| Streptococcus suis is an important Gram-positive pathogen responsible for enormous economic losses to the swine industry worldwide, which is made up of many serotypes（1 to 32 and 1/2）. the most frequently isolated bacterial strain SS2 also is a zoonotic agent, which colonizes in tosils of host, breach the mucosal barrier, cause bacteremia, invades different tissues and cause meningitis, arthritis, pneumonia, septic shock and other symptoms. In recent years, S.suis has play more and more important role in scholars, many virulence factors have been identified and characterized, such as CPS, Ssn A, suilysin and so on, the exact mechanisms by which this bacterium invades the host, evades immune system and causes infections are still poorly known. Therefore, we consider SS2 SsPepO as a object, the plasminogen-binding protein SsPepO can aid in colonization and adherence to host, immune invasion. The objective of this work is to provide more theoretical basis for preventing and controlling SS2 and interaction of pathogen with host.Dr.Tan constructed a delete mutant strain of the ΔSsPepo（SSU050153） in our group, research showed that the virulence of ΔSsPepo on piglets decrease significantly and the adhesion ability of Hep-2 cells derease compared to the wild strain SC19, so the SsPepo gene is an important virulence factor of SS2. Dr Li identified a novel recombinant SsPepO by prokaryotic expression, which protected mice and pig against lethal doses of SS2 infection and inhibit growth of SS2 in whole blood. It showed that SsPepO can be used as a subunit vaccine candidates. In view of the above background, we try to further explore the function of SsPepO and main content are as follows: 1. Using bioinformatics analysis of SsPepOAcorrding to the sequence analysis of the virulent strain 05ZYH33 SsPepo gene published on NCBI, it is a zinc metalloprotease. Blast comparative analysis found that the protein SsPepO in all SS2 strains are highly conserved, belonging to the family of M13 peptide enzyme. SsPepO shares 29% homology with pneumococcal plasminogen and fibronectin binding PepO. 2. Cloning, expression and purification of SsPepOThe full SsPepo gene was amplified by PCR acrroding to the chromosomal DNA of the virulent strain SC19 with primers, the amplified 1893 bp fragment was digested with Nde I and XhoI and connected to the vector pET-30 a, then transformed into Escherichia coli DH5α with correctly identification and transform into a Escherichia coli（DE3） for prokaryotic expression. The SsPepO was purified by Ni-NTA column according to instructions, the purity of tagert protein was more than 90%. 3. Identification of SsPepO binds plasminogen and fibronectinELISA demonstrated that SsPepO binds specifically to human plasminogen and fibronectin in a dose-dependent manner. Binding of plasminogen did not compete with fibronectin, the SsPepO-plasminogen interaction is affected by ionic strength and is mediated by lysine residues. Bound to SsPepO, plasminogen activated by urokinase-type plasminogen activator, plasmin was able to degrade C3 b thus assisting in complement control. 4. Researh on the physiological characteristics of ΔSsPepo in vivoCompared to the wild-type strain SC19, ΔSsPepo mutant strain showed decreased in invasion and adherence to rat brain microvascular endothelial cell and adherence to human brain microvascular endothelial cell. We compare violence of ΔSsPepo mutant strain with wild strains by different dosage and injection methods, it shows the difference of intraperitoneal challenged group is not obvious, in the intravenous injection group, ΔSsPepo mutant strains was attenuated. In vivo colonization experiment, the colonization of ΔSsPepo mutant strain in the lungs, brain organ has no significant difference in comparison to wild strain after 24 h post-infection.Taken together, SsPepO is plasminogen and fibronectin binding protein, which could play a important role in dissemination of S.suis and contribute to immune evasion. |
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