Dank Family Protein Dnaj Of Streptococcus Suis Type2is Involved In Heat Stress And Adhesion To Host Cells

Streptococcus suis is an economically important disease. S. suis type2（SS2） is the most virulent and prevalent in pigs and humans. A large body of recent research has uncovered a number of putative virulence factors, such as suilysin, autolysin, su-peroxide dismutase, enolase, and the like. Adherence to host cells is the first and key step for successful infection. Virulence factors involved in adhesion identified so far in SS2include glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, sortase A, di-peptidyl peptidase IV and glutamine synthetase. A recent study shows that heat shock protein DnaK might be an adhesion molecule of SS2. DnaK family proteins DnaK family proteins belong to heat-shock proteins （HSPs） and are conserved in SS2strains. However, their functions other than DnaK itself in the pathogenesis of SS2infection remain largely unknown. This study aimed to find out:（1） the surface abundance of DnaK family proteins in SS2, and （2） their roles in resisting heat stress and bacterial adhesion to host cells.Biological characteristics of SS2strains JX0811and HA9801isolated from clin-ically infected pigs in Zhejiang and Jiangsu provinces were initially compared. There was apparent difference between the two strains in their adhesion to HEp-2cells, HA9801being significantly more adherent （7.6%） than JX0811（0.2%）（P<0.01）. In-direct ELISA using whole bacterial coating （HA9801） and hyperimmune sera to re-combinant DnaK family proteins revealed the surface abundance of DnaJ protein over the other proteins examined. The DnaJ protein was significantly higher in the strain HA9801than JX0811（P<0.01）. The rabbit anti-DnaJ polyclonal antibody could effec-tively antagonize SS2adhesion to HEp-2cells. Therefore, DnaJ is an important sur-face protein of SS2and has a role in adhesion. This provides a basis for further ex-ploration of the mechanisms of SS2adhesion.Exposure of bacteria to environmental and host factors may lead to the heat shock response such as increased synthesis of HSPs. We attempted to construct mutant strains lacking the genes encoding the DnaK family proteins from the strain HA9801to analyze their functions on cell adhesion, thermotolerance and their mutual rela-tionship. Except for the deletion mutant ASSU05₀301（gene encoding a hypothetical protein）, we failed to obtain the deletion mutants of the genes hrcA, grpE, dnaK, dnaJ and the hrcA-dnaJ cluster although double cross-over was seen with all these putative mutants. However, these genes appeared to be down-regulated （named as knock-down strains, abbreviated "kd"）. Compared to the wild type strain, dnaK（kd）, dnaJ（kd）, hrcA-dnaJ（kd） and△SSU05₀301strains exhibited decreased adhesion to HEp-2cells （P<0.05）. Heat stress had certain influence to all mutant strains. However, the dnaJ（kd） strain was the most susceptible to heat stress. Heat stress for3hours at42℃increased the expression level and surface abundance of DnaJ protein in the strain HA9801. And the treated HA9801also had enhanced adhesion to HEp-2cells.In conclusion, heat shock protein DnaJ in the DnaK family proteins of S. suis type2is functional in themotolerance and adhesion to host cells. With clinical cases due to systemic infection, the febrile condition could be a facilitating factor to help this bac-terium improve its adhesion to host cells by raising expression of the DnaJ protein. Since DnaJ is highly immunogenic, it could also be a putative molecule for develop-ment of subunit vaccines against S. suis type2infection.