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Membrane vesicles derived from streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

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Journal of Integrative Agriculture 2024年第4期23卷 1338-1353页

作者： Keda Shi Yan Li Minsheng Xu Kunli Zhang Hongchao Gou Chunling Li Shaolun Zhai Institute of Animal Health Guangdong Academy of Agricultural Sciences/Key Laboratory of Livestock Disease Prevention ofGuangdong Province/Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of GuangdongProvinceMinistry of Agriculture and Rural AffairsGuangzhou 510640China Maoming Branch Guangdong Laboratory for Lingnan Modern Agricultural Science and TechnologyMaoming 525000China College of Animal Science&Technology Zhongkai University of Agriculture and EngineeringGuangzhou 510225China

streptococcus suis serotype 2(*** 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,*** 2 must colonize the host,break the blood barrier,and cause exaggerated *** the last few years,most studies have focused on a single virulence factor and its influences on the *** vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host ***-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell ***,little is known about the effect of the MVs from *** 2(Gram-positive bacteria without LPS)on cell ***,we investigated the molecular mechanism by which *** 2 MVs participate in endothelial cell *** this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by *** ***,we demonstrated that *** 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were ***,we showed that MVs were transported to cells via mainly dynamin-dependent *** *** 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 *** importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by *** 2 *** results in this study suggest for the first time that MVs from *** 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,

关键词： streptococcus suis serotype 2 membrane vesicles endocytosis pyroptosis NLRP3 inflammasomes mitochondrial damage endothelial cell

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In vitro Transcriptome Analysis of Two Chinese Isolates of streptococcus suis serotype 2

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Genomics, Proteomics & Bioinformatics 2014年第6期12卷 266-275页

作者： Dake Zhang Nan Du Sufang Ma Qingtao Hu Guangwen Lu Wei Chen Changqing Zeng CAS Key Laboratory of Genomic and Precision Medicine Beijing Institute of GenomicsChinese Academy of Sciences CAS Key Laboratory of Pathogenic Microbiology and Immunology Institute of MicrobiologyChinese Academy of Sciences University of Chinese Academy of Sciences

The streptococcus suis serotype 2（S. suis 2） isolates 05ZYH33 and 98HAH33 have caused severe human infections in China. Using a strand-specific RNA-seq analysis, we compared the in vitro transcriptomes of these two Chinese isolates with that of a reference strain（P1/7）. In the89 K genomic island that is specific to these Chinese isolates, a toxin–antitoxin system showed relatively high levels of transcription among the S. suis. The known virulence factors with high transcriptional activity in these two highly-pathogenic strains are mainly involved in adhesion, biofilm formation, hemolysis and the synthesis and transport of the outer membrane protein. Furthermore,our analysis of novel transcripts identified over 50 protein-coding genes with one of them encoding a toxin protein. We also predicted over 30 small RNAs（s RNAs） in each strain, and most of them are involved in riboswitches. We found that six s RNA candidates that are related to bacterial virulence, including csp A and rli38, are specific to Chinese isolates. These results provide insight into the factors responsible for the difference in virulence among the different S. suis 2 isolates.

关键词： streptococcus suis serotype 2 Transcriptome Novel transcripts s RNA Virulence factor

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Prokaryotic Expression of Gene Encoding Glutamate Dehydrogenase of streptococcus suis serotype 2 and Preparation of Polyclonal Antibodies against Its Expressed Products

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Animal Husbandry and Feed Science 2011年第5期3卷 15-19页

作者： XIA Xiao-jing SHEN Zhi-qiang JIANG Shi-jin LI Shu-guang WU Li-zhi MA Zhen-qian CHENG Li-kun College of Animal Science & Veterinary Medicine Shandong Agricultural University Taian 271018 China Shandong Binzhou Animal Science & Veterinary Medicine Academy Binzhou 256600 China Shandong Lvdu Biotechnology Co. Ltd. Binzhou 256600 China College of Animal Science and Technology Huazhong Agricultural University Wuhan 430070 China College of Animal Science and Veterinary Medicine Shenyang Agricultural University Shenyang 110161 China

[ Objective] To obtain detection antigen for diagnosis of streptococcus suis infection. [ Method] The complete ORF of glutamate dehy- drogenase （GDH） gene was amplified from the genomic DNA of streptococcus suis serotype 2 strain SC22 isolated in Sichuan Province by poly- merase chain reaction （PCR）. The resulting product was cloned into the prokaryotic expression vector pET-30a, which was then transformed into E. coil BL21 （DE3）. The identified positive transformants were screened for expression induced by IPTG. The expression products were subjected to SDS-PAGE and the recombinant protein was purified by nickel ion-agarose affinity chromatography. New Zealand rabbits were immunized with the purified recombinant GDH protein to prepare polyclonal antibodies. Titers of the anti-serum were determined by indirect ELISA and Western blot assay. [ Result] The recombinant GDH protein was effectively expressed in the host bacteria, and highly pure recombinant protein was obtained by nickel ion-agarose affinity chromatography. High-titer anti-serum against the recombinant protein was obtained. As evidenced by western blot as- say, the sera could react specifically with the lysates of all detected streptococcus suis strains. In addition, the recombinant GDH protein could re- act specifically with serum samples collected from five pigs experimentally infected by strain SC22. [ Conclusion] The expressed GDH fusion protein has some common epitopes of natural GDH and can be used as detection antigen to develop ELISA and other diagnostic methods.

关键词： streptococcus suis serotype 2 Glutamate dehydrogenase Prokaryotic expression Western blot

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Cloning,expression and immunocompetence analysis of gene encoding the glutamate dehydrogenase of streptococcus suis serotype 2

Cloning,expression and immunocompetence analysis of gene enc...

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中国畜牧兽医学会畜牧兽医生物技术学分会暨中国免疫学会兽医免疫分会第八次学术研讨会

作者： YANG Wei-jun~(1,3*),YANG Zhi-biao~(1*),CAI Xue-hui~2,HAO Yong-qing~3,ZHENG Sheng-bo~1, LAN Dao-liang~1,GU Jiang-ping~1,XING Rui~1,YUAN Cong-li~1,CUI Li~1,HUA Xiu-guo~1 (1.Shanghai Key Laboratory of Veterinary Biotechnology,College of Agricultural and Biology,Shanghai Jiaotong University, Shanghai 200240,China 2.Division of Swine Infectious Diseases,State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,Harbin 150001,China 3.College of Animal Science and Animal Medicine,Inner Mongolia Agricultural University,Huhhot 010018,China)

This study aimed to study the immune activity of glutamate dehydrogenase(GDH) of streptococcus suis serotype 2 and detect its ***:The GDH gene was cloned and *** GDH protein was expressed in a prokaryotic expression system and purified by affinity *** of the resulting protein was analyzed by western blot with anti-GDH monoclonal *** fusion protein was efficiently *** blot analysis confirmed that the resulting protein was *** GDH gene of *** 2 was successfully cloned and the high expressed,which facilitated the further studies on the development of immunological detection and vaccine design.

关键词： streptococcus suis serotype 2 GDH prokaryotic expression Immunocompetence

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A Method for Detecting Adhesive Related-Factors of streptococcus suis serotype 2 by Real-time PCR

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Agricultural Science & Technology 2013年第10期14卷 1378-1382页

作者：汪伟何孔旺倪艳秀周俊明张雪寒俞正玉吕立新茅爱华温立斌王小敏李彬郭容莉江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心江苏南京210014

[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.

关键词： streptococcus suis serotype 2 Adhesive related-factors (adhesins) Real- time PCR

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Factor H specifically capture novel Factor H-binding proteins of streptococcus suis, and contribute to the adherence and invasion of epithelial cells and promote mouse infection

Factor H specifically capture novel Factor H-binding protein...

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第十一届全国免疫学学术大会

作者： Li Quan Zhang Wei Key Lab of Animal Bacteriology OIE Reference Lab for Swine StreptococcosisCollege of Veterinary MedicineMinistry of AgricultureNanjing Agricultural University

Background: Factor H(FH), a regulatory protein of the complement system, can bind specifically to factor H-binding proteins(FHBPs) of streptococcus suis serotype 2(SS2), which contribute to evasion of host innate immune defenses. In the present study, we aimed to identify novel FHBPs and characterize the biological functions of FH in SS2 ***: Here, a method that combined proteomics and Far-western blotting was developed to identify the surface FHBPs of SS2. we sought to characterize whether SS2 interact specifically with mouse FH and evaluated whether FH could influence the adhesion and invasion of SS2 to HEp-2 cells. To evaluate the roles of FH in vivo, we infected a specific pathogen free mouse model to evaluate the pathogenesis of SS2 in the presence of human ***: With this method, fourteen potential novel FHBPs were identified among SS2 surface proteins. We selected eight newly identied proteins and further confirmed their binding activity to FH. The binding of SS2 to immobilized FH decreased dramatically after pre-incubation with anti-FHBPs polyclonal antibodies. We showed for the first time that SS2 also interact specifically with mouse FH. Furthermore, we found that FH play an important role in adherence and invasion of SS2 to HEp-2 cells. Additionally, using a mouse model of intraperitoneal challenge, we confirmed that SS2 preincubated with FH enhanced bacteremia and brain invasion, compared with SS2 not pretreated with ***: Taken together, this study provides a useful method to characterize the host-bacteria interactions. These results firstly indicated that binding of FH to the cell surface improved the adherence and invasion of SS2 to HEp-2 cells, promoted SS2 to resist killing and enhanced virulence.

关键词： streptococcus suis serotype 2 factor H-binding proteins adherence invasion interaction virulenc

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