survival ability, most bacteria are able to grow in the external pH range of 5.5–9.0 and maintain the cytoplasmic pH within a suitable range of 7.4–7.8 [41]. On the basis that the phosphorylation of FadR enhances the acid resistance of WT SS2, we speculated that phosphorylation of FadR may affect the intracellular pH of WT SS2. To eliminate the influence of acidic stimuli on bacterial death, a weakly acidic environment (pH 5.5) was chosen, and the survival of Δadi was significantly lower than that of WT SS2 (S13A Fig). The survival ability of Δadi in PBS at pH 5.5 at different time points was tested, and the results revealed that Δadi presented little change up to 30 minutes but significantly decreased at 40 minutes after treatment (S13B Fig). We first used extracellular buffer to balance the pH inside the SS2 cells to establish a standard curve, and the results revealed an almost linear relationship between the intracellular fluorescence and pH (S12B Fig). Indeed, when bacteria were quickly switched from pH 7.6 to pH 5.5 and incubated at 37°C for 1 h, the decrease in the intracellular pH of WT SS2 was ~ 1 unit, which was more than 0.5 units greater than that experienced by FadR-T230E and ADI^Eno (Fig 6D). Additionally, the intracellular pH of ΔfadR, FadR-T230A, Δadi, FadR-T230E-Cadi, and ADI^Imp strains also decreased significantly (Fig 6D). These results indicate that adi can assist SS2 in maintaining stable intracellular pH.

Many pathogens have the ability to resiliently survive in the acidic microenvironment created by macrophages [38]. Such as, bacteria can utilize intracellular arginine to resist acid-mediated killing [30]. To further mimic in vivo infections, all strains were co-incubated with RAW264.7 macrophages for 3 h. Compared to WT SS2 strain, the intracellular arginine levels were significantly increased in cells infected with ΔfadR, FadR-T230A, and Δadi strains, the intracellular arginine levels were significantly decreased in cells infected with FadR-T230E and ADI^Eno strains, while no significant differences in arginine content were observed in cells infected with FadR-T230E-Cadi and ADI^Imp strains, suggesting that the ADI pathway can consume intracellular arginine (Fig 6E). Given that arginine in macrophages is also an important source of nitric oxide (NO), we detected the concentration of NO in the cells. The results showed that the production of NO were significantly increased in cells infected with ΔfadR, FadR-T230A, and Δadi strains compared to WT SS2 strain, the production of NO were significantly decreased in cells infected with FadR-T230E and ADI^Eno strains, while no significant differences for NO production were observed in cells infected with FadR-T230E-Cadi and ADI^Imp strains (Fig 6F). Importantly, ADI^Eno strain had a similar phenotype to FadR-T230E strain. Therefore, these data further show that the enhanced virulence of SS2 resulting from FadR phosphorylation is due to increased transcription of adi. In summary, our study demonstrated that FadR phosphorylation upregulates the transcription level of adi, thereby enhancing the ability of SS2 to acquire arginine from host cells, ultimately leading to increased ammonia production that enhances acid tolerance and intracellular survival capacity.

Meanwhile, we tested the acid tolerance of knockdown strain ADI^Imp and overexpression strain ADI^Eno of ADI. The results showed that compared to WT SS2 strain, the survival rate of ADI^Eno strain was significantly increased, while the survival rate of ADI^Imp strain was similar to that of WT SS2 strain (Fig 6G). We further examined the intracellular survival capacity of ADI^Imp and ADI^Eno strains. The results indicated that, compared to WT SS2 strain, the survival rate of ADI^Eno strain was significantly improved, whereas the survival rate of ADI^Imp strain was similar to that of WT SS2 strain (Fig 6H). The research results indicated that the enhanced acid tolerance and intracellular survival capacity of FadR-T230E strain are attributed to the increased transcription of adi.

We further investigated the link between STK and adi, as STK has been implicated in the acid resistance process of Streptococcus suis [35]. Our study showed that the acid resistance of Δstk strain significantly decreases, while the acid resistance of CΔstk strain has basically recovered to WT SS2 strain level (S14A Fig). To further demonstrate the regulatory effect of STK on adi in acidic conditions, WT SS2, Δstk, and CΔstk strain were incubated at 37°C for 1 h in TYH at pH 5.5, and then RNA-seq technology was used to detect the transcription levels of adi in WT SS2 strain, Δstk, and CΔstk strain. The RT‒qPCR results showed that, in comparison to WT SS2 strain, the transcription level of adi is significantly decreased in Δstk strain, while the transcription level of adi in CΔstk strain was restored (S14B Fig). In addition, Western blotting was used to assess the expression level of ADI protein. The results indicated that, in comparison to WT SS2 strain, the expression level of ADI protein is significantly reduced in Δstk strain, while the expression of ADI protein in CΔstk strain was decreased (S14C Fig). These results suggested that STK affect the transcription and expression levels of adi, thereby participating in the SS2 acid resistance process. Altogether, the STK indirectly enhances the transcript level of adi by phosphorylating FadR, which in turn enhances bacterial acid tolerance and virulence.

Ethics statement

All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Nanjing Agricultural University, China (approval number NJAU.No20210510065 and NJAU.No20211005144). The Chinese National Laboratory Animal Guideline for Ethical Review of Animal Welfare adhered to animal care and protocol.

Bacterial strains, plasmids, cell lines, and growth conditions

The bacterial strains and plasmids used in this study are listed in the S4 Table. The SS2 strain ZY05719 was isolated from dead pigs infected with Streptococcus suis in Sichuan Province, China. SS2 was grown at 37°C in THY (Todd-Hewitt Broth [THB, Becton, Franklin Lakes, NJ, USA] supplemented with 2% yeast extract [Oxoid Ltd., UK]) or in THA (THY containing 1.5% agar). E. coli DH5α was used as the host for the preparation of plasmid DNA and was cultured at 37°C in lysogeny broth (LB, Oxoid Ltd.) or LB containing 1.5% agar. E. coli BL21 (DE3) was used for protein prokaryotic expression and was cultured at 37°C in lysogeny broth (LB, Oxoid Ltd.) or LB containing 1.5% agar. Spectinomycin (Spc) was used at 50 μg/mL for E. coli and 100 μg/mL for SS2. Gentamicin (100 μg/mL) and penicillin (10 μg/mL) were used to kill the extracellular SS2. RAW264.7 cells were grown in DMEM containing 10% fetal calf serum in an incubator at 37°C and 5% CO₂.

Strain construction

In this study, a deletion vector and a replacement vector were constructed by homologous recombination. With the ZY05719 genome as a template, the upstream and downstream homologous arm fragments of FadR were amplified by PCR with the primers ΔfadR-F1/ΔfadR-R1 and ΔfadR-F2/ΔfadR-R2. The two PCR products were purified by gel recovery, and the two purified products were subsequently mixed at a ratio of 1:1 and amplified by fusion PCR with the using primers ΔfadR-F1/ΔfadR-R2. The fusion fragment was subsequently cloned and inserted into the temperature-sensitive shuttle vector pSET4s for SS2 ZY05719 and E. coli with the ClonExpress II One Step Cloning Kit (Vazyme Biotech Co., China; item number: C112-01) to construct the recombinant vector pSET4s-fadR. In addition, the plasmids pSET4s-adi and pSET4s-stk were obtained by the same method. With the ZY05719 genome as a template, the fadR and its upstream and downstream homologous arm fragments full-length were amplified by PCR with the primers fadR-F/fadR-R. The template plasmid pSET4s-(homologous arm)-fadR for in situ point mutation was obtained by the same method. With the plasmid pSET4s-(homologous arm)-fadR as a template, PCR amplification was performed with the primers FadR-T230A-F/R and FadR-T230E-F/R, respectively. The template circular plasmid in the PCR product was digested with DpnI enzyme, after which pSET4s-fadR-T230A and pSET4s-fadR-T230E constructs were obtained. The pSET4s-fadR, pSET4s-adi, and pSET4s-stk were electroporated into SS2 ZY05719 with the Gene Pulser XCell electroporation system (Bio-Rad, Hercules, CA, USA) (voltage: 2300 V, capacitance: 25 μF, resistance: 200 Ω, cuvette: 1 mm). To obtain ΔfadR and Δadi, it was necessary to first perform subculturing in a 28°C incubator and then perform PCR identification, which is consistent with previous methods [55,56]. The pSET4s-fadR-T230A and pSET4s-fadR-T230E plasmids were electroporated into ΔfadR strain, and the in situ mutant strain of fadR was obtained by the above method.

The complementary strain of the ΔfadR mutant was constructed as follows. With the SS2 ZY05719 genome as a template, PCR amplification was performed with the primers CΔfadR-F/CΔfadR-R to obtain the fadR fragment, which was then cloned and inserted into the plasmid pSET2. Subsequently, the recombinant plasmid was electroporated into the ΔfadR strain to obtain the CΔfadR strain. The CΔfadR-flag and CΔadi strain was obtained in the same manner. The ADI overexpressing strains were constructed as follows. With the SS2 ZY05719 genome as a template, the adi fragment sequence was amplified by PCR and fused to the promoter sequences of impdh and enolase, respectively. Subsequently, these two fusion fragments were separately cloned into pSET2 plasmid and electroporated into Δadi to obtain low expression strain ADI^Imp and overexpression strain ADI^Eno of ADI. In addition, the impdh-adi was cloned into pSET2 plasmid and electroporated into FadR-T230E-Δadi to obtain transcriptional recovery strain FadR-T230E-CΔadi of ADI. All strains and plasmids used are listed in the S4 Table. All primers used are listed in the S5 Table.

Phosphoproteomics analysis

WT SS2 and Δstk strain were cultured under shaking at 37°C until logarithmic growth stage (OD₆₀₀= 0.4 ~ 0.6), 8000 rpm, 4°C, and centrifuged for 10 minutes to collect bacterial cells. Each strain provides three biological replicate samples. The bacterial cell precipitation was washed twice with ice cold PBS, and then stored at low temperature and transported to Hangzhou PTM Bio-Tech company for phosphoproteomics analysis. Identification of phosphorylated proteins and their corresponding phosphorylation sites in SS2 using phosphorylation modified 4D Label free quantitative proteomics, and comparison of whole protein phosphorylation status between two bacterial strains.

Protein expression, purification, and preparation of polyclonal antibodies

The stk gene fragment sequence was amplified by PCR from the ZY05719 genome with the primers nSTK-F/nSTK-R, and subsequently cloned and inserted into the expression vector pGEX4T-1. With the SS2 ZY05719 genome as a template, the gene sequences of fadR and adi were amplified by PCR using primers B-FadR-F/B-FadR-R and ADI-F/ADI-R, respectively. Subsequently, the amplified sequences were cloned and inserted into the expression vector pET28a, resulting in the construction of plasmids pET28a-fadR and pET28a-adi. Plasmids pET28a-gapdh and pET28a-groel were obtained by using the same method. With the plasmid pET28a-fadR as a template, PCR amplification was performed with the primers B-FadR-T230A-F/R and B-FadR-T230E-F/R. The template circular plasmid in the PCR product was digested with DpnI enzyme, after which pET28a-FadR-T230A and pET28a-FadR-T230E constructs were obtained. All the plasmids were first transformed into E. coli DH5α, after which the positive plasmids were extracted and transformed into E. coli BL21 (DE3) to obtain recombinant expression strains. The GST-tagged proteins were purified with GST-tag purification resin (Beyotime, Shanghai, China), and the His-tagged proteins were purified with HisTrap HP (5 mL; GE Healthcare, Piscataway, NJ, USA). BALB/c mice were immunized by subcutaneous injection of 100 μg of the purified FadR protein, ADI protein, GAPDH protein, and GroEL protein emulsified with adjuvants. All immunizations were administered every 2 weeks for a total of 3 times, and serum samples were collected from the mice a week after the third immunization. All strains and plasmids used are listed in the S4 Table. All primers used are listed in the S5 Table.

In vitro phosphorylation assays

The experiment was conducted using 5 μg of purified recombinant substrate protein and 15 μg of purified nSTK to react in phosphorylation buffer (100 mM Tris HCl pH 8.0, 10 mM MgCl₂, 25 mM NaCl, 100 mM ATP, and 1 mM DTT) at 37°C in a 50 μL system for 2 h. To accurately detect whether the recombinant proteins could be phosphorylated by nSTK in vitro, two control groups were used. In one group, only recombinant protein samples were added, and in the other group, only the nSTK protein was added. The experimental group contained both recombinant protein and nSTK protein. Protein loading buffer was added to all the samples immediately after the reaction, and the samples were boiled at 100°C for 10 min. The proteins were then separated by standard Tris-glycine‒SDS polyacrylamide gel electrophoresis (PAGE) gels and Phos-tag‒SDS polyacrylamide gels containing 100 μM phos-tag solution (ApexBio Technology LLC, Houston, TX, USA) and 200 μM MnCl₂. After electrophoresis, the separated protein was observed by staining with Coomassie Brilliant Blue for 1 h and then decolorizing for a period of time.

In vivo phosphorylation assays

WT SS2, Δstk, CΔstk, and FadR-T230A strain were cultured in THY liquid medium by shaking until the logarithmic phase and then washed three times with fresh sterile PBS. The bacteria were resuspended in 0.01 M PBS containing lysozyme (1 mg/mL), protease inhibitor cocktail (1:100), and phosphatase inhibitor cocktail (1:100). After being incubated at 37°C for 1 h, ultrasonic fragmentation treatment was performed on ice (20 min: 4 s on, 6 s off). After ultrasonic fragmentation, the samples were centrifuged at 12000 × g for 30 min at 4°C to remove cell debris. FadR polyclonal antibodies were added to the supernatant and incubated at 4°C for 2 h, followed by the addition of A/G agarose (Beyotime) and overnight rotation at 4°C for immunoprecipitation. After the agarose protein complex was washed three times with precooled PBS buffer, the agarose was boiled in 5 × SDS loading buffer for 10 min. The proteins were detected through Western blot analysis with FadR polyclonal antibodies and anti-phosphorylation (threonine) antibodies (Santa Cruz, USA). The experiment was performed three times.

Stressor sensitivity assay

To explore the effects of stressors on the phosphorylation level of FadR, WT SS2 strains were grown in 40 mL of THY at 37°C until the OD₆₀₀ reached ~0.6. The strains were subsequently washed three times with PBS, placed in pH 5.5 THY adjusted by hydrochloric acid, THY supplemented with 10 mM H₂O₂, and THY supplemented with 0.2 M NaCl, and incubated at 37°C for 30 min. Meanwhile, the survival rates of SS2 were calculated on the basis of the number of bacterial colonies before and after different treatment. Then, the strains were collected by centrifugation at 5000 × g and 4°C for 10 min. A protease inhibitor and phosphatase inhibitor were added to the strains, and the strains mixture was sonicated (power 300 w, ultrasonication for 4 s, 6 s interval, 10 min) in an ice bath and centrifuged at 12000 × g for 10 min at 4°C. Two microliters of FadR polyclonal antibody was added to the supernatant, and the mixture was incubated at 4°C for 2 h. Then, 50 μL of protein A/G agarose beads was added, and the mixture was incubated overnight at 4°C. The next day, the beads were collected by centrifugation at 4°C and 2500 × g for 5 min. After the precipitate was washed with precooled PBS three times, a certain amount of 5 × SDS‒PAGE loading buffer was added, and after boiling for 10 min, SDS‒PAGE gel electrophoresis and Western blot analysis were performed. For each experiment, at least three biological replicates were performed.

Animal experiments

All 4‒6-week-old female mice were purchased from the Comparative Medicine Center of Yangzhou University (Yangzhou, China). To study the survival curves of the mice, the animals were randomly divided into different experimental groups, and intraperitoneally injected with WT SS2, fadR variants, Δadi, or FadR-T230E-Cadi strains at a dose of 2 × 10⁸ CFUs. Sterile PBS was used as a blank control. Survival rates and clinical manifestations were closely monitored for each mouse within 7 days after inoculation with the bacteria. To investigate the colonization ability of different strains of bacteria in the internal organs of BALB/c mice, these bacteria were injected at a dose of 2 × 10⁷ CFUs. The bacterial loads in the blood, liver, spleen, lungs, and brain were measured 24 h after each strain was injected. To conduct pathological analysis of various tissues, the liver, spleen, lungs, and brain of infected BALB/c mice were first fixed and embedded in paraffin, then sectioned in paraffin, and finally stained with hematoxylin and eosin. All stained sections were scanned by white light through a panoramic scanner, and the lesions were observed. The blind method was used to evaluate the microscopic lesions in 5 random areas of each tissue slice, and scoring was performed as follows: 0, no lesions; 1, minimal; 2, mild; 3, moderate; and 4, severe.

RNA-seq analysis

WT SS2 and ΔfadR strains were cultured at 37°C until OD₆₀₀ = 0.8. Each strain was cultured respectively in triplicate and then mixed in equal amounts [57]. Total RNA was extracted from bacteria using Total RNA Extraction Reagent (Vazyme Biotech Co., China) according to the manufacturer’s instructions. RNA-seq was performed at Genepioneer Biotechnologies (Nanjing, People’s Republic of China). Differential expression analysis between the two groups was conducted using the DESeq R package 1.18.0. The resulting P values were adjusted using Benjamini and Hochberg’s method to control the false discovery rate. Genes with an adjusted P value of ≤ 0.05 found by DESeq were assigned as differentially expressed.

EMSA

A 6% nondenaturing polyacrylamide gel was used for electrophoresis to detect the binding of the FadR protein to the adi promoter. The online tool BProm program (SoftBerry) was used to predict the promoter sequence of adi, amplify the target fragment by PCR, and purify the obtained product with a gel electrophoresis kit. The negative control was a DNA fragments ~197 bp in length from the enolase promoter region. The purified DNA fragments were incubated with FadR or point mutant protein in binding buffer (10 mM Tris-base, 50 mM KCl, 5 mM MgCl₂, 1 mM DTT, 0.05% Nonidet P-40, and 2.5% glycerol, pH 7.5) for 30 min at 37°C. Protein‒DNA complexes were added to a 6% nondenaturing polyacrylamide gel, which was then electrophoretically separated at 150 V in 0.5 × TBE buffer for 2.5 h and observed by ethidium bromide staining.

RNA isolation and RT‒qPCR assays

Total RNA was extracted from bacteria with Total RNA Extraction Reagent (Vazyme Biotech Co., China). Bacterial cDNA samples were obtained with a HiScript II Q RT SuperMix Kit (Vazyme Biotech Co., China). All cDNA was used as a template for real-time quantitative PCR on a 7300 Real-Time PCR System (Applied Biosystems, Foster City, California, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., China). The primer sequences are listed in the S5 Table. The gapdh gene was used as an internal control, and the relative fold changes in transcript levels were calculated by the 2^-ΔΔCt method. The assays were performed in triplicate, and the qRT‒PCR experiments were repeated 3 times for each group.

β-Galactosidase activity assays

The promoter of adi was amplified with the primers shown in the S5 Table. The obtained PCR products were fused with the empty plasmid pTCV-lacZ and then electroporated into WT SS2, ΔfadR, FadR-T230A, and FadR-T230E. The determination of β-galactosidase activity was carried out by previously reported methods [58]. Each test was performed in triplicate and repeated at least three times.

ChIP assay

After CΔfadR-flag was cultured to the logarithmic growth stage, it was washed three times with 0.01 M PBS, fixed at room temperature with 1% formaldehyde for 10 min, and finally crosslinked with 0.125 M glycine for 5 min. The bacterial cells were collected, centrifuged at 5000 × g at 4°C, washed three times with precooled 0.01 M PBS, and resuspended in cold lysis buffer. The genome was sonicated and fragmented into DNA fragments within the range of 0.5–1.0 kb. The sonicated sample was centrifuged at 12000 × g for 30 min at 4°C, and the supernatant was collected. Fifty microliters of the supernatant was retained. As an input, anti-flag antibody (Engibody, WI, USA) or negative mouse IgG (Beyotime, Shanghai, China) was added to the remaining supernatant for immunoprecipitation, and the mixture was incubated overnight on a rotating shaker at 4°C. Protein A/G beads (Beyotime) were added and incubated on a rotating shaker at 4°C for 2 h. The immunoprecipitation complexes were collected and washed three times each with ChIP washing buffer and TE solution, followed by elution with ChIP elution solution. All protein and RNA were removed by adding proteinase K and RNase (TAKARA, Dalian, China) to the eluent, and DNA fragments were recovered with a pure DNA fragment kit (Omega, USA). The input group and the recovered DNA fragment products were used as templates, and primers were designed on the basis of the adi promoter DNA sequence for PCR amplification and identification analysis. Each test was repeated at least three times.

Western blots

Electrophoretically separated proteins were transferred onto polyvinylidene fluoride (PVDF, Millipore) membrane using a Semi-Dry Transfer Device (Bio-Rad) and run at 10 V for 20 min. The membrane was blocked in 5% non-fat milk or 1% bovine serum albumin for protein detection in TBST for 2 h at room temperature. The membranes were then incubated with primary antibodies at 4°C overnight. The primary antibodies used were as follows: Mouse anti-phosphotyrosine antibodies (Santa Cruz Biotechnology, Cat. No. sc-5267) at a 1:1000 dilution and mouse polyclonal anti-serum against FadR, ADI, GAPDH, and GroEL at a 1:1000 dilution.

The membranes were washed with TBST and then incubated with Anti-mouse IgG conjugated to HRP (1:5,000; Abmart, Cat. No. M21001S) at room temperature for 1 h. The membranes were incubated with ECL Femto-Detect Western Blotting Substrate (Engibody Biotechnology) and exposed using a ChemiDoc Touch Imaging System (Bio-Rad), and the resulting images were analyzed using Image Lab software. The immunoblots represent at least three independent replicates.

Stressor sensitivity assay

The survival ability of bacteria under acid stress was determined according to the method of Roy S with slight modifications [59]. In general, SS2 was cultured to an OD₆₀₀ of ~0.6. After centrifugation, the bacteria were washed 3 times in PBS and resuspended in 1 mL of PBS (pH 5.0) for 1 h at 37°C. The survival rates of bacteria were calculated on the basis of the number of bacterial colonies before and after acidic treatment. Each test was repeated three times.

Intracellular survival assays

The intracellular survival assays of SS2 were performed with slight modifications compared with the method outlined in previous reports [60]. RAW264.7 cells were uniformly cultured in 24-well plates containing DMEM supplemented with 10% serum. When the cells were cultured to a density of 80%, bacteria were added at an initial multiplicity of infection multiple (MOI) of 10:1 and centrifuged at 400 × g at room temperature to promote sufficient direct contact between bacteria and cells. For the CQ group, RAW264.7 cells were treated with 40 μM CQ at the same time that bacterial infection. The cells were then cultured at 37°C and 5% CO₂ for 1 h. After coculturing, the cells were washed three times with 0.01 M PBS, and then DMEM containing 100 µg/mL gentamicin and 10 µg/mL penicillin was added, followed by continuous cultivation for 1 h and 3 h. After cultivation, the cells were washed three times with PBS and then lysed with sterile water for 10 min. The lysate was continuously diluted and evenly dropped onto THY agar plates, followed by colony counting. The cell viability was calculated as CFU_3h/CFU_1h× 100%. All experiments were repeated 3 times, with 3 independent samples each time.

Determining the intracellular pH in SS2

The calibration of the intracellular pH was carried out with using the manufacturer’s provided intracellular pH buffer calibration kit (Invitrogen, California, USA) as described previously [61]. The cells were treated with the intracellular pH calibration buffers by adding valinemycin (1 mM) and nigericin (1 mM) to ensure consistency in pH between the intracellular and extracellular environments, and then, the pH was measured by adding the intracellular fluorescent pH indicator BCECF-AM (Beyotime, Shanghai, China) and incubating at 37°C for 30 min. The fluorescence intensity was measured under 488 nm excitation and 535 nm excitation using a standard microplate reader. The calibration curve for SS2 was determined in the presence of various pH values ranging from 4.5–7.5 and was used in subsequent measurements to determine the intracellular pH in different strains of bacteria.

To measure the intracellular pH in SS2, the intracellular pH was measured with the indicator BCECF-AM as described previously. Bacteria were grown in THY media to an OD₆₀₀ of ~0.6, washed with PBS 3 times, suspended in PBS (pH 7.6) with the indicator BCECF-AM and incubated at 37°C for 30 min. After 30 min, the fluorescence value was measured once, and then, the bacteria were switched from PBS medium with a pH value of 7.6 to PBS medium with a pH of 5.5 and incubated at 37°C for 1 h [62]. After incubation, the fluorescence value was measured once. Finally, the changes in the intracellular pH of the bacteria were compared. All spectra were measured for three biological replicates at the intracellular pH of each bacteria in 96-well black microplates (Beyotime). The experiment was repeated 3 times.

Extraction and quantitative analysis of arginine

SS2 was cultured in THY medium to an OD₆₀₀ of ~ 0.6, harvested by centrifugation (5 min; 5,000 × g; 4°C), washed 3 times, and subsequently resuspended in 0.01 M PBS at pH 5.5 for 1 h. Bacterial arginine concentrations were determined with an arginine assay kit (Solarbio, Beijing, China). The bacteria were disrupted by ultrasound in an ice bath (power 300 W, ultrasonication for 4 s, 6 s interval, 10 min) and centrifuged at 12000 × g for 10 min. A total of 800 μL of the supernatant was collected, after which 150 μL of extract solution 2 was slowly added, followed by blowing and mixing until no bubbles formed. The mixed mixture was centrifuged at 12000 × g for 10 min at 4°C, and the supernatant was removed for testing according to the instructions.

The different strains were added into the RAW264.7 cell holes at an MOI of 100:1 incubating for 1 h. The hole without strain was used as the control. The culture medium in these wells was replaced with DMEM containing gentamicin (100 µg/mL) and penicillin (10 µg/mL). The arginine content produced by RAW264.7 was determined using an arginine assay kit (Solarbio, China) after incubating for 3 h.

In addition, we incubated the recombinant protein of the ADI enzyme with the arginine standard at 37°C in reaction buffer (50 mM Tris, pH 7.6, 10 mM MgCl₂, 1 mM DTT, and 5 mM ATP) and measured the arginine content every 5 min for a total of 30 min to better determine whether arginine could be broken down by ADI. Each assay was performed three times independently.

Preparation of cell extracts and measurement of ammonia concentrations

The bacterial ammonia concentrations were determined with an ammonia assay kit (Abcam, UK). This reagent reacts with ammonia/ammonium and forms a fluorescent product. The fluorescence intensity (λ_ex/em = 360/450 nm) is proportional to the ammonia concentration in the sample, and the standard curve was should be established first. Similarly, when S. suis was cultured in THY medium to an OD₆₀₀ of approximately 0.6, it was washed three times with PBS and then incubated at 37°C for 1 h in PBS (pH 5.5). After cultivation, 10 μL of the supernatant was directly centrifuged to determine the ammonia content. The remaining precipitate was washed three times with PBS and then sonicated (power 300 W, ultrasonication for 4 s, 6 s interval, 10 min) in an ice bath. After crushing, the mixture was centrifuged at 12000 × g for 10 min, and 10 μL of the supernatant was collected to measure the ammonia content. All ammonia content measurements were performed according to the manufacturer’s method.

Determination of nitric-oxide production

The different strains were added into the RAW264.7 cell holes at an MOI of 100:1 incubating for 1 h. The hole without strain was used as the control. The culture medium in these wells was replaced with DMEM containing gentamicin (100 µg/mL) and penicillin (10 µg/mL). The NO content produced by RAW264.7 cells was determined using a micro NO content assay kit (Solarbio, China) after 24 h.

ATR assays

The experimental method of ATR has been modified as described previously [16]. The bacteria were cultured to the logarithmic phase and then incubated at 37°C in THY with different pH values for 2 h to obtain the sublethal pH and lethal pH of WT SS2. For non-acid-induced conditions, bacterial cells were first grown in neutral THY at 37°C, and when cultures reached OD_600nm ~ 0.6, 100 μL aliquots were taken and added to 900 μL of THY (lethal pH) and incubated for 2 h at 37°C. In parallel, to determine survival under acidic-induced conditions, bacterial cells were grown in neutral THY until OD_600nm ~ 0.6, centrifuged at 5,000 g for 5 min, resuspended in THY (sublethal pH) and incubated for 2 h at 37°C. Then, 100 μL aliquots were taken and added to 900 μL of THY (lethal pH) and incubated for 2 h at 37°C. The survival rates of bacteria were calculated on the basis of the number of bacterial colonies before and after acidic treatment.

Data analysis

GraphPad Prism software, version 8.0 (La Jolla, CA, USA), was used for data analysis in this study. All bar charts were analyzed with the mean ± standard deviation (SD) calculated from at least three biological replicates. All the data were analyzed with an unpaired t test (comparison of 2 groups) and one-way ANOVA or two-way ANOVA (comparison of survival curves) to analyze the statistical significance. A P value of < 0.05 was considered to indicate a significant difference.