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Streptococcus suis serotype 2 is a significant zoonotic pathogen that poses a serious threat to the health of humans and various animals. Ribosomal protein S1 (RPSA), acting as a key virulence factor, mediates SS2 adhesion to host cells and facilitates its penetration of the blood–brain barrier. In this study, we expressed the SS2 RPSA recombinant protein and used it to immunize BALB/c mice. Through screening, we obtained a high-affinity hybridoma clone and produced a specific anti-RPSA monoclonal antibody (designated mAb-RPSA-5E2). We found that mAb-RPSA-5E2 exhibited potent antibacterial activity, significantly inhibiting the proliferation of SS2 in vitro. In SS2-challenged mice, treatment with mAb-RPSA-5E2 significantly reduced mortality and alleviated pathological damage in the lung and brain tissues. Furthermore, evaluating the immunoprotective effect of the SS2-RPSA protein as a subunit vaccine revealed that this vaccine provided good protection against SS2 infection. These findings indicate that RPSA is a promising candidate target for developing SS2 subunit vaccines and targeted therapeutic biologics. Key points • The SS2 RPSA monoclonal antibody can inhibit the proliferation of SS2 in vitro. • The SS2 RPSA monoclonal antibody can reduce the mortality rates of mice. • The SS2-RPSA subunit vaccine provides significant immunoprotective effects in mice.

Streptococcus suis serotype 2 (SS2) is a major zoonotic pathogen responsible for severe infections in both pigs and humans. Despite its clinical significance, no effective vaccine is currently available, and the influence of capsular polysaccharide (CPS2) structural features on host immunity remains unclear. Here, we identified a fast-growing SS2 strain from a panel of 40 clinical isolates and established a novel, reliable CPS2 purification method. This approach combines polyethylenimine-mediated nucleic acid precipitation with phenol-based protein denaturation, eliminating the need for column chromatography and enabling gram-scale CPS2 recovery per liter of culture while preserving structural integrity. To examine the role of sialic acid, we compared antibody responses induced by native and desialylated CPS2 conjugates in mice, which revealed no significant differences. Based on this finding, a native CPS2 conjugate vaccine was subsequently evaluated in pigs, where it induced robust IgG responses and conferred significant protection against virulent SS2 challenge. The effects of different adjuvants and dosages were also investigated. Collectively, these results establish an efficient CPS2 production platform, clarify the limited impact of sialic acid on conjugate-induced antibody responses, and highlight the potential of CPS2-based conjugates as promising vaccine candidates against SS2 infection. •Developed a streamlined method for high-purity CPS2 purification.•Identified sialic acid as key for immune recognition but not vaccine efficacy.•Constructed a CPS2–protein conjugate vaccine using biotin–rhizavidin chemistry.•Vaccine elicited strong IgG responses and protected against virulent SS2.•Provides a promising strategy for SS2 vaccine development.

Streptococcus suis serotype 2 (SS2) is a prominent pathogen that impacts swine and presents a zoonotic threat to humans; it is a cause of bacterial meningitis, a severe condition linked to neurological impairment and elevated mortality rates. For SS2 to access the central nervous system, it must traverse the blood–brain barrier (BBB); however, the precise mechanisms underlying this process remain incompletely elucidated. In this study, we demonstrate that the RTX family exoprotein A (RfeA), which is secreted by SS2, can be internalized by human brain microvascular endothelial cells (hBMECs) via a caveolae/lipid raft-dependent pathway. RfeA subsequently induces pyroptosis through the NLRP3/Caspase-1 pathway, a process attributed to the increase in mitochondrial reactive oxygen species (mtROS). The interaction between the N-terminus of RfeA and voltage-dependent anion channel 1 (VDAC1) leads to mtROS production, which can be suppressed by a VDAC1 oligomerization inhibitor. RfeA-induced pyroptosis results in disruption of the BBB in both the hBMEC monolayer model and the mouse infection model, thereby promoting bacterial infection of the brain. These findings elucidate a novel mechanism by which SS2 induces pyroptosis to breach the BBB, suggesting a potential target for the prevention and treatment of SS2 infection.

Streptococcus suis serotype 2 (SS2) frequently colonizes the swine upper respiratory tract and can cause Streptococcal disease in swine with clinical manifestations of pneumonia, meningitis, and septicemia. Previously, we have shown that vimentin, a kind of intermediate filament protein, is involved in the penetration of SS2 through the tracheal epithelial barrier. The initiation of invasive disease is closely related to SS2-induced excessive local inflammation; however, the role of vimentin in airway epithelial inflammation remains unclear. Here, we show that vimentin deficient mice exhibit attenuated lung injury, diminished production of proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and the IL-8 homolog, keratinocyte-derived chemokine (KC), and substantially reduced neutrophils in the lungs following intranasal infection with SS2. We also found that swine tracheal epithelial cells (STEC) without vimentin show decreased transcription of IL-6 , TNF- α, and IL-8 . SS2 infection caused reassembly of vimentin in STEC, and pharmacological disruption of vimentin filaments prevented the transcription of those proinflammatory cytokines. Furthermore, deficiency of vimentin failed to increase the transcription of nucleotide oligomerization domain protein 2 (NOD2), which is known to interact with vimentin, and the phosphorylation of NF-κB protein p65. This study provides insights into how vimentin promotes excessive airway inflammation, thereby exacerbating airway injury and SS2-induced systemic infection.

Background Ribosomal protein SA (RPSA) of human brain microvascular endothelial cells (HBMECs) can transfer from the cytosol to the cell surface and act as a receptor for some pathogens, including Streptococcus suis serotype 2 (SS2), a zoonotic pathogen causing meningitis in pigs and humans. We previously reported that SS2 virulence factor enolase (ENO) binds to RPSA on the cell surface of HBMECs and induces apoptosis. However, the mechanism that activates RPSA translocation to the cell surface and induces ENO-mediated HBMEC apoptosis is unclear. Results Here, we show that RPSA localization and condensation on the host cell surface depend on its internally disordered region (IDR). ENO binds to the IDR of RPSA and promotes its interaction with RPSA and vimentin (VIM), which is significantly suppressed after 1,6-Hexanediol (1,6-Hex, a widely used tool to disrupt phase separation) treatment, indicating that ENO incorporation and thus the concentration of RPSA/VIM complexes via co-condensation. Furthermore, increasing intracellular calcium ions (Ca 2+ ) in response to SS2 infection further facilitates the liquid-like condensation of RPSA and aggravates ENO-induced HBMEC cell apoptosis. Conclusions Together, our study provides a previously underappreciated molecular mechanism illuminating that ENO-induced RPSA condensation activates the migration of RPSA to the bacterial cell surface and stimulates SS2-infected HBMEC death and, potentially, disease progression. This study offers a fresh avenue for investigation into the mechanism by which other harmful bacteria infect hosts via cell surfaces’ RPSA.

Streptococcus suis is one of the most important zoonotic pathogens causing serious diseases in both pigs and humans, especially serotype 2. In northern Thailand, there is a notable prevalence of S. suis infection in humans and transmission has occurred mainly through the consumption of raw pork products. Despite the continued practice of consuming raw pork in this region, limited data exist regarding S. suis contamination in such products. Therefore, this study aimed to assess the prevalence of S. suis and S. suis serotype 2 in retail raw pork meat and edible pig organs sold in Chiang Mai city, Thailand. A total of 200 samples, comprising raw pork meat and edible pig organs, were collected from nine fresh markets in Chiang Mai city between May and July 2023. Samples were prepared and cultured in Todd-Hewitt broth. Bacterial DNA was extracted and tested for any serotypes of S. suis and serotype 2 using loop-mediated isothermal amplification (LAMP) techniques. The study revealed contaminations of S. suis and serotype 2 at rates of 84% and 34%, respectively, with a higher prevalence observed in pig organs compared to raw pork. Both S. suis and serotype 2 were detected across all nine fresh markets investigated. The prevalence of S. suis remained consistently high throughout the study period, whereas serotype 2 showed peaks in May and July. These high rates of contamination indicate that people who consume or work in close contact with raw pork or edible pig organs are at a high risk of S. suis infection. Urgent implementation and maintenance of food safety campaigns and public health interventions are crucial for disease prevention and control.

serotype 2 is a major pathogen of swine streptococcicosis, which result in serious economic loss worldwide. SS2 is an important zoonosis causing meningitis and even death in humans. Neutrophil extracellular traps (NETs) constitute a significant bactericidal strategy of innate immune. The battle between SS2 and NETs may account for the pathogenicity of SS2. However, the molecular mechanism underlying release of SS2-induced NETs remains unclear. In this study, SS2 was found to induce NETs within 2-4 h, and was dependent on reactive oxygen species (ROS) from NADPH oxidase. Moreover, SS2 could activate neutrophil p38 MAPK and ERK1/2. Blockage of p38 MAPK or ERK1/2 activation decreased SS2-induced NETs formation by 65 and 85%, respectively. In addition, NADPH oxidase derived ROS inhibition negatively affected phosphorylation of p38 MAPK and ERK1/2 in SS2 induced neutrophils. Both TLR2 and TLR4 were significantly up-regulated by SS2 infection in blood cells and neutrophils , which indicates these two receptors are involved in SS2 recognition. Blocking TLR4 signaling could further inhibit the activation of ERK1/2, but not p38 MAPK; however, TLR4 signaling inhibition reduced NETs formation induced by SS2. In conclusion, SS2 could be recognized by TLR2 and/or TLR4, initiating NETs formation signaling pathways in a NADPH oxidase derived ROS dependent manner. ROS will activate p38 MAPK and ERK1/2, which ultimately induces NETs formation.

Streptococcus suis serotype 2 (S. suis 2) is a highly invasive pathogen in pigs and humans that can cause severe systemic infection. Sepsis and meningitis are the most common clinical manifestations of S. suis 2 infection. However, the mechanisms of S. suis 2 surviving in human blood remains unclear, so to identify novel virulence factors in evasion of polymorphonuclear leukocyte (PMN)-mediated innate immunity play important roles in developing therapies against S. suis 2 infection. Here, we found that S. suis 2 can escape phagocytic clearance by adenosine synthesis in blood. Through bioinformatics-based analyses we identified a cell wall-anchored protein harbors a 5'-nucleotidase signature sequence and evidence strongly indicated that it can convert adenosine monophosphate (AMP) to adenosine. It was designated as Ssads (the adenosine synthase of S. suis 2). Furthermore, we found that Ssads could impair PMN's defense against S. suis 2 with decreasing of oxidative activity and degranulation of PMNs in human blood via A2a receptors. Additionally, this enzyme-deficient mutant was found to have diminished virulence in the piglet infection model. Taken together, these results indicate that Ssads play an important role in S. suis 2 escaping human innate immunity in the context of inhibiting PMN's activity by synthesis of adenosine.

Host proteins interacting with pathogens are receiving more attention as potential therapeutic targets in molecular medicine. Streptococcus suis serotype 2 (SS2) is an important cause of meningitis in both humans and pigs worldwide. SS2 Enolase (Eno) has previously been identified as a virulence factor with a role in altering blood brain barrier (BBB) integrity, but the host cell membrane receptor of Eno and The mechanism(s) involved are unclear. This study identified that SS2 Eno binds to 40S ribosomal protein SA (RPSA) on the surface of porcine brain microvascular endothelial cells leading to activation of intracellular p38/ERK-eIF4E signalling, which promotes intracellular expression of HSPD1 (heat-shock protein family D member 1), and initiation of host-cell apoptosis, and increased BBB permeability facilitating bacterial invasion. This study reveals novel functions for the host-interactional molecules RPSA and HSPD1 in BBB integrity, and provides insight for new therapeutic strategies in meningitis.

Streptococcus suis serotypes 2 and 14 are the most common zoonotic strains, but previous identification methods made distinguish these two serotypes from other S. suis serotypes difficult. To effectively prevent and control them, there is an urgent need for a highly sensitive and specific method to identify these two serotypes. In this study, a fluorescent probe was designed for the single nucleotide polymorphism site at cpsK 483 of Streptococcus suis type 2 and type 14 compared with other serotypes, and an enzyme-activated probe quantitative PCR (EA-probe qPCR) method was established for the detection of Streptococcus suis type 2 and type 14 by combining with the specific hydrolysis characteristics of the RNase H2 enzyme. The results showed that the optimal probe concentration for this method was 0.5 µM and the optimal RNase H2 enzyme concentration was 25 mU.This method showed no reactivity with genomic DNA from Streptococcus suis strains 1/2, 5, 7, 9, 23, 28, 29, and 31, confirming its high specificity. And its sensitivity can reach 18.4 CFU. In addition, 19 clinical strains of Streptococcus suis type 2 or type 1/2 were tested. The results showed 100% agreement with the gene sequencing method. In conclusion, this method can meet the needs of accurate laboratory testing of Streptococcus suis serotypes 2 and 14 and has value for clinical prevention.