Two-Component Signal Transduction System VraSR Contributes to Neuroinammation in Streptococcus Suis Meningitis

Background: Streptococcus suis (S. suis) is an important zoonotic pathogen that can cause high morbidity and mortality in both humans and swine. As the most important life-threatening infection of the central nervous system (CNS), meningitis is an important symptom of S. suis infection. The VraSR is a critical two-component signal transduction system that affects S. suis ability to resist against host innate immune system and promotes the ability of S. suis to adhere to hBMEC. Whether and how VraSR contributes to the development of S. suis meningitis are currently unknown. Methods: The in vivo colonization, in vivo BBB permeability, histopathological examination and immunohistochemistry were applied to compare and characterize the degree of destruction of brain tissue in response to wild type SC19 and mutant ΔvraSR. Western blotting and real-time PCR were combined to identify the breakdown of tight junction proteins (TJ proteins). The secretion of proinammatory cytokines and chemokines in the serum were detected on a BD FACSVerse ow cytometer. Results: We found an important role of VraSR regulatory system in S. suis SC19-induced meningitis. A mouse infection model demonstrated that ΔvraSR had signicantly attenuated inammatory lesions in the brain tissues compared with wild-type S. suis. In vitro, we characterized that SC19 could increase the blood-brain barrier (BBB) permeability through downregulating the TJ proteins compared with mutant ΔvraSR. Moreover, we found signicant generation of proinammatory cytokines and chemokines in the serum including IL-6, TNF-α, MCP-1, and IL-12p70 compared with ΔvraSR infected mice. Conclusions: For the rst time, our work investigated the VraSR regulatory system of S. suis played an important role in streptococcal meningitis and revealed VraSR to be an important contributor to the disruption of TJ proteins. Characterization of these BBB disruption will facilitate further study of meningitis mechanisms in humans, thereby offering the development Future studies will aim to understand the precise molecular regulated by VraSR which is critical for the development of meningitis induced by S. suis. Our study of the role of VraSR in S. suis meningitis indicates that therapies targeted at VraSR TCS could contribute to preventing the development of streptococcal meningitis.


Background
Streptococcus suis is a major swine pathogen that can cause serious diseases including septicemia, arthritis, endocarditis, pneumonia, meningitis, endophthalmitis, as well as sudden death, and it results in serious economic losses in the porcine industry worldwide [1,2]. S. suis can be transmitted to humans through direct contact with contaminated raw pork products or infected pigs, resulting in streptococcal toxic shock-like syndrome (STSLS) and meningitis [3][4][5]. So far, S. suis infections in humans have been reported in Asia, Europe, America, Oceania [2,6,7]. However, two large-scale human cases of S. suis infection in China, the one case was 14 deaths in Jiangsu in 1998, and the another was 204 cases with a fatality rate reaching 20 % in Sichuan in 2005 [2,5,8]. Among the 29 S. suis serotypes, S. suis 2 is the most prevalent in pigs and humans [9][10][11]. In southern Vietnam and Thailand, S. suis was the most frequent pathogen responsible for bacterial meningitis [12,13]. However, the mechanisms that S. suis pass across the blood-brain barrier (BBB) to cause meningitis are poorly understood.
Bacterial meningitis, an in ammation of meninges, continues to be an important life-threatening infection with high mortality and morbidity throughout the world. It could affect the pia, arachnoid, and subarachnoid space, and most survivors sustain neurological sequelae such as permanent deafness [14,15]. It is prerequisite that pathogens invade and traverse across the BBB for central nervous system (CNS) infection. The BBB, a structural and functional barrier, which can maintain CNS homeostasis by regulating the passage of molecules in and out of the brain tissue and protect the brain from pathogens and toxins into circulation. It is formed by brain microvascular endothelial cells (BMECs), astrocytes and pericytes [14,16]. Pericytes and astrocytes are responsible for maintaining the BBB properties. As the indispensable structural component of BBB, BMECs are linked by cytoplasmic zonala-occludin family members (such as ZO-1, ZO-2, and ZO-3) and tight junction (TJ) proteins (such as β-catenin, Occludin, and Claudins) [17,18]. Decreasing or destroying TJ proteins could increase the permeability of BBB, which is an indicator of BBB dysfunction [19]. Now, how the S. suis causes meningitis and STSLS and leads to high mortality and morbidity remains unclear. Previous literature has demonstrated that S. suis in humans could induce the generation of interleukin (IL)-1β, IL-6, IL-8, IL-12, tumor necrosis factor-alpha (TNF-α) [20]. An investigation showed high IL-6, IL-1α, monocyte chemoattractant protein-1 (MCP-1), MIP-2 and chemokine (C-X-C motif) ligand 1 (CXCL1/GRO-α) levels in the blood and brain of mice with meningitis [13]. The excessive production of proin ammatory cytokines was con rmed to be an important cause of septicemia, STSLS, and meningitis [21,22]. S. suis virulence factors, such as SsPA, SLY, CPS, MRP, have been reported to mediate the release of proin ammatory cytokines, and contribute to the occurrence of meningitis [23][24][25]. Under the stimulation of S. suis, hBMEC can secrete arachidonic acid which would help pathogens enter brain tissue and regulate the local in ammation [26]. A report found that EGFR transactivation contributed to CNS infection in S. suis meningitis [13]. However, the mechanism of S. suis meningitis is as yet poorly understood.
Two-component signal transduction systems (TCSs) are an important mechanism that bacteria monitor, respond and adapt to environmental changes. TCSs typically consist of a membrane-bound sensor histidine kinase (HK) and a cytoplasmic response regulator (RR) [27,28]. TCSs have been shown to be related to bacterial resistance, bio lm synthesis and virulence [29][30][31]. For example, SaeS, an important sensor, could detect α-defendin1 (HNP-1) and response to neutrophil-derived stimuli in staphylococcus aureus [32]. Among 15 TCSs of S. suis, CiaRH, VirR/VirR, Ihk/Irr, NisK/NisR, SalK/SalR, and CovR have been con rmed to regulations of virulence [11]. The 1910HK/RR signal transduction system can not only promote the adhesion of S. suis to HEp-2 cells, but also promote immune escape, and had an important impact on the pathogenic ability of mice and piglets [33]. Knockout of ItdR gene in group B streptococcus resulted in a signi cant increase in bacterial invasion in hBMEC, as well as disruption of BBB and meningitis in vivo [34]. Ihk/Irr had important effects on cell adhesion, anti-macrophage killing, oxidative stress, and pathogenicity [35]. Further studies had shown that Ihk/Irr directly regulated metal endopeptidase SsPepO, and SsPepO is an important virulence factor for meningitis caused by S. suis [24]. We have found that disruption of VraSR resulted decrease in the ability to resist neutrophil killing and phagocytosis, as well as a signi cant decrease in cell adhesion and animal experiments also veri ed that the virulence of the missing strain decreased [11]. However, whether the VraSR TCS plays a role in the process of streptococcal meningitis is still unknown.
In this study, we provided evidences that VraSR is an essential TCS for S. suis meningitis. Moreover, S. suis infection led to disruption of TJ proteins, resulting in the increase of BBB permeability. Bacterial infection also leaded to production of chemokines and proin ammatory cytokines, which further accelerated BBB disruption. These ndings provide evidences supporting the role of VraSR in S. suis mediated CNS dysfunction, which broaden our horizons on streptococcal meningitis.

Methods
Bacterial strains and cell culture S. suis strain SC19 was originally isolated from a disease pig brain during the Sichuan Province S. suis outbreak in 2005 [36]. ΔvraSR mutant was a deletion of VraSR gene in SC19 through homologous recombination. And CΔvraSR was the complementary strain of ΔvraSR [11]. All strains were cultured in TSB broth (BD) or plated on TSA (BD) with 10 % (vol/vol) fetal bovine serum (FBS) at 37 ℃. Spectinomycin (100 μg/mL) was incorporated into the growth medium when required.

In vivo colonization
For the in vivo colonization assay, the ve-week-old female CD1 mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China), were used for induction of hematogenous bacterial meningitis. Mice were injected intravenously with 2 × 10 8 CFUs in sterile PBS. At the indicated time points, mice were anesthetized and blood was collected for quantitative circulating bacterial cultures and serum collection. Mice were subsequently perfused as previously described [39]. The brains were homogenized and plated to determine the bacterial counts.

Evaluation of BBB Permeability
Evan's blue was used to assess BBB permeability, which binds to serum albumin and not enter into the CNS when the BBB is integral. Brie y. Mice were injected with 2 × 10 8 CFUs or PBS, at 72 hours postinfection, 300 µL Evan's blue (EB) solution (1 % in PBS) was injected intraperitoneally into the mice for 1 hour. Mice were anesthetized and perfused transcardially with sterile PBS. And then, brains were removed, and photographed [24].

Histopathological examinations and IHC
The brain samples were xed in 4 % paraformaldehyde solution followed by embedding in para n.
Sections were mounted on adhesive glass slides, dewaxed in xylene, and rehydrated in descending graded ethanol for the hematoxylin and eosin (H&E) histopathological staining [40]. For IHC, the sections were depara nized in xylene, rehydrated in ethanol, incubated in 3 % hydrogen peroxide to quench endogenous peroxidase and performed in 10 mM citrate buffer. Then the sections were blocked with 5 % BSA for 1 h at room temperature, and incubated with antibody at 4 ℃ overnight. After that, the sections were incubated with secondary antibodies and Diaminobenzidine (DAB) was utilized for color development [24].

Western blotting
Infected and uninfected hBMEC were collected and lysed in RIPA buffer supplemented with a protease inhibitor, sonicated and centrifuged at 10,000 g for 10 min at 4 °C to remove insoluble cell debris. The protein concentration in supernatant was measured using BCA protein assay kit (Beyotime, Shanghai, China). Cell lysates were then separated on 8 %-12 % SDS-PAGE, and transferred to PVDF membranes (Bio-Rad, CA, USA). The blots were blocked with 5 % BSA in Tris-buffered saline with Tween 20 at room temperature for 1-2h and incubated overnight at 4 °C with primary antibodies against β-actin, ZO-1, β-Catenin, Occludin, Claudin-5. After that, the blots were washed and incubated with HRP-conjugated antirabbit or anti-mouse IgG at 37 °C for 1h, and visualized with ECL reagents (Meilunbio, Dalian, China). The densitometric analysis was performed using Image J software (Bio-Rad).
RNA extraction and quantitative real-time PCR Total RNA from cells were extracted using the TRIpure reagent (Aidlab biotechnologies CO. Ltd, Beijing, China). 1 μg of total RNA was used for cDNA synthesis using the HiScript II Q RT SuperMix for qPCR with gDNA Eraser (Vazyme, Nanjing, China). Quantitative real-time PCR was performed with ViiATM 7 Real-Time PCR System (Applied BioSystems, Foster City, CA, USA) using Power SYBR Green PCR master mix (Vazyme, Nanjing, China) according to the manufacturers' instructions. Primers for real-time PCR are listed in Table 1 [16]. Expression levels of target genes were normalized to GAPDH by 2 −ΔΔCT method. Each assay was performed in triplicate. Cytokine measurement assays CD1 mice were injected with 2 × 10 8 CFUs of SC19, ΔvraSR or CΔvraSR as described above. At each indicated time. Serum was prepared from the blood samples and stored at -80 °C. The concentration of in ammatory cytokines in the serum was measured on a BD FACSVerse ow cytometer using a CBA mouse in ammation kit (BD, USA) according to the manufacturers' instructions. The data were analyzed with FCAP Array software.

Statistical analysis
Data were expressed as the mean ± SD. Signi cance of the differences between each group was analyzed by Student's t test and GraphPad Prism version 8.0 (GraphPad Software Inc., La Jolla, CA, USA). For all tests, P < 0.05 (*) was considered signi cant, and p < 0.01 (**), as well as < 0.001 (***) were all considered extremely signi cant.

VraSR contributes to development of meningitis in vivo
We rst analyzed S. suis SC19 and ΔvraSR infection of the brain in vivo. 5-week-old CD1 mice were intravenously injected at dosage of 2 × 10 8 CFUs SC19 or ΔvraSR for 48 hours to analyzed bacterial colonization in the brain and blood. The bacterial load in the blood and brains of SC19-infected and CΔVraSR-infected mice were signi cantly higher than of ΔvraSR-infected group (Fig. 1a), these results con rmed the weak ability of mutant ΔvraSR in forming bacteremia and colonizing brain. And the SC19infected mice showed severe symptoms, such as trembling, circling, paddling and opisthotonos, but these neurological symptoms were not observed in ΔvraSR infected mice (date not shown). Histologic examination of the brain tissue infected with SC19 showed classic histopathological changes of meningitis, such as meningeal thickening, in ammatory cell accumulation, and meningorrhagia, but this was not obvious in ΔvraSR infected mice. The presence of WT S. suis SC19 in the brains was observed by immunohistochemical analysis, but the ΔvraSR mutant was rarely found, meanwhile, we could observe the CΔvraSR in brain tissue section (Fig. 1b). In addition, the production of proin ammatory cytokines and chemokines in serum were detected at the indicated time points. As shown, the cytokines and chemokines (including IL-6, TNF-α, IL-12p70, MCP-1, IFN-γ) increased rapidly in infected with either SC19 or the mutant ΔvraSR compared with uninfected controls as early as 2h, and the average concentration of these cytokines (IL-6, MCP-1, IL-12p70, TNF-α) in SC19 infected mice were obviously higher than that of ΔvraSR-infected group. The production of IL-6 and TNF-α increased sharply to the highest at 2 hours postinfection (PI), and TNF-α rapidly decreased to nearly basal levels at 18h PI. We also measured the production of the anti-in ammatory cytokine IL-10, there was no signi cant difference in SC19-infected and ΔvraSR-infected mice, they all rapidly raised at 2h PI and decreased to the initial level at 18h PI (Fig.  1c). Taking together, these results demonstrate that VraSR contributes to the pathogenesis of meningitis.

VraSR contributes to the increased BBB permeability
We next investigated if mutant ΔvraSR could induce the disruption of BBB. Evan's blue which binds to serum albumin to be protein tracer and permeate into the damaged tissues, was used to evaluate the change of BBB permeability after S. suis infection. It was obvious that Evan's blue penetrated to the brains of SC19-infected mice more than the brains of ΔvraSR-infected mice (Fig. 2). Together, these observations directly indicated that VraSR might contribute to increase BBB permeability during the development of S. suis meningitis.
VraSR contributes to the BBB permeability via downregulating and disrupting the TJ proteins As the most important components of BBB, the TJ proteins determined the paracellular permeability. Therefore, we determined the alteration of these TJ proteins (ZO-1, β-catenin, Occludin, and Claudin-5) in the hBMEC of SC19-infected or ΔvraSR-infected hBMEC by real-time PCR and western Blotting in vitro. It was found that the expression of these junction associated genes in SC19-infected hBMEC were obviously decreased compared with ΔvraSR-infected hBMEC (Fig. 3a-3d). Meanwhile, the translation of these TJ proteins in SC19-infected hBMEC also showed the trends of downregulation (Fig. 3e). Therefore, these in vitro ndings suggest that S. suis VraSR induces BBB disruption via downregulating the expression of the TJ proteins.

Discussion
As an important zoonotic pathogen that causes public health problems and heavy economic losses in swine husbandry around the world, S. suis has been recognized as the fatal organism causing meningitis and streptococcal toxic shock-like syndrome [1,41]. There many articles have reported the mechanism of streptococcal meningitis. Adenosine of S. suis was reported to contribute to the activation of A1 adenosine receptor signaling cascade and cytoskeleton remodeling, thus promoting S. suis penetration across BBB [42]. Suilysin has been shown to remodel cytoskeleton of hBMEC by activating Rac1 GTPase and RhoA, therefore contributing to the breakdown of BBB [43]. In addition, recent study has demonstrated that S. suis 2 Enolase could bind to RPSA promoting the expression of HSPD1 and leading to the destroy of BBB integrity [44]. Studies also have reported the important roles of host targets in development of streptococcal meningitis, for example, TRIM32 is the key role that positively regulated the production of proin ammatory cytokines and chemokines including IL-18, TNF-α, IL-6, MIP-1α, RANTES, and MCP-1 secretion in mice, and was found to upregulated hemorrhage and bacterial loads in the brains [45]. S. suis infection could induce the transactivation of EGFR, thus triggered the MAPK-ERK1/2 and NF-κB signaling pathway, which contributing to the development of streptococcal meningitis [13]. TCSs which present in all domains of life, are an important signal transduction protein for bacteria to monitor and respond to environmental stimuli [46,47]. Some TCSs (e.g., 1910HK/RR, VirR/VirS, VraSR) have been identi ed to have important impacts on the ability to virulence and pathogenicity [11,48]. And studies also found that some TCSs deletion strains had lower capability to adhere to hBMEC [11]. However, there are very few studies on meningitis caused by TCSs.
During the induction of streptococcal meningitis, bacterial adherence of hBMEC is an essential step for disruption of BBB [14,24,49]. Our previous study found that ΔVraSR had lower adherence ability to hBMEC compared with wild S. suis SC19, bacterial burden experiments also showed that there are fewer bacteria in the brain of ΔVraSR-infected mice compared with SC19-infected mice [11]. Moreover, there are SC19-infected mice not ΔVraSR-infected mice had obvious neurological symptoms in the process of animal experiments (date not shown). These results implied that VraSR had an important role in S. suis induction of meningitis. In a mouse meningitis model, we found signi cant differences in the number of bacteria from blood and brains. Furthermore, in CD1 mice, infection with ΔVraSR strain showed there was less bacterial invasion and neutrophil in ltration in the brain tissue.
As a pathogen characterized by STSLS and meningitis, S. suis infection can stimulate in ammatory cytokines production (e.g., MCP-1, TNF-α, INF-γ, IL-12p70, IL-6), which might be responsible for a strong in ammatory response, nally leading to sudden death or BBB breakdown [1,5,13]. Previous study had found that high levels of IL-6, IL-12, IFN-γ, TNF-α, MCP-1, CXCL1, and CCL5 cytokines caused by S. suis 2 might be responsible for the sudden death of animals [21]. Neutrophil can be activated by cytokines such as TNF-α and IFN-γ [50]. Here, a similar situation was observed with SC19, the cytokine increased obviously in the blood at 2 h PI, indicating an acute in ammation response in mice. IL-6 and TNF-α reached a peak after 2 h of infection. Meanwhile INF-γ and IL-12p70 reached a peak at 6 h PI. Likewise, we observed the high production of chemokines and proin ammatory in ΔVraSR-infected mice. But there is signi cant difference between SC19-infected and ΔvraSR-infected mice, the in ammatory response caused by ΔvraSR was signi cantly lower than wild group. MCP-1 is a potent chemokine that recruits monocyte and exacerbate in ammation, and previous studies reported decreased BBB leakage, macrophage/microglia accumulation, and an increased neuronal density [51,52]. MCP-1 had been reported to be able to alter expression of TJ proteins in brain microvascular endothelial cells [53]. We found that MCP-1 in blood caused by SC19 infection was higher than that of ΔVraSR-infected group, this nding implies MCP-1 might be an important role in streptococcal meningitis. Whether MCP-1 plays an important role in streptococcal meningitis remains to be further investigated. Taken together, our observations supported that VraSR TCS plays an important role in S. suis induced in ammation storm.

suis induction of BBB disruption is a complex process between the host and pathogen. Many studies
have reported that as the most important junctional structure of BBB, the TJ proteins play important roles in maintain BBB integrity and stability of CNS microenvironment [17,54,55]. Recent

Conclusions
Our results showed that VraSR regulatory system is vital for S. suis to downregulate TJ proteins, and to increasing BBB permeability. Moreover, SC19-induced high level of cytokines and chemokines are also important contributors in the disruption of BBB. Future studies will aim to understand the precise molecular regulated by VraSR which is critical for the development of meningitis induced by S. suis. Our study of the role of VraSR in S. suis meningitis indicates that therapies targeted at VraSR TCS could contribute to preventing the development of streptococcal meningitis. SC19 infection induced a strong neuroin ammation compared with ΔvraSR. a Bacterial loads in the blood (CFU/ml of blood), and in the brain (CFU/g of tissue). b 5-week-old CD1 mice were injected intravenously with 2×108 CFUs SC19 strain, ΔvraSR or CΔvraSR. Brain histopathological changes in infected mice with neurological signs were examined by H&E staining (a, b, d, e, g, h, j, k). S. suis in the brains of mice was detected by IHC (c, f, i, l). Scale bar = 50 μm. c Serum was harvested at indicated time point, and the concentrations of cytokines were measured by a CBA Mouse in ammation Kit. Results were expressed as the mean ± SD from ve infected CD1 mice at each time point. Statistical analysis was carried out between the SC19-infected group at each time point and the ΔvraSR-infected group.

Figure 2
S. suis infection increased the blood-brain barrier permeability of mice. CD1 mice were challenged by S. suis at 3 days postinfection, Evan's blue was injected to evaluated the integrity of the BBB. S. suis infection enhanced the blood-brain barrier permeability of mice via inducing downregulation of the TJ proteins. a-d Real-time PCR analysis of the TJ proteins transcription in RNAs from infected hBMEC. GAPDH was used as the internal reference for the cellular RNAs in vitro. Analyzed data are presented as mean ± SD from three independent assays. e Western blotting analysis of the TJ proteins in hBMEC in response to S. suis infection. β-actin was used as the loading control, and densitometry was performed to analyze the difference.