Effects Of DNA Nuclease On The Virulence Of Streptococcus Suis Serotype 9 And The Identification Of Small RNA Targets Of Streptococcus Suis

Streptococcus suis(SS)is an important zoonotic pathogen which causes meningitis,endocarditis,pneumonia,arthritis,sepsis and even acute death in pigs.Based on the difference of antigenicity of capsular polysaccharide,SS can be divided into 33 serotypes so far,of which serotype 2(SS2)is the most prevalent and virulent.However,serotype 9(SS9)is also a prevalent serotype isolated from diseased pigs in China and some countries in Europe.The first case of human infection caused by SS9 has been reported recently in Thailand.More than 80 virulent factors of SS have been identified.They play essential roles in bacterial infection.In addition to surface proteins,small RNAs(sRNAs)are also considered as important virulent factors.They can regulate the expression of several virulence associated genes to affect the bacterial virulence.In this study,the effects of DNA nuclease on the virulence of SS9 were investigated and the targets of virulence related sRNAs rss04 and rss06 were identified.These findings will pave the way for further understanding the virulence characteristics of this pathogen.1.The relationship between DNA nuclease and SS9 virulencePrevious studies have shown that DNA nuclease(SsnA)exists in SS9 virulent strains and is located on the surface of bacteria.However,SsnA does not exist in SS9 avirulent strains.To study the biological function of ssnA,we constructed the ssnA deletion strain?ssnA by shuttle plasmid pSET-4s.Then the zebrafish virulence test,HEp-2 cell adhesion,pig whole blood survival and enzyme activity test were conducted to compare differences of virulence between wild type strain and AssnA.In a zebrafish infection experiment,the 50%lethal dose value of ?ssnA was 11.2-fold higher than that of wild type strain.Ihe adhesion rate of AssnA to HEp-2 cells was 60.61%of the wild type strain.The survival rate of ?ssnA in whole blood was 71.88%of the wild type strain.Enzyme activity test showed that SsnA degrades both linear and circular DNA.SsnA contributes to SS9 virulence in a zebrafish infection model,the adherence to HEp-2 cells and the survival in pig blood.SsnA is indeed an essential virulence factor for SS9.2.Identification targets of small RNA in SSThe identification of sRNA target is critical to reveal the regulation mechanism of sRNA.However,the related studies are few in gram-positive bacteria.In the previous study,29 sRNAs were found in the SS2 meningitis reference strain P1/7,of which rss04 and rss06 are associated with SS virulence.In this study,we constructed the plasmids pAT18-MS2-rss04 and pAT18-MS2-rss06 for transcribing fusion sRNAs MS2-rss04 and MS2-rss06,respectively.By MS2 recognition sequence purification combining with bio-information analysis,11 candidate targets of rss04 and 15 candidate targets of rss06 were identified,respectively.Then SSU0057,one of candidate targets of rss06,was selected to verify our results.Flag tag fusion expression and Western blot anylysis showed that rss06 can significantly inhibit the expression of SSU0057 in translation level,which confirmed SSU0057 is a direct target of rss06.These findings contribute to better understanding the sRNA regulation mechanism in SS.