Screening, Identification And Characterization Of Infection Related Factors Of Streptococcus Suis Serotype 2

Streptococcus suis is an important swine pathogen that causes many pathological conditions,such as arthritis,endocarditis,meningitis,pneumonia,and septicemia.It is also an important zoonotic agent for humans in contact with colonized,otherwise healthy pigs or their by-products,causing meningitis and endocarditis.Thirty-five serotypes(types 1 to 34,and 1/2) based on capsular antigens are currently known.Type 2 is considered the most virulent and prevalent type in diseased pigs.The mechanisms involved in the pathogenesis and virulence of S.suis are not completely understood.Attempts to control the disease are still hampered by the lack of sufficient knowledge about the pathogenesis of the disease and the lack of effective vaccines and sensitive diagnostic methods.In this study,In vivo-induced antigen technology(IVIAT) has been employed to study SS2 infection-related factors.1 Construction of Streptococcus suis serotype 2 genomic expression librariesReferred to published European SS2 strain P1/7 genomic DNA sequence,software DNASTAR was used to select proper restriction enzyme for construct SS2 genomic expression library.Vector DNA was respectively digested with BamHI,gel-purified by using a gel extraction kit,and treated with alkaline phosphatase.Genomic DNA from strain ZY05719(isolate from Sichuan SS2 outbreak) was partially digested with Sau3AI,and fractionated on a 0.7%agarose gel.DNA fragments ranging from 0.1 to 5.0 kbp(insert DNA) were recovered and cloned into the three BamH I-digested and dephosphorylated vectors,then each library was electroporated into competent E.coli DH5αand BL21(DE3) to create three genomic expression libraries,pET30 vectors specific primers were used to detect the of efficiency of insert ratio.2 Selection and identification of infection-associated factors from Streptococcus suis serotype 2 expression libraries with swine convalescent seraSera from swine convalescing from SS2 challenge were collected,adsorbed against in vitro-grown SS2 strain ZY05719(such as bacterial whole cells,cell lysates and extracellular proteins),then used to screen SS2 genomic expression libraries,after rigorous selection,which output 64 positive clones.Sequencing analysis revealed 60 clones encoded 48 proteins,which had homology to proteins involved in various aspects of cell surface structure,molecule synthesis/degradation,energy metabolism,regulation,transport systems, and unknown functions.Align these sequences with P 1/7 whole genomic sequence to learn about the distribution of infection-related factors in genome.Alignment result some of them showed high similarity with 89/1589 but not in P1/7,or vice versa,about 44 insert DNA sequence can be located on P1/7 genome.3 Development of a real-time fluorescent quantitative polymerase chain reaction for assessment of differential expression of newly identified infection-related factors between in vitro and in vivo conditionVirulence factors and the pathogenesis of SS2 infection are not well understood.In this study,13 out of 48 newly identified infection-related factors(trag,ysirk,abc56,sdh,srt, abc15,orf1616,hprk,nlpa,hp10,ptss,cwh,flps) were selected to analyze their differential expression between in vitro and in vivo condition,gapdh was used reference gene.In vitro, ZY05719 was harvested at OD₆₀₀ of 0.1,0.2,0.4,0.6 and 0.8 respectively.In vivo, specific-pathogen free piglets(SPF Bama minipigs) were used as experimental model to leam about mRNA expression difference at in vivo condition.SPF Bama minipigs were challenged with ZY05719 intravenous(i.v.) or intramuscular(i.m.) injection,then bacterial cells were recovered from blood at 12h,24h and 36h(i.v.) and 24h and 48h(i.m.).RNA was isolated and used as template for assess their differential expression by quantitative real-time PCR between in vitro and in vivo.During i.v.approach orf1616,trag,nlpa,abc56 and flps upregulated continuously;abc15,ysirk,hp10 and ptss upregulated before 24h then went down;cwh,hprk,srt and sdh went down from 12h.During i.m.approach, orf1616,trag,hprk,sdh and flps climbed up from 24h to 48h,and abc15,nlpa,srt, ysirk,abc56,cwh,hp10 and ptss went down form 24h to 48h.Results mentioned above suggesting that these genes may play a role during SS2 invasive course.4 Distribution of newly identified SS2 infection-related factors in Streptococcus suis strainsStreptococcus suis serotype 2 is an important pathogen for pig and human.About 48 infection-related factors had been selected by IVIAT in previous study.In this study,13 factors(trag,ysirk,abc56,sdh,srt,abe15,orf1616,hprk,nlpa,hp10,ptss,cwh,flps) were selected to analyze their distribution in Streptococcus suis strains from different sources, regions and times.According to published SS2 genomic sequence of European strain P1/7 or Canadian strain 89/1591,primers for these 13 genes have been designed and used to detect their distribution.Genomic DNA of SS2 outbreak represent strains(Jiangsu isolate HA9801 and Sichuan isolate ZY05719),reference strain ATCC43765,SS2 isolates from different sources,and other serotypes or group(SS1,SS1/2,SS7,SS9 and C group) were used as template for polymerase chain reaction(PCR).Results showed that these 13 factors widely distributed in most SS2 strains,but SS2-N and non-virulent strain T15 didn't have. In addition,these factors could be detected in other serotypes but each serotype had different distribution,and negative in Lancefield group C.5 Identification and characterization of a novel infection-related factor(cell wall hydrolase/autolysin) of Streptococcus suis serotype 2Cell wall autolysins have been implicated in various biological functions,including cell separation,cell wall turnover,restructuring of cell walls,and bacterial autolysis.In this study,the complete autolysin genes of HA9801,ZY05719,ATCC43765,260 and 11611 were amplified by PCR and cloned into T-vector for sequence,then their sequences were analyzed by software.Results showed that open reading frame(orf) length of SS2 autolysin is 3108bp,they were consisted of 6 repeated domain "GBS_Bsp-like" and 1 domain "N-acetylmuramoyl-L-alanine amidase",while 89/1591 only has 5 repeated domain "GBS_Bsp-like".Alignment results showed that autolysins of strain HA9801 and ZY05719 showed high homologue to that of European strain P1/7(99.8%),but differ from Canada strain 89/1589.Based on published N-acetylmuramoyl-L-alanine amidase tertiary structure, we forecast N-acetylmuramoyl-L-alanine amidase tertiary structure of these five strains and Canadian strain 89/1591 and European strain P1/7.Tertiary structures of these five strain were similar with each other,and showed high homology with that of P1/7,but differ from that of 89/1591 at partial domains.Partial gene of autolysin was cloned and inserted into expression vector pET30a(+),and induced by IPTG to express recombinant autolysin. SDS-PAGE and western blot were used to analyze its antigenicity.6 Identification and characterization of a novel infection-related factor HprK/P of Streptococcus suis serotype 2P-Ser-HPr is the central regulator of carbon metabolism in Gram-positive bacteria,but also plays a role in virulence development of certain pathogens.In this study,the complete hprk genes of HA9801,ZY05719,ATCC43765,11611(isolated from pig) and 260(isolated from human) were amplified by PCR and cloned into T-vector for sequence,then their sequences were analyzed by software.Results showed that open reading frame(orf) length of SS2 HprK/P is 933bp,and HprK/P molecular weight is about 35kDa.Alignment results showed that HprK/P is highly conserved at Genbank database,SS2 HprK/P demonstrated high similarity with HprK/P of almost all bacteria.Based on published HprK/P tertiary structure,we forecast HprK/P tertiary structure of these five strains and Canadian strain 89/1591 and European strain P1/7.Results showed that tertiary structures of these five strain were similar with each other,and showed high homology with that of P 1/7,but differ from that of 89/1591 at partial domains.In addition,signal peptide of SS2 HprK/P were analyzed by software,result showed that it's a mature protein,has no signal peptide.7 Identification and characterization of a novel function-unknown infection-related factor ORF1616 of Streptococcus suis serotype 2ORF1616 is a putative cell surface protein,but its function unknown,may play a role during SS2 infection.In this study,the complete orf1616 genes of HA9801,ZY05719, ATCC43765,11611(isolated from pig) and 260(isolated from human) were amplified by PCR and cloned into T-vector for sequence,then their sequences were analyzed by software. Results showed that open reading frame(orf) length of SS2 orf1616 is 3366bp,ORF1616 is consisted of 1121 amino acids(Aa) and molecular weight is about 122kDa.ORF1616 sequence information is limited at Genbank database,Alignment results showed high homologue to that of European strain P1/7(99.8%),but differ from Canada strain 89/1589, the latter only have 917Aas.In addition,signal peptide of SS20RF1616 were analyzed by software,result showed that it harbor 37Aa signal peptide,was cleaved between Ala and Asp.8 Construction of mutation plasmid of three infection-realted factors(cwh,hprk and orf1616)Three genes of cwh,hprk and orf1616 has been studied in previous study.In this study, to construct the homologous recombination plasmids of cwh,hprk and orf1616 of Streptococcus suis serotype 2 so as to provide the basis for further study of their function during infection course.5'-upstream and 3'-upstream of cwh,hprk and orf1616 and their flanking fragments were amplified and cloned into streptococcal integration vector pfw5,to construct pfw5-cwh,pfw5-Ahprk and pfw5-orf1616.