Exploring the destructive synergy between IL-33 and Suilysin hemolysis on blood-brain barrier stability.

Streptococcus suis type 2 (SS2) is a zoonotic pathogen capable of eliciting meningitis, presenting significant challenges to both the swine industry and public health. Suilysin (Sly), one of SS2 most potent virulence determinants, releases a surfeit of inflammatory agents following red blood cell lysis. Notably, while current research on Sly role in SS2-induced meningitis predominantly centers on its interaction with the blood-brain barrier (BBB), the repercussions of Sly hemolytic products on BBB function have largely been sidestepped. In this vein, our study delves into the ramifications of Sly-induced hemolysis on BBB integrity. We discern that Sly hemolytic derivatives exacerbate the permeability of Sly-induced in vitro BBB models. Within these Sly hemolytic products, Interleukin-33 (IL-33) disrupts the expression and distribution of Claudin-5 in brain microvascular endothelial cells, facilitating the release of Interleukin-6 (IL-6) and Interleukin-8 (IL-8), thereby amplifying BBB permeability. Preliminary mechanistic insights suggest that IL-33-driven expression of IL-6 and IL-8 is orchestrated by the p38-mitogen-activated protein kinase signaling, whereas matrix metalloproteinase 9 mediates IL-33-induced suppression of Claudin-5. To validate these in vitro findings, an SS2-infected mouse model was established, and upon intravenous administration of growth stimulation expressed gene 2 (ST2) antibodies, in vivo results further underscored the pivotal role of the IL-33/ST2 axis during SS2 cerebral invasion. In summation, this study pioneerly illuminates the involvement of Sly hemolytic products in SS2-mediated BBB compromise and spotlights the instrumental role and primary mechanism of IL-33 therein. These insights enrich our comprehension of SS2 meningitis pathogenesis, laying pivotal groundwork for therapeutic advancements against SS2-induced meningitis.IMPORTANCEThe treatment of meningitis caused by Streptococcus suis type 2 (SS2) has always been a clinical challenge. Elucidating the molecular mechanisms by which SS2 breaches the blood-brain barrier (BBB) is crucial for the development of meningitis therapeutics. Suilysin (Sly) is one of the most important virulence factors of SS2, which can quickly lyse red blood cells and release large amounts of damage-associated molecular patterns, such as hemoglobin, IL-33, cyclophilin A, and so on. However, the impact of these hemolytic products on the function of BBB is unknown and ignored. This study is the first to investigate the effect of Sly hemolytic products on BBB function. The data are crucial for the study of the pathogenesis of SS2 meningitis and can provide an important reference for the development of meningitis therapeutics.

Orphan response regulator CovR plays positive regulative functions in the survivability and pathogenicity of Streptococcus suis serotype 2 isolated from a pig.

BACKGROUND: Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen. Orphan response regulator CovR plays crucial regulative functions in the survivability and pathogenicity of S. suis 2. However, research on the CovR in S. suis 2 is limited. RESULTS: In this study, the regulative functions of CovR in the survivability and pathogenicity were investigated in S. suis 2 isolated from a diseased pig. The deletion of CovR significantly weakened the survivability and pathogenicity of S. suis 2. Compared with the wild-type strain, ΔcovR showed slower growth rates and thinner capsular polysaccharides. Moreover, ΔcovR showed reduced adhesion and invasion to Hep-2 cells as well as anti-phagocytosis and anti-killing ability to 3D4 cells and anti-serum killing ability. In addition, the deletion of CovR significantly reduced the colonisation ability of S. suis 2 in mice. The survival rate of mice infected with ΔcovR was increased by 16.7% compared with that of mice infected with S. suis 2. Further, the deletion of CovR led to dramatic changes in metabolism-related pathways in S. suis 2, five of those, including fructose and mannose metabolism, glycerolipid metabolism, ABC transporters, amino sugar and nucleotide sugar metabolism and phosphotransferase system, were significantly down-regulated. CONCLUSIONS: Based on the results, CovR plays positive regulative functions in the survivability and pathogenicity of S. suis 2 SC19 strain isolated from a pig.

Transcriptome and metabolome profiling to elucidate the mechanism underlying the poor growth of Streptococcus suis serotype 2 after orphan response regulator CovR deletion.

The deletion of orphan response regulator CovR reduces the growth rate of Streptococcus suis serotype 2 (S. suis 2). In this study, metabolome and transcriptome profiling were performed to study the mechanisms underlying the poor growth of S. suis 2 caused by the deletion of orphan response regulator CovR. By comparing S. suis 2 (ΔcovR) and S. suis 2 (SC19), 146 differentially accumulated metabolites (upregulated: 83 and downregulated: 63) and 141 differentially expressed genes (upregulated: 86 and downregulated: 55) were identified. Metabolome and functional annotation analysis revealed that the growth of ΔcovR was inhibited by the imbalance aminoacyl tRNA biosynthesis (the low contents of L-lysine, L-aspartic acid, L-glutamine, and L-glutamic acid, and the high content of L-methionine). These results provide a new insight into the underlying poor growth of S. suis 2 caused by the deletion of orphan response regulator CovR. Metabolites and candidate genes regulated by the orphan response regulator CovR and involved in the growth of S. suis 2 were reported in this study.

Flanking N- and C-terminal domains of PrsA in Streptococcus suis type 2 are crucial for inducing cell death independent of TLR2 recognition.

Streptococcus suis type 2 (SS2), a major emerging/re-emerging zoonotic pathogen found in humans and pigs, can cause severe clinical infections, and pose public health issues. Our previous studies recognized peptidyl-prolyl isomerase (PrsA) as a critical virulence factor promoting SS2 pathogenicity. PrsA contributed to cell death and operated as a pro-inflammatory effector. However, the molecular pathways through which PrsA contributes to cell death are poorly understood. Here in this study, we prepared the recombinant PrsA protein and found that pyroptosis and necroptosis were involved in cell death stimulated by PrsA. Specific pyroptosis and necroptosis signalling inhibitors could significantly alleviate the fatal effect. Cleaved caspase-1 and IL-1ß in pyroptosis with phosphorylated MLKL proteins in necroptosis pathways, respectively, were activated after PrsA stimulation. Truncated protein fragments of enzymatic PPIase domain (PPI), N-terminal (NP), and C-terminal (PC) domains fused with PPIase, were expressed and purified. PrsA flanking N- or C-terminal but not enzymatic PPIase domain was found to be critical for PrsA function in inducing cell death and inflammation. Additionally, PrsA protein could be anchored on the cell surface to interact with host cells. However, Toll-like receptor 2 (TLR2) was not implicated in cell death and recognition of PrsA. PAMPs of PrsA could not promote TLR2 activation, and no rescued phenotypes of death were shown in cells blocking of TLR2 receptor or signal-transducing adaptor of MyD88. Overall, these data, for the first time, advanced our perspective on PrsA function and elucidated that PrsA-induced cell death requires its flanking N- or C-terminal domain but is dispensable for recognizing TLR2. Further efforts are still needed to explore the precise molecular mechanisms of PrsA-inducing cell death and, therefore, contribution to SS2 pathogenicity.

Deletion of lacD gene affected stress tolerance and virulence of Streptococcus suis serotype 2.

Streptococcus suis type 2 (S. suis type 2, SS2), an infectious pathogen which is zoonotic and can induce severely public health concern. Our previous research identified a newly differential secreted effector of tagatose-bisphosphate aldolase (LacD) mediated by VirD4 factor within the putative type IV secretion system of SS2, whereas the functional basis and roles in virulence of LacD remain elusive. Here in this study, the LacD was found enzymatic and can be activated to express under oxidative stress. Gene mutant and its complemental strain (ΔlacD and cΔlacD) were constructed to analyze the phenotypes, virulence and transcriptomic profiles as compared with the parental strain. The lacD gene deletion showed no effect on growth capability and cells morphology of SS2. However, reduced tolerance to oxidative and heat stress conditions, increased antimicrobial susceptibility to ciprofloxacin and kanamycin were found in ΔlacD strain. Further, the LacD deficiency led to weakened invasion and attenuated virulence since an easier phagocytosed and more prone to be cleared of SS2 in macrophages were shown in ΔlacD mutant. Distinctive transcriptional profiling in ΔlacD strain and typical down-regulated genes with significant mRNA changes including alcohol dehydrogenase, GTPase, integrative and conjugative elements, and iron ABC transporters which were mainly involved in cell division, stress response, antimicrobial susceptibility and virulence regulation, were examined and confirmed by RNA sequencing and real time qPCR. In summary, the results demonstrated for the first time that LacD was a pluripotent protein mediated the metabolic, stress and virulent effect of SS2.

Development of a recombinase polymerase amplification assay for rapid detection of Streptococcus suis type 2 in nasopharyngeal swab samples.

Streptococcus suis serotype 2 (SS2), an emerging zoonotic pathogen, may induce severe infections and symptoms manifested as septicemia, meningitis and even death both in human and pigs. The aim of this article was to develop a new methodology as real-time recombinase polymerase amplification (RT-RPA) assay targeting cps2J gene for the detection of SS2 (or SS1/2). The sensitivity and reproducibility of RT-RPA results were evaluated and compared with a real-time quantitative PCR (RT-qPCR). The established RT-RPA reaction could be completed in 20 minutes with distinguishable specificity against the predominant S. suis infection serotypes of 3, 4, 5, 7, 9, 14, and 31. Lower detection limit for RT-RPA was 102 genomic DNA copies per reaction. The specimen performance of RT-RPA was tested in nasopharyngeal swab samples with the sensitivity and specificity as 97.5% and 100%, respectively. Thus, this RT-RPA method is a rapid and potential molecular diagnostic tool for SS2 detection.

A serine/threonine phosphatase 1 of Streptococcus suis type 2 is an important virulence factor.

Streptococcus suis is regarded as one of the major pathogens of pigs, and Streptococcus suis type 2 (SS2) is considered a zoonotic bacterium based on its ability to cause meningitis and streptococcal toxic shock-like syndrome in humans. Many bacterial species contain genes encoding serine/threonine protein phosphatases (STPs) responsible for dephosphorylation of their substrates in a single reaction step. This study investigated the role of stp1 in the pathogenesis of SS2. An isogenic stp1 mutant (Δstp1) was constructed from SS2 strain ZJ081101. The Δstp1 mutant exhibited a significant increase in adhesion to HEp-2 and bEnd.3 cells as well as increased survival in RAW264.7 cells, as compared to the parent strain. Increased survival in macrophage cells might be related to resistance to reactive oxygen species since the Δstp1 mutant was more resistant than its parent strain to paraquat-induced oxidative stress. However, compared to parent strain virulence, deletion of stp1 significantly attenuated virulence of SS2 in mice, as shown by the nearly double lethal dose 50 value and the lower bacterial load in organs and blood in the murine model. We conclude that Stp1 has an essential role in SS2 virulence.

Roles of the Putative Type IV-like Secretion System Key Component VirD4 and PrsA in Pathogenesis of Streptococcus suis Type 2.

Streptococcus suis type 2 (SS2) is a zoonotic pathogen causing septic infection, meningitis and pneumonia in pigs and humans. SS2 may cause streptococcal toxic shock syndrome (STSS) probably due to excessive release of inflammatory cytokines. A previous study indicated that the virD4 gene in the putative type IV-like secretion system (T4SS) within the 89K pathogenicity island specific for recent epidemic strains contributed to the development of STSS. However, the functional basis of VirD4 in STSS remains unclear. Here we show that deletion of virD4 led to reduced virulence as shown by about 65% higher LD50, lower bacterial load in liver and brain, and lower level of expression of inflammatory cytokines in mice and cell lines than its parent strain. The ΔVirD4 mutant was more easily phagocytosed, suggesting its role as an anti-phagocytic factor. Oxidative stress that mimic bacterial exposure to respiratory burst of phagocytes upregulated expression of virD4. Proteomic analysis identified 10 secreted proteins of significant differences between the parent and mutant strains under oxidative stress, including PrsA, a peptidyl-prolyl isomerase. The SS2 PrsA expressed in E. coli caused a dose-dependent cell death and increased expression of proinflammatory IL-1ß, IL-6 and TNF-α in murine macrophage cells. Our data provide novel insights into the contribution of the VirD4 factor to STSS pathogenesis, possibly via its anti-phagocytic activity, upregulation of its expression upon oxidative stress and its involvement in increased secretion of PrsA as a cell death inducer and proinflammatory effector.

Superoxide dismutase of Streptococcus suis serotype 2 plays a role in anti-autophagic response by scavenging reactive oxygen species in infected macrophages.

Streptococcus suis serotype 2 (SS2) causes septic shock and meningitis. However, its pathogenesis is still not well-understood. We have recently shown that superoxide dismutase sodA of SS2 is a virulence factor probably by increasing resistance to oxidative stresses. Reactive oxygen species (ROS) are products of the respiratory burst of phagocytic cells and have been shown to activate autophagy. We wanted to know if and how SS2 explores its sodA to interfere with cell autophagic responses. A sodA deletion mutant (Δsod) was compared with its parent and complemented strain in autophagic response in the murine macrophage cell line RAW264.7. We found that the Δsod mutant induced significant autophagic responses in infected cells, shown as increased LC3 lipidation (LC3-II) and EGFP-LC3 punctae, than those infected by its parent or complemented strain at 1 or 2h post-infection. Co-localization of the autophagosomal EGFP-LC3 vesicles with lysosomes was seen in cells infected with Δsod mutant and its parent strain, indicating that SS2 infection induced complete autophagic responses. Reduced autophagic responses of cells infected with the wild-type strain might be related to decreased ROS by the scavenging effect of its sodA, as shown by increased superoxide anion or ROS level in cells infected with the Δsod mutant and in the cell free xanthine oxidase-hypoxanthine ROS-generating system, as compared with its parent or complemented strain. Taken together, SS2 makes use of its sodA for survival not only by scavenging ROS but also by alleviating the host autophagic responses due to ROS stimulation.

Assessment of the pathogenesis of Streptococcus suis type 2 infection in piglets for understanding streptococcal toxic shock-like syndrome, meningitis, and sequelae.

Streptococcus suis type 2 (SS2) is an zoonotic pathogen that had caused outbreaks in 1998 and 2005 in China. It is still not very clear how the disease progresses into the streptococcal toxic shock-like syndrome (STSLS) or meningitis, as well as the sequelae from the survivals. The present study used piglets as infection model to systematically investigate the pathogenesis of the infection caused by the SS2 strain 05ZYH33. The infected piglets showed joint swelling, lameness, and crouch at beginning, then developed into septic-like shock syndrome (SLSS) or prostration syndrome, at last the survivals showed physical activity impairment. The morbidity and mortality were 100% (71% for SLSS, 29% for prostration syndrome) and 29%, respectively. The pigs exhibiting SLSS had deep invasive infections in tissues and organs, and displayed more severe bacteremia and cytokine secretion in the bloodstream and organs than pigs with prostration syndrome. Moreover, the polymorphisms in the toll-like receptor 1 (TLR1) and TLR2 genes varied between the pigs affected with SLSS and prostration syndrome. Several lines of evidence indicated that SS2 infection progression into SLSS or relatively lighter prostration syndrome in pigs is closely related to the degrees of bacteremia and cytokine storm, which may be inherently determined by the diversity of innate immunity-associated genes. Furthermore, brain lesions, such as venous thrombosis, may directly contribute to the sequelae in human cases, were identified in the pigs. These results might help us to further understand the pathogenesis of SS2 in humans.

Catabolite control protein A is an important regulator of metabolism in Streptococcus suis type 2.

Streptococcus suis (S. suis) type 2 is an extremely important Gram-positive bacterial pathogen that can cause human or swine endocarditis, meningitis, bronchopneumonia, arthritis and sepsis. Catabolite control protein A (CcpA) is a major transcriptional regulator in S. suis type 2 that functions in catabolite control, specifically during growth on glucose or galactose. The regulation of central metabolism can affect the virulence of bacteria. In the present study, a metabolomics approach was used along with principal components analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA) models and 37 metabolites were found that differed substantially between native S. suis and a mutant lacking CcpA. These results showed that CcpA is an important protein in S. suis type 2 for studying bacterial protein function.

Prevalência de Streptococcus suis tipo 2 por meio da técnica de reação em cadeia da polimerase em suínos abatidos no Estado do Mato Grosso / Prevalence of Streptococcus suis type 2 using the polimerase chain reaction in slaughtered pigs in the State of Mato Grosso, Brazil

Streptococcus suis é um patógeno que a afeta a produção industrial de suínos em todo o mundo. É de extrema importância, pois está associado a doenças em suínos e humanos. O objetivo deste estudo foi determinar a prevalência do Streptococcus suis tipo 2 em 201 amostras de tonsilas de animais clinicamente sadios a partir da técnica de PCR. As amostras positivas foram submetidas à pesquisa do gene codificador do fator extracelular (ef). Os resultados demonstraram que a prevalência (23,38 por cento) foi maior que em outro estudo recentemente realizado no mesmo Estado, indicando que a PCR é um método mais sensível em relação ao isolamento bacteriano. Houve baixa ocorrência do gene ef* (1,49 por cento), o que mostra uma grande importância para população analisada, pois cepas negativas são potencialmente menos virulentas que cepas positivas.

Streptococcus suis is a pathogen that affects the industrial production of swine worldwide. It is extremely important, because it is associated with pigs and humans diseases. The aim of this study was to determine the prevalence of Streptococcus suis type 2 in 201 samples of tonsils from clinically healthy animals by the PCR technique. The samples positive for S. suis type 2 were tested for the gene encoding extracellular factors (ef). The results showed that the prevalence (23.38 percent) was higher than other recent survey in the State, demonstrating that the PCR is a more sensitive method in relation to the bacterial isolation. There was a low occurrence of ef* gene in samples (1.49 percent) showing great importance to local swine population, because negative strains are potentially less virulent that positive strains.