| Streptococcus suis(S.suis)is considered an important zoonotic agent responsible for a wide variety of diseases in pigs and human,including meningitis,septicemia,arthritis and endocarditis.S.suis serotype 2(S.suis 2,SS2)is one of the most virulent,hazardous and frequently isolated serotype among 33 serotypes,causing important economic losses to porcine industry and endangering public health security.Pathogenic mechanism of SS2 is too complex to explain despite numorous of virulence factors had been reported.In this study,we identified genes involed in anti-phagocytosis by using technology of transposon mutagenesis.Biological function of gene hsdS was analyzed by multiple experiments.This study will lead to a better understanding of the anti-phagocytosis mechanism of SS2.1.Selection of anti-phagocytosis-related genes in Streptococcus suis serotype 2Anti-phagocytosis is an essential feature of SS2 to escape host innate immune system and cause Streptococcus suis diease.This research was aimed at identifying genes involed in features of anti-phagocytosis by constructing SS2 mutant library and phagocytosis test.Finally,we successfully constructed a mutant library containing 2000 mutant strains and selected 4 mutants with high phagocytic rate which means weakened ability in anti-phagocytosis.Genes inserted by transposon in the 4 mutants are as follows:ZY05719_RS02540,ZY05719_RS06850,ZY05719_RS08285 and ZY05719_RS03750,with product of permease,type I restriction endonuclease subunit S,1,4-dihydroxy-2-naphthoate octaprenyltransferase and branched-chain amino acid ABC transporter,respectively.This study will provide a foundation to the research of anti-phagocytosis mechanism of SS2.2.Verification of anti-phagocytic function of hsdS in Streptococcus suis serotype 2hsdS is a gene in type I R-M system gene cluster of SS2,encoding protein HsdS,forming retriction endonuclease and methyltransferase.In this study,we verified the anti-phagocytic function of hsdS by constructing complementation strain CMhsdS using a shuttle vector,pSET2.Results of phagocytosis test showed that the anti-phagocytic ability of complementation strain CMhsdS was recovered for 45.4%compared to mutant strain MhsdS(P<0.001).In addition,compared to wild strain ZY05719,mutant strain MhsdS was significantly different in vability in microglia,pig whole blood,acid condition of pH5 and oxygen stress with 1mM H2O2,and ability in stimulate production of cytokines and nitric oxide by microglia(P<0.05).Biological characteristics of complementation strain CMhsdS were partly recovered.Transcriptome analysis showed that there were 56 genes expressed differently between wild strain and mutant strain,among which 33 genes were up-expressed while 23 were down-expressed.In summary,hsdS is indeed a gene related to function of anti-phagocytosis,and also a gene contributed to many biological characteristics in SS2.3.Analysis of anti-phagocytic function of type Ⅰ restriction-modification system in Streptococcus suis serotype 2Type Ⅰ R-M system studied in our research contains four genes:hsdR,hsdM,hsdS and hsdS’.hsdS is the inserted gene by transposon TnYLB-1,verified to be anti-phagocytic related.In order to study the function of type I R-M system in anti-phagocytosis,six defective mutant strains(△hsdM,△hsdR,AhsdSp,△hsdSf,△hsdS’p and △hsdS’f)were constructed by temperature-sensitive shuttle vector pSET4s.Compared with wild strain ZY05719,mutant strains had no significant difference in growth rate,but were partly different in phagocytic rate and ability in stimulate production of cytokines by microglia.Therefore,the anti-phagocytic function of hsdS may related to the biological functions of type Ⅰ R-M system. |
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