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

Streptococcus suis serotype 2(S.suis 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,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-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 pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 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,leading to the pyroptosis of endothelial cells.

A Method for Detecting Adhesive Related-Factors of Streptococcus suis Serotype 2 by Real-time PCR

[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.

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

[ 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.

Advances in pathogenesis of Streptococcus suis serotype 2

Streptococcus suis is one of the major pathogens of swine streptococcosis. Among them, the strongest virulence and highest rate of clinical isolation serotype is S. suis serotype 2（SS2）. Moreover, SS2 is also an important zoonosis pathogen, which caused severe public health issues in China. It has been reported that SS2 has several virulence factors, including muramidase released protein, extracellular factors, capsule, fibronectin-binding protein, enolase, hemolysin, small RNA, biofilm, two-component regulatory systems, STK/STP, etc., whose functions involved in adhesion, anti-phagocytosis, inflammatory pathway activation, invasion, etc. Actually, SS2 has developed a variety of ways to escape from host immune system during evolution. In particularly, capsule could resist phagocytosis through inhibiting sphingosine dependent immune cell recognition, which plays an important role in escaping host inflammation response; moreover, superoxide dismutase encoding by sod A enables SS2 escaping reactive oxygen species（ROS） in host immune cells; besides, binding complement factor h with Fhb could suppress the activation of complement alternative pathway and bactericidal effect. And SS2 could also hinder the formation of neutrophil extracellular traps（NETs） to avoid trapping by swine neutrophils, while host immune globulin could be degraded by Ig A1 hydrolase and Ig M protease. In addition, SS2 could escape host immune defense with the help of multiple transcriptional factors and micro-RNA. So far, the pathogenesis of meningitis, arthritis caused by SS2 infection, is still unclear, and the virulence regulatory mechanism of phosphorylation, micro-RNA need to be further clarified. Importantly, the study of interaction mechanism of pathogen and host contribute to further demonstration the pathogenesis of SS2.

Isolation and characterization of dengue virus serotype 2 from the large dengue outbreak in Guangdong, China in 2014

Dengue has been well recognized as a global public health threat,but only sporadic epidemics and imported cases were reported in recent decades in China.Since July 2014,an unexpected large dengue outbreak has occurred in Guangdong province,China,resulting in more than 40000 patients including six deaths.To clarify and characterize the causative agent of this outbreak,the acute phase serum from a patient diagnosed with severe dengue was subjected to virus isolation and high-throughput sequencing(HTS).Traditional real-time RT-PCR and HTS with Ion Torrent PGM detected the presence of dengue virus serotype 2(DENV-2).A clinical DENV-2 isolate GZ05/2014 was obtained by culturing the patient serum in mosquito C6/36 cells.The complete genome of GZ05/2014 was determined and deposited in Gen Bank under the access number KP012546.Phylogenetic analysis based on the complete envelope gene showed that the newly DENV-2 isolate belonged to Cosmopolitan genotype and clustered closely with other Guangdong strains isolated in the past decade.No amino acid mutations that are obviously known to increase virulence or replication were identified throughout the genome of GZ05/2014.The high homology of Guangdong DENV-2 strains indicated the possibility of establishment of local DENV-2 circulation in Guangdong,China.These results help clarify the origin of this epidemic and predict the future status of dengue in China.

In vitro Transcriptome Analysis of Two Chinese Isolates of Streptococcus suis Serotype 2

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.