The Study On The Molecular Mechanism Of Surface Protein Hp0272of Streptococcus Suis Serotype2in Evasion Of Host Innate Immunity

Streptococcus suis (S.suis), belonging to cluster R with Lancefield classification, is an important zoonotic pathogen of human and pig. Among the35serotypes of S.suis, serotype2(SS2) is the most opular and pahogenic. The surface hypothetical protein0272(Hp0272) of S.suis, as a adherance ligand molecular with strong immunogenicity, can be candidate of vaccine molecular, and has the action in evasion of host innate immunity and anti-phagocytosis but the molecular mechanism have not been well understood.In this study, the interaction model between S.suis and human serum with fluorescence activted cell sorter (FACS) was constructed successfully, then studied the deposition of complement on the surface of SS2strong virulent strain wild type05ZYH33, mutant Δ0272and its complemented strain CA0272. The role of Hp0272in S.suis for complement activation by alternative pathway was analyzed. Hp0272binding with a negative regulator of human alternative complement pathway (human Factor H, hFH) was identified, and the effection of the interaction on inhibition of complement activation in human serum was revealed. Then the GST (glutathione S-transferase)-tag fusion protein Hp0272of SS2was recombinanted and purified. The binding model of Hp0272and hFH was studied by GST-Pulldown, thus the molecular mechanism of evasion of host innate immunity and anti-phagocytosis of Hp0272were revealed. The results are as follows:1. Influence of Hp0272on complement in human serum activation by s.suis.The deposition of C3b/iC3b and iC3b on the surface of SS2strong virulent strain WT05ZYH33, mutant Δ0272and its complemented strain CΔ0272were analyzed by FACS. Results showed that C3b/iC3b deposited onto the surface of mutant Δ0272((fluorescence index, FI)=(10.7±5.0)×107)was more than WT05ZYH33(FI=(3.8±0.2)×107) and complemented CΔ0272strainFI=(4.6±2.1)×107) significantly at50%serum, however, the iC3b appeared opposite trend. The results suggested that Hp0272may inhibite the human serum complement activation by S.suis. Moreover, the phenomenon the iC3b binding to the surface of mutant Δ0272(FI=(3.5±0.2)X106) was less than WT05ZYH33(FI=(5.7±0.0) X106) and complemented CΔ0272strain (FI=(4.2±0.0)×106) significantly suggests that HpO272seems to have interaction with negative regular of complement activation pathway, and degrated C3b into iC3b.2. The analysis of the activation pathway of complement by streptococcus suis.The detection model of S.suis with flow cytometry (FACS) was contructed and optimized. Inhibition experiments with inhibitors of classical pathway, alternative pathway and MBL pathway respectively by FACS found that the inhibitors of classical pathway and MBL pathway couldn’t play an inhibitory effect, while the inhibitors of alternative pathway could reduce more than90%of C3b/iC3b deposited to the surface of S.suis, indicating S.suis activates host complement system mainly through the alternative pathway, so the regular interacted with HpO272likely to be hFH.3. The identification of Hp0272binding with hFH.Ligand blotting result showed that HpO272binding hFH in human serum specifically but not purified hFH, which suggested that HpO272combined with hFH indirectly. The validation assay of the in stu strain level by FACS showed that hFH binding to Δ0272(FI=(0.7±0.2)×106) surface is less than05ZYH33(FI=(1.4±0.3)×106) and CΔ0272(FI=(2.4±0.6)×106) significantly; the specific anti-hFH antibody blocking assay revealed that the presence of anti-hFH polyclonal antibody strongly increased deposition of C3b/iC3b on WT bacteria (increase fold=2.2) and on complemented CΔ0272bacteria (increase fold=2.6) but not on Δ272bacteria. These data confirm that Hp0272-bound hFH inhibits complement deposited to the surface of S.suis activation.4. The identification of the binding mode of Hp0272and hFH and Hp0272binding with other plasma protein ligand.The prokaryotic expression vector pGEX-4T-1-0272was constructed, which was transformed into E.coll BL21, then the GST-tag fusion protein Hp0272was expressed and purified. GST-Pulldown with high specificity between GST-Hp0272and human plasma obtained2diffirent bands.The results of LC-MS showed that one is complex of FH and C3d, which suggesting C3d mediates the binding of Hp0272and hFH, the other is homo kininogen. What’s more, Hp0272can bind with hIgG and hIgA and the binding sites both located on the N terminal (41-318aa).