Isolation And Identification Of Streptococcus Suis NCL Strains And Genomic Analysis Of NCL4 Strain WUSS351

As a zoonotic pathogen,Streptococcus suis has caused serious damage to the development of the pig industry in the world.According to the antigenicity of its capsular polysaccharide(CPS),S.suis can be divided into 29 traditional serotypes,Chz serotype,26 new capsular gene clusters(NCL)strains and some non-typed strains.However,the information about the pathogenic characteristic of NCL strains is very limited.To remedy this,S.suis strains were isolated and identified by collecting swine tonsils from a slaughterhouse in Jiangsu Province,and the virulence of NCL strains was evaluated in a zebrafish model.The genome of NCL4 strain WUSS351 was sequenced and a new VII secretion system was found in this strain.The results not only provide a reference for the prevention and control of S.suis infections in the region,but also enrich the knowledge of pathogenic characteristics of S.suis.1.Isolation,identification and pathogenicity analysis of S.suis NCL strainsTo isolate and identify S.suis,the total of 378 samples of tonsils were collected from a slaughterhouse in Jiangsu Province between 2017 and 2018.Isolates were identified as S.suis strains by the presence of S.suis specific genes recN and gdh and 268 samples were indentified as S.suis(70.8%).In 2017,there were 184 samples,of which 141 were positive(76.6%);in 2018,there were 194 samples,of which 127 were positive(65.4%).Single colony was isolated from S.suis positive tonsil samples,and the S.suis isolates were classified by PCR.A total of 339 strains of S.suis were isolated,including 296 strains belonging to the traditional 29 serotypes,accounting for 87.3%.There were 20 NCL strains,accounting for 5.9%.23 strains were unknown serotypes,accounting for 6.7%.Among all the isolated NCL strains,12 strains were isolated in 2017,and eight strains were isolated in 2018.The percentage of NCLl was the highest at 25%(5/20),followed by NCL25(20%,4/20).NCL3,NCL7,NCL11,NCL17 and NCL20 all accounted for 5%(1/20).Except that the virulence of NCL4 strain WUSS351 was similar to that of S.suis serotype 2 strain SC070731,the virulence of other NCL strains was significantly lower than that of SC070731 in a zebrafish infection model.2.Genome analysis of NCL4 strain WUSS351The whole genome of NCL4 strain WUSS351 was sequenced.The genome size was 2.28 Mb,and there was no plasmid.The average GC content was 41.3%.The sequence of cps gene cluster in WUSS351 showed 98.93%nucleotide identity to that of genes in NCL4 strain YS33 whose sequence is available on NCBI.The capsule of WUSS351 strain was observed by transmission electron microscopy.Phylogenetic tree analysis based on the whole genome sequence showed that WUSS351 was closely related to strains HA1003,0061,1081 and serotype 5 strain HN105,but distantly related to serotype 2 strains.Compared with the whole genome sequences of 42 strains of S.suis published on NCBI,WUSS351 contains a new VII secretory system gene cluster,which is located in a 25K island.Further analysis of the VII secretory system gene cluster showed that there were five candidate effector molecules in this gene cluster:two proteins with 100%homology belonging to the SACOL2603 family(1962 and 1956)and three proteins containing LXG domain(1960,1954 and 1948).pSET2 plasmid was used to construct the expression vectors of SACOL2603 family protein 1962 and three proteins containing LXG domain(1948,1954,1960).The recombinant plasmid was electrotransformed into WUSS351,and Western blot confirmed that all four proteins could be expressed and secreted.pBADhisA plasmid was used to construct an expression vector for those proteins and the recombinant plasmids were transformed into Escherichia coli.By measuring the growth curve,it was found that two proteins,1954 and 1960,could significantly inhibit the growth of E.coli,and the toxic effects of the two proteins were confirmed.The immune proteins of toxic effector molecules were searched in the vicinity of these two proteins.By measuring the growth curve,it was found that 1953 protein could significantly inhibit the toxicity of 1954 to E.coli,indicating that 1953 was the immune protein of 1954 toxic effector molecule.