The Influence Of Streptococcus Suis Serotype 2 Dnak In Macrophage Phagocytosis Resistance

Streptococcus suis serotype 2(SS2)is an important zoonotic pathogen.Two large outbreaks caused by SS2 occured in 1998 Nantong Jiangsu and in 2005 Ziyang,Sichuan,which resulted in human-swine infection and caused serious casualties.SS2 severely endangered the China's pig industry and public health safety.Streptococcus suis type 2 could cause Streptococcallike toxic shock syndrome(STSS)in human and septicemia and meningitis in pigs,resulting in acute infection and acute death.Phagocytosis is an important part of host's innate immunity.After phagocytes,such as monocytes,macrophages,and neutrophils,internalized the pathogenic bacteria,they can eliminate the bacteria by bactericidal substances such as lysosomal proteases,reactive oxygen species,and reactive nitrogen.A Streptococcus suis serotype 2 ZY05719 transposon mutant library was conserved in this lab,which was based on a thermosensitive suicide shuttle plasmid pMar4s.The pMar4s plasmid contains the TnYLB-1 tranposon,Himarl C9 transposase and thermosensitive fragment repATs.Transposon mutant library has been widely used in screening for novel virulence factors,which provided an effective means for elucidation mechanism of bacterial pathogenesis.In this study,the transposon mutant library was screened for mutants with decreased anti-phagocytosis ability to RAW264.7 cells,inserted genes were identified and their anti-phagocytosis mechanism were studied.This study provides new ideas for further understanding the pathogenic mechanism of Streptococcus suis type 2.1 Screening and identification of anti-phagocytosis related genes via ZY05719 transposon mutant libraryBy screening the mutant library of SS2 ZY05719,13 mutant strains with decreased phagocytic resistance ability to RAW264.7 cells were identified.Through inverse PCR and sequencing,putative transposon insertion sites were discovered.Through bioinformatic analysis and normal PCR assay,the 13 inserted genes were identified,and the disrupted genes were Cps2F,WalR,TehB,rpiA,prs,HsdM,proB,and upstream region of dnaK,GNAT family N-acetyltransferase encoding gene,S-transferase encoding gene and 3 genes belonging to ABC transporters.Except for Cps2F,a component associated with capsular polysaccharide biosynthesis,other genes were not elucidated for their antiphagocytic ability.The survival ability in macrophages of randomly selected 4 transposon mutants reduced significantly compared with wild-type strain.The survival ability in whole blood of randomly picked 3 mutants were significantly impaired compared with ZY05719.The virulence of the mutant strains M319,M431 and M137 was attenuated in a mouse infection model more or less.WalR transposon mutant was used to verify its anti-phagocytosis function.The transcription of HP1065,which is a cell wall hemostasis related gene,decreased significantly compared with wild-type strain,meanwhile,the anti-phagoctosis ability of ?HP 1065 decreased significantly.This indicated through regulation of HP1065 expression,WalR contributed to the anti-phagocytosis of SS2.2 The transcriptomic analysis of RAW264.7 cells treated with DnaK protein of Streptococcus suis serotype 2DnaK is a moonlighting protein.Cytosolic DnaK participated in revising misfolded proteins and degrading denatured proteins,while the extracellular DnaK could act as adhesin or chaperokine,which was involved with triggering the innate immunity and adaptive immunity and induction of pro-inflammatory cytokines.In this study,to investigate the regulatory mechanism of SS2 DnaK protein on macrophage phagocytosis and immune process,RAW264.7 cells were treated with recombinant SS2 DnaK protein and a transcriptome analysis was conducted.A total of 8282 differentially expressed genes were detected,in which 4406 were up-regulated and 3876 were down-regulated.Among the up-regulated genes,multiple phagocytic related and immune response-related pathways were enriched.The down-regulated genes enrichment was mainly involved with such as metabolism of fatty acids,DNA replication,carbon metabolism,and cell cycle pathways.DnaK protein promotes the up-regulation of multiple cytokines in RAW264.7 cells,such as Il12b,Il1b,116 and tnf.DnaK protein also affected the transcription of multiple pattern recognition receptors(PRR),leading to the down-regulation of multiple Toll-like receptor genes,such as TLR2,TLR4 and TLR7,while some other PRRs,such as CD36,Nod2,Dectin-1 and SR-A,were up-regulated.Through high-throughput transcriptomic analysis,this study deepened the understanding of the immune regulatory function of SS2 DnaK protein.3 Analysis of Streptococcus suis serotype 2 DnaK in phagocytosis resistance by RAW264.7 cellsHeat shock protein DnaK is a bacterial homolog to eukaryotic Hsp70.Knockdown of the dnaK gene(dnaK-KD SS2)was constructed in this study.dnaK-KD SS2 led to attenuated anti-phagocytosis ability and anti-opsonophagocytosis ability.dnaK-KD SS2 also showed decreased survival ability in H2O2 and 0.3 M NaCl environment,as well as whole blood.In addition,dnaK mutant strain showed increased surface located DnaK protein compared to wild-type ZY05719.As dnaK-KD SS2 showed decreased phagocytosis resistance,to explore the mechanism of the anti-phagocytosis of DnaK,the transcription of putative DnaK receptors TLR2,TLR4 and LRP1 were examined.LRP1,an Hsp70 receptor,increased significantly in dnaK-KD SS2-infected macrophages compared to ZY05719-treated cells.LRP1 is a multi-functional receptor,implicated in endocytosis and signal transduction.LRP1-mediated endocytosis is involved with tyrosine and serine/threonine phosphorylation in its cytoplasmic domain and interactions with adaptor proteins.The pan PKC inhibitor Go6983 inhibited the ingestion of dnaK-KD SS2 by macrophages,with no significant influence on wild-type strain.LRPAP,the antagonist of LRP1,could block the phagocytosis of dnaK-KD SS2.These results suggested that anti-phagocytosis resistance of DnaK was LRP1 associated.Further,SS2 DnaK and SS2 infection both could inhibit LRP1 expression.Thus,dnaK-KD SS2 infected RAW264.7 cells showed increased LRP1 level compared with WT infection group,leading to increased phagocytosis rate.However,through Pull-down assay,the direct interaction between DnaK and LRP1 was negative,which suggested that LRP1 did not act as DnaK receptor.Therefore,DnaK modulation of LRP1 expression may be mediated by other molecules.It was reported that TLR7 can positively regulate the expression of LRP1.In this study,it was showed that DnaK protein can inhibit the transcription of TLR7 in RAW264.7 cell.Hence,in this study,SS2 DnaK protein might indirectly inhibit the expression of LRP1 through repression of TLR7,which resulted in antiphagocytosis of SS2.In summary,by screening the mutant library of SS2 ZY05719,13 mutant strains with decreased phagocytic resistance ability to RAW264.7 cells and the corresponding genes were identified,dnaK gene included.Through transcriptome analysis,differentially expressed genes of SS2 DnaK treated RAW264.7 cells were screened,searching for the phagocytic related and immune related genes.In this study,SS2 DnaK was shown to inhibit LRP1,which contributed to the phagocytosis resistance of SS2.