Lawsonia intracellularis is an important obligate intracellular bacterium. It localizes in close proximity to mitochondria within host cells. Mitochondria regulate diverse physiological processes in host cells, thereby significantly affecting the intracellular proliferation of bacteria. However, how L. intracellularis interacts with mitochondria to promote its own proliferation remains unclear. In this study, the mitochondrial morphology of McCoy cells infected with L. intracellularis was examined by indirect immunofluorescence assay (IFA), and L. intracellularis infection was found to induce mitochondrial elongation. Subsequently, the intracellular L. intracellularis load was assessed by IFA and qPCR following the alteration of mitochondrial morphology. The results showed that the intracellular L. intracellularis load was increased in cells with elongated mitochondria and decreased in cells with fragmented mitochondria. Western blot analysis revealed that the L. intracellularis outer membrane protein Omp2 inhibited ERK1/2 phosphorylation. This inhibition led to a reduction in Drp1 protein levels, thereby inducing mitochondrial elongation. Furthermore, Omp2 interacted with macrophage migration inhibitory factor (MIF), an upstream regulator of ERK1/2, and Omp2 suppressed MIF-induced ERK1/2 phosphorylation, suggesting that MIF is involved in the Omp2-mediated regulation of ERK1/2 phosphorylation.

In 2024, an outbreak of piglet diarrhea occurred in Guangxi Zhuang Autonomous Region, China. RT-PCR assay of clinical samples confirmed that porcine deltacoronavirus (PDCoV) was the primary pathogen. In this study, the PDCoV GX/2024 strain was successfully isolated in PK-15 cells with optimized trypsin treatment conditions. The cytopathic effect (CPE) of this strain gradually intensified during serial passages. After plaque purification, the strain achieved stable proliferation with high titer, reaching a peak titer of 107·2 TCID50/mL, which indicated its potential as an inactivated vaccine candidate. Phylogenetic analysis demonstrated that the strain belonged to the Chinese lineage, with nucleotide similarity ranging from 97.89% to 99.76% compared with other domestic and international strains. Analysis of the Spike protein (S protein) revealed that the strain harbored critical amino acid substitutions in the S1 domain and the S1/S2 cleavage site, which led to conformational changes in the protein surface and might be associated with viral infection efficiency. Animal experiments verified that PDCoV GX/2024 exhibited strong pathogenicity in newborn piglets. Diarrhea, vomiting and other symptoms appeared within 24 h post-infection. The virus mainly colonized the epithelial cells at the tip of intestinal villi and caused severe pathological damage. This study provides important theoretical basis for the genetic evolution, pathogenic mechanism and transmission potential of PDCoV, and offers reference value for disease prevention and control as well as vaccine development.

Schmallenberg virus (SBV) is an emerging arbovirus that causes fever, diarrhea, abortion, and congenital malformations in ruminants. The disease spreads rapidly and poses significant control challenges. Upon its identification, SBV disseminated across Europe, causing major economic losses in livestock production and international trade. Although vaccines exist in certain regions, effective treatment options remain limited, highlighting the need for rapid, early, and accurate detection. In this study, we report the development and preliminary validation of a rapid point-of-care detection method for SBV and closely related viruses based on reverse transcription recombinase-aided amplification (RT-RAA) combined with lateral flow dipstick (LFD) technology. A conserved S gene region was selected as the target, and a single-nucleotide modification was introduced into the primer-probe set to reduce false-positive signals. Due to the absence of naturally infected samples in China, sheep fetal tissues spiked with defined amounts of plasmid standards were used to simulate clinical specimens, and in vitro-transcribed SBV RNA was incorporated to verify performance at the RNA level. After optimization, the method achieved a sensitivity of 5 copies/μL. Compared with a commercial qPCR kit, the assay is faster, simpler, and does not require specialized laboratory equipment, making it well suited for on-site testing in farms, quarantine stations, and regional laboratories. As the assay detects both SBV and closely related viruses, positive results require confirmation by specific PCR or sequencing to accurately identify the virus. These results provide proof-of-concept for the assay and support its potential application in rapid SBV and related virus monitoring and control.

ABSTRACT As a zoonotic opportunistic pathogen, adhesion to the porcine upper respiratory tract epithelium is a prerequisite for Streptococcus suis serotype 2 (SS2) to breach epithelial barriers and cause systemic infections under certain conditions. In this study, we first demonstrated the adhesive function of the autolysin Atl of SS2 to swine tracheal epithelial cells. We found that Atl functions as a molecular bridge for epithelial adhesion. The Bsp-like domain (AtlBsp) specifically binds bacterial lipoteichoic acid, anchoring to the bacterial surface, while the C-terminal hydrolase domain (AtlCOOH) directly interacts with host epithelial receptor Fibronectin (Fn). With the purified recombinant Atl (rAtl), we demonstrated that both membrane-bound and secreted forms of Atl could mediate adhesion to the epithelial cells. Moreover, the rAtl was able to restore the adhesive capacity of the Atl-deficient SS2 mutant (Δatl) to both porcine and murine upper respiratory tract epithelium. This study reveals a novel function of the autolysin Atl in promoting SS2 adhesion to the upper respiratory tract epithelium, which elucidates the underlying molecular mechanism and identifies the key functional domains involved. These findings offer new insights into autolysin biology and provide a theoretical basis for developing anti-adhesion strategies against streptococcal infections in pigs.

Avian mycoplasmosis has a considerable financial impact on the global chicken industry with Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) being the dominant causes. Currently, inactivated vaccines provide minimal cross-protection and can't induce high levels of mucosal immunity, indicating the need to develop more effective immunisation platforms. This study aimed at designing and testing rationally engineered live recombinant vaccines using Salmonella enterica serovar Typhimurium expressing MS- and MG-specific antigens, GrpE and CrmA, to produce a robust immune response. Three recombinant Salmonella ΔphoPQ strains, including SG1 (GrpE), SG2 (CrmA), and SFGs (GrpE + CrmA), were designed and verified by western blotting. The humoral (IgG), mucosal (IgA), cytokine (IFN- α, IL-1β, TNF- α), and innate immune (TLR15, iNOS) responses were determined using ELISA and qRT-PCR. The effectiveness of protection was determined by the use of clinical indicators, bacterial load measurements, histological examination, and survival parameters following the homologous challenge of MS or MG. The results showed that SG1 and SG2 significantly increased mucosal IgA titers (OD: 0.82 ± 0.02 and 0.77 ± 0.01, respectively) and antigen-specific IgG (mean OD: 1.66 ± 0.03 and 3.17 ± 0.06, respectively) relative to inactivated vaccine and control groups (p < 0.0001). The IgG (1.24 ± 0.04) and IgA (0.73 ± 0.01) of SFGs were moderate, indicating a dual expression of antigens in them. These reactions were accompanied by the upregulation of IFN-alpha, IL-1beta, TNF-alpha, TLR15, and iNOS gene expression, indicating the activation of Th1-biased and innate immune responses. Recombinant strains showed strong protective performance, with a survival rate of 90 to 95% after challenge and also reduced shedding of bacteria as well as histological changes. As a result, strains of Recombinant Salmonella expressing GrpE and CrmA resulted in robust humoral, innate, and mucosal immune responses, which provide significant protection against MS and MG.

The phagolysosomes of macrophages play a crucial role in eradicating pathogenic microorganisms, but bacteria have evolved sophisticated mechanisms to survive in the acidic environment of phagolysosomes, leading to host infection and subsequent dissemination. However, it is largely unknown how bacteria sense the extracellular stimuli and regulate their acid tolerance capacity to resist the killing by host immune cells. Here, we report the new substrate FadR of the serine/threonine kinase (STK) in Streptococcus suis serotype 2 (SS2) and demonstrate that the phosphorylation site is Thr230. Notably, FadR phosphorylation significantly enhances the acid resistance of SS2, leading to an increase in the lethality of SS2 in mice, and a marked increase in bacterial load in the blood and various organs, and more severe pathological changes in various organs of the mice. Interestingly, this study further indicated that FadR protein can bind to the promoter of arginine deiminase (adi), and FadR phosphorylation enhances its binding ability to the adi promoter and increases adi transcription levels. The increase of ADI in SS2 promotes the metabolism of arginine and increases the ammonia content, thus enhancing the acid resistance and intracellular survival capacity of the bacteria in macrophages. Altogether, the research reveals an acid resistance regulatory mechanism that bacteria can utilize the STK-FadR signaling axis to sense changes in the external acidic environment, and then manipulate the ADI system to enhance bacterial resistance to acidic environment or host immunity.

Glaesserella parasuis (G. parasuis) is an important pathogen, which can cause systemic inflammatory response in pigs and bring huge economic losses to the global swine industry. G. parasuis can induce a strong inflammatory response in the lungs under environmental changes and certain stress conditions. However, the underlying mechanism of this adverse response has not been thoroughly studied. In this study we demonstrated that G. parasuis serotype 5 strain (GPS5-SQ) has the potential to induce pyroptosis in 3D4/21 cells. GPS5-SQ could degrade the expression of Cav-1. Knockdown or overexpression of Cav-1 promoted or reduced the occurrence of pyroptosis, respectively. These results suggested that Cav-1 is involved in pyroptosis induced by GPS5-SQ in 3D4/21 cells. In addition, overexpression of Cav-1 suppressed the activation of NLRP3 inflammasome by inhibiting ASC oligomerization, resulted in reducing pyroptosis. In general, we found that GPS5-SQ infection could promote pyroptosis by degrading the expression of Cav-1. The results of the study revealed the new mechanism of inflammation induced by GPS5-SQ in 3D4/21 cells.

Streptococcus suis serotype 2 (SS2), a significant zoonotic pathogen, causes streptococcal toxic shock-like syndrome (STSLS) in humans and acute septicemia in pigs, highlighting its ability to evade host immune clearance during early infection. Evasion of macrophage phagocytosis is a critical strategy for bacterial immune escape. However, the mechanisms by which SS2 subverts cytoskeletal remodeling to inhibit phagocytosis remain elusive. Here, we identified a TCS named Ihk/Irr as a key regulator of SS2 resistance to macrophage phagocytosis through screening SS2 TnYLB-1 mutant library. Phosphorylation of histidine at position 246 (H246) in Ihk and aspartate at position 52 (D52) in Irr enhances SS2 anti-phagocytic capacity and virulence. EMSA revealed that Irr directly binds to the pepO promoter; however, the phosphorylation-site inactivation protein Irr-D52A loses this binding ability. Interestingly, SS2 directly regulates PepO expression by sensing extracellular stimuli through Ihk/Irr phosphorylation. PepO subsequently activates the p38 and ERK1/2 pathways via TLR2, leading to enhanced phosphorylation of p38 and ERK1/2. This phosphorylation ultimately inhibits actin polymerization, impairing phagocytosis and thereby enhancing SS2's phagocytosis resistance. Our study elucidates a novel immune evasion mechanism that SS2 exploits Ihk/Irr-PepO axis to resist macrophage phagocytosis, providing new insights into SS2 pathogenesis and potential therapeutic targets for combating bacterial infections.

Background/Objectives: Lawsonia intracellularis (L. intracellularis) is an important intestinal pathogen that causes porcine proliferative enteropathy (PPE) in swine production worldwide. Currently, only a few commercially available vaccines are available for PPE prevention. Methods: In this study, an attenuated L. intracellularis variant of JS-G90 was obtained through subculturing of L. intracellularis JS isolates in McCoy cells for 90 generations, and its immune response was evaluated in pigs. Results: The results demonstrated that pigs who underwent intragastric administration of JS-G90 had lower fecal bacterial shedding and no histopathological lesions, indicating that it was safe in pigs. Therefore, JS-G90 was selected to develop the attenuated PPE vaccine. The immune response of JS-G90 in pigs was further evaluated based on fecal bacterial shedding, histopathological lesions, and humoral and cell-mediated immune responses following challenge with pathogenic L. intracellularis. The results revealed that JS-G90 significantly decreased the copies of L. intracellularis in rectal swabs containing feces and ileum infection (p < 0.001), reduced histopathological lesions in the ileum, and elicited non-specific humoral (IgG and sIgA) and cell-mediated immune responses (p < 0.001) compared with the challenge control and mock groups. Conclusions: In conclusion, the attenuated vaccine JS-G90 is safe and induced humoral and cell-mediated immune responses in pigs against pathogenic L. intracellularis infection. It may serve as an effective strategy for preventing and controlling PPE.

Streptococcus suis serotype 2 (SS2) is an emerging zoonotic agent responsible for a variety of diseases. The septicemia caused by SS2 suggests that it can evade the bactericidal effects of innate immune cells. However, the mechanisms by which SS2 evades innate immunity remain largely unknown. Neutrophils are critical components of innate immunity for antimicrobial defense. In this study, we identified a cell surface protein (CSP) insertion mutant in an SS2 transposon mutant library that significantly induces neutrophil extracellular traps (NETs) and shows poor survival compared to the parent SS2 in the bloodstream. CSP deletion mutant (ΔCSP) was constructed and it exhibits a defective polysaccharide capsule and enhanced biofilm formation. Although ΔCSP induces increased NET release and ROS formation, its survival and proliferation within neutrophils and NETs are significantly reduced. Additionally, ΔCSP stimulates extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in neutrophils, which may enhance neutrophil bactericidal activity. Importantly, purified polysaccharide capsule from SS2 can inhibit neutrophil bactericidal effects. This study identifies bacterial virulence factors that prevent SS2 clearance by neutrophils, offering potential antigens or drug targets for the prevention and control of swine streptococcosis.

Bovine mastitis, caused by Streptococcus agalactiae (Group B Streptococcus; GBS), poses significant economic challenges to the global dairy industry. Mouse models serves as valuable tools for assessing GBS-induced infections as an alternative to large animals. This study aimed to investigate the LD50 dose, organ bacterial load, and quantification of peritoneal leukocyte populations for GBS serotypes Ia and II isolates from China and Pakistan. Additionally, we measured indicators such as lactoferrin, albumin, and myeloperoxidase (MPO) activity. Pro-inflammatory cytokines (TNF-alpha, IL-1 beta, IL-6, and IL-2) and anti-inflammatory cytokines (IL-10 and TGF-beta) in serum and tissue samples were evaluated using ELISA and qPCR, respectively. BALB/c mice (4 mice per group) received individual intraperitoneal injections of 100 mu l containing specific bacterial inoculum concentrations (ranging from 105 to 109 CFU per mouse) of Chinese and Pakistani GBS isolates (serotypes Ia and II). Control groups received 100 mu L of sterile PBS. Results revealed that the LD50 bacterial dose causing 50 % mortality in mice was 107 CFU. The highest bacterial load in all experimental groups was quantified in the peritoneum, followed by blood, mammary gland, liver, spleen, lungs, and brain. The most significant bacterial dissemination was observed in mice inoculated with Pakistani serotype Ia at 24 h, with a subsequent notable decline in bacterial counts at day 3. Notably, infection with Pakistani serotype Ia showed a trend of increased total leukocyte counts, significantly higher than Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II. A substantial influx of neutrophils and lymphocytes was observed in response to all tested serotypes, with Pakistani serotype Ia inducing a significantly higher influx compared to other groups (Pakistani serotype II, Chinese serotype Ia, and Chinese serotype II). Furthermore, TNF-alpha, IL-1 beta, IL-2, and IL-6 expressions were significantly increased in mice one day after infection with the Pakistani serotype Ia. Compared to mice infected with the Pakistani serotype II, Chinese Serotype Ia, and Chinese serotype II, those infected with the Pakistani serotype Ia isolate exhibited the highest production of IL-10 and TGF-beta, along with significantly increased concentrations of lactoferrin, albumin, and MPO. These findings suggest that the persistence and severity of infection caused by the Pakistani serotype Ia may be linked to its ability to spread to deeper tissues. This study enhances our understanding of the clinical characteristics of bovine mastitis caused by S. agalactiae in China and Pakistan.

Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin β1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-β signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.

Glaesserella parasuis (G. parasuis) is a common Gram-negative commensal bacterium in the upper respiratory tract of swine that can cause Glässer's disease under stress conditions. Pyroptosis is an important immune defence mechanism of the body that plays a crucial role in clearing pathogen infections and endogenous danger signals. This study aimed to investigate the mechanism of G. parasuis serotype 5 SQ (GPS5-SQ)-induced pyroptosis in swine tracheal epithelial cells (STECs). The results of the present study demonstrated that GPS5-SQ infection induces pyroptosis in STECs by enhancing the protein level of the N-terminal domain of gasdermin D (GSDMD-N) and activating the NOD-like receptor protein 3 (NLRP3) inflammasome. Furthermore, the levels of pyroptosis-related proteins, including GSDMD-N and cleaved caspase-1 were considerably decreased in STECs after the knockdown of retinoic acid inducible gene-I (RIG-I) and mitochondrial antiviral signaling protein (MAVS). These results indicated that GPS5-SQ might trigger pyroptosis through the activation of the RIG-I/MAVS/NLRP3 signaling pathway. More importantly, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) repressed the activation of the RIG-I/MAVS/NLRP3 signaling and rescued the decrease in Occludin and zonula occludens-1 (ZO-1) after GPS5-SQ infection. Overall, our findings show that GPS5-SQ can activate RIG-I/MAVS/NLRP3 signaling and destroy the integrity of the epithelial barrier by inducing ROS generation in STECs, shedding new light on G. parasuis pathogenesis.

GntR transcription factor of Streptococcus suis serotype 2 (SS2) is a potential substrate protein of STK, but the regulation mechanisms of GntR phosphorylation are still unclear. This study confirmed that STK phosphorylated GntR in vivo, and in vitro phosphorylation experiments showed that STK phosphorylated GntR at Ser-41. The phosphomimetic strain (GntR-S41E) had significantly reduced lethality in mice and reduced bacterial load in the blood, lung, liver, spleen, and brain of infected mice compared to wild-type (WT) SS2. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments demonstrated that the promoter of nox was bound by GntR. The phosphomimetic protein GntR-S41E cannot bind to the promoter of nox, and the nox transcription levels were significantly reduced in the GntR-S41E mutant compared to WT SS2. The virulence in mice and the ability to resist oxidative stress of the GntR-S41E strain were restored by complementing transcript levels of nox. NOX is an NADH oxidase that catalyzes the oxidation of NADH to NAD+ with the reduction of oxygen to water. We found that NADH is likely accumulated under oxidative stress in the GntR-S41E strain, and higher NADH levels resulted in increased amplified ROS killing. In total, we report GntR phosphorylation could inhibit the transcription of nox, which impaired the ability of SS2 to resist oxidative stress and virulence.

Porcine proliferative enteropathy (PPE), an important infectious disease in pig production caused by an obligate intracellular bacterium Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs widespread. An accurate method for detecting L. intracellularis is particularly important for preventing and controlling PPE. Heat shock protein 60 (Hsp60) is an immunodominant bacterial antigen found in all eukaryotic and prokaryotic organisms. Thus, the purpose of the current investigation was to produce a novel L. intracellularis Hsp60 monoclonal antibody (mAb) useful for immunodiagnostics. Three hybridomas secreted anti-Hsp60 termed 3E5, 4E2, and 9G6 were generated, and the titers of ascitic fluids of 3E5, 4E2, 9G6 were 1:1 024 000, 1:2 048 000 and 1:2 048 000, respectively. The Western blotting analysis demonstrated that recombinant Hsp60 (rHsp60) was recognized by mAbs 3E5, 4E2 and 9G6. Subsequently, analyses of specificity showed all the mAbs were highly specific to L. intracellularis while could not significantly react with other enteric bacteria commonly found in the ileum of pigs, such as Escherichia coli, Salmonella Choleraesuis, Salmonella Typhimurium, and Brachyspira hyodysenteriae. Furthermore, the mAbs were useful for detecting L. intracellularis in the infected monolayer cells and histological sections of the ileum from PPE-affected pigs. Our research will provide a foundation for the development of immunological diagnostic tests.

Coinfection of Porcine circovirus type 2 (PCV2) and Glaesserella parasuis type 4 (GPS4) is widespread clinically, resulting in high morbidity and mortality, however, interactions between the two pathogens during coinfection and the coinfection pathogenesis are poorly understood. In this study, a piglet model coinfected with PCV2 and GPS4 was established; coinfection of the piglets' group showed more obvious symptoms, such as high fever and emaciation, and more severe histological lesions appeared in various organs. Importantly, piglets in the coinfection group produced lower levels of PCV2 and GPS4 antibodies, and showed high levels of inflammatory cytokines, TLR2, and TLR4, while the levels of CD4, CD8, MHC II, costimulatory molecules, and IL-12p40 were decreased. In addition, a model of macrophage 3D4/21 cells coinfection with PCV2 and GPS4 was established, coinfected cells exhibited increased expression of the cytokines IL-6, IL-8, TNF-α, IL-1β, and the receptors TLR2, TLR4, while decreased MHC II. We further demonstrate that cytokine production is associated with the activation of NF-κB and NLRP3 inflammasome signaling pathways, and TLR4 is also involved. Altogether, our findings suggest that coinfection with PCV2 and GPS4 exacerbates the inflammatory response, resulting in severe tissue damage, and probably impaired macrophage antigen presentation and T cell activation, resulting in immune dysregulation, aggravating host infection.

Background: Brucella melitensis RM57, a mutant of B. melitensis M1981, is an effective vaccine candidate due to the reduced virulence. This study was aimed to explore the molecular mechanism of virulence reduction in Brucella melitensis RM57.Methods: The differentially expressed genes (DEGs) between samples from M1981 and RM57 were determined based on gene expression profiles and were used for protein subcellular localization (SCL) analysis. Then based on the SCL results, proteinprotein interaction (PPI) network analysis was performed, followed by function enrichment analysis and gene-drug interaction prediction.Results: A total of 403 DEGs were identified between the RM57 and M1981 groups. Then, 58 proteins were predicted based on the NCBI protein database, including 3 membrane proteins (nuoK, exbD, and cydB), 31 cytoplasmic proteins, and 2 periplasmic proteins. A PPI network was constructed, which consisted of 27 nodes, such as rpoB, rpoA, rpsP, rpoC, RpsA and nuoK, and 146 interactions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the proteins in the PPI networks showed that they were mainly enriched pathways of ribosome, and RNA polymerase, and functions related to biosynthetic process and metabolic process. Mitoxantrone, adriamycin, and daunorubicin were predicted to be target drugs of RpsA.Conclusions: The membrane proteins of nuoK, exbD, and cydB, and cytoplasmic proteins, such as rpoB, rpoA and rpsP, may contribute to the virulence reduction of the RM57 strain. RpsA may induce an immune response against brucellosis infection through ribosome pathway. These proteins may be targets for novel vaccine development.

旨在探究副猪格拉瑟菌(Glaesserella parasuis,GPS)突破猪呼吸道上皮屏障引起系统性感染的机制.通过超速离心和密度梯度离心提取副猪格拉瑟菌外膜囊泡(outer membrane vesicles,OMVs),OMVs经SDS-PAGE显示,蛋白质条带分布在55～100 ku,经透射电子显微镜(TEM)观察,OMVs的粒径在100～200 nm,纳米粒子直径分析(NTA)结果显示,样品在100～200 nm处的粒子数目最多.进而用制备的HbpA及OmpP2多克隆抗体对OMVs及不含OMVs的细菌上清进行Western blot验证,证实所提取的样品为外膜囊泡,且进一步结果证明细胞致死性膨胀毒素(CDT)在GPS培养物中主要以OMVs的形式存在.用OMVs或CdtB处理猪气管上皮细胞(swine tracheal epithelial cells,STEC)36 h,检测 STEC 中 cleaved-caspase3、ZO-1 和 Occludin 的蛋白表达水平,并用FITC-葡聚糖(FD-4)检测STEC单层细胞的细胞旁通透性.结果发现,OMVs与CdtB处理后凋亡相关蛋白cleaved-caspase3表达水平升高,ZO-1和Occludin的表达水平降低,FD-4渗透率升高,同时,乳酸脱氢酶(LDH)细胞毒性检测试验发现,当CdtB与STEC单层细胞共孵育24 h、OMVs与STEC单层细胞共孵育36 h后,STEC存活率显著降低.而Pifithrin-α预处理STEC可抑制OMVs和CdtB引起的细胞凋亡和屏障通透性的增加.另外,用免疫磁珠捕获的方法去除 OMVs 中的 CdtB(OMVs-△CdtB),OMVs-4CdtB 处理 STEC 36 h 后 cleaved-caspase3、ZO-1、Occludin表达水平与对照组相比无显著差异.综上,成功提取并纯化GPS OMVs,且试验证明GPS可通过OMVs转运CdtB诱导STEC凋亡和屏障损伤,为副猪格拉瑟菌突破呼吸道上皮屏障的机制提供新思路.

Synthesis of capsular polysaccharide (CPS), an important virulence factor of pathogenic bacteria, is modulated by the CpsBCD phosphoregulatory system in Streptococcus. Serine/threonine kinases (STKs, e.g. Stk1) can also regulate CPS synthesis, but the underlying mechanisms are unclear. Here, we identify a protein (CcpS) that is phosphorylated by Stk1 and modulates the activity of phosphatase CpsB in Streptococcus suis , thus linking Stk1 to CPS synthesis. The crystal structure of CcpS shows an intrinsically disordered region at its N-terminus, including two threonine residues that are phosphorylated by Stk1. The activity of phosphatase CpsB is inhibited when bound to non-phosphorylated CcpS. Thus, CcpS modulates the activity of phosphatase CpsB thereby altering CpsD phosphorylation, which in turn modulates the expression of the Wzx-Wzy pathway and thus CPS production.

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.