Preliminary Study On The Biological Function Of Type 2 Streptococcus Suis Cell Division Gene DivIVA

Streptococcus suis is an important zoonotic pathogen responsible for a wild range of life-threatening diseases in pigs and human.Among the 33 strains,S.suis 2 is considered to be the most virulent strain,which causes severe human infections clinically featuring with varied diseases/syndromes?such as meningitis,septicemia,endocarditis,and Streptococcal toxic shock syndrome?.Proliferation and cell division is the basic pathogenic mechanism for pathogenic bacteria.The DivIVA protein,located at the division septum and controling the site specificity of cell division,is responsible for synthesis of new peptidoglycan.In streptococcus pneumonia,lack of the DivIVA protein will lead to defective cell division and virulence reduction.However,the cell division mechanism in Streptococcus suis serotype 2 remains unkown.On the basis of whole genome sequencing and functional annotation for the China strong virulent strain 05ZYH33,our group found that the transcript protein of gene SSU05₀487 is homologous of DivIVA.The bioinformatics method suggeststhat the function of this gene may be involved in cell division.This study purified the recombinant DivIVA protein by prokaryotic expression technology,and successfully obtained polyclonal antibodies by immunizing the New Zealand white rabbit,and constructed a mutant strain ? divIVA gene based on the principle of homologous recombination.The main results were as follows:1.The bioinformatics analysis of SSU05₀487 encoding DivIVA proteinBioinformatics analysis shows the gene of SSU05₀487 consists of 687 nucleotides,and encodes a protein that has no signal peptide and transmembrane domain,but a typical DivIVA protein domain.The sequence similarityof DivIVA protein was 67%between the S.suis 2 and the Streptococcus pneumoniae.Alpha helix predominate the the secondary structure.2.Recombinant prokaryotic expression and polyclonal antibody preparation of DivIVA protein in S.suis 2The Whole DivIVA encodingDNA sequencewas amplified from the genome DNA of 05ZYH33 and cloned into the expression pET28a vecter to construct the recombinat plasmid pET28a-divIVA.Thereafter,pET28a-divIVA was transformed into E.coli BL21.The culture was induced by adding 1mMIPTG,then the induced cells was collected and subjectd NTA chromatography,the DivIVA protein was purified by 6XHis tag columnThe rabbit anti-DivIVA serum was obtained by immunizing theNew Zealand rabbit with DivIVA protein,and the titer of the serum was 1:409600 by ELISA.Finally the serum could be specifically reacted with the recombinant protein demonstrated by Western blot.3.Construction the ?divIVA mutantTo further study the biological functions of divIVA gene in S.suis 2,the gene knock-out plasmid pUC18::divIVA was constructed and transformed into the 05ZYH33 competent cells by electropotation.Colonies were screened by addingspectinomycin resistance.The fidelity of the double-crossover recombination of mutants were confirmed by PCR using primers inside the homologous regions,then further confirmed by a series of PCR,reverse transcription PCR and sequenceing,the successfully constructed mutant was coined ?divIVA.4.The characterization of ?divIVA mutant in biological functions,cells phenotype and pathogenicityThe basic biological properties of the wild type strain 05ZYH33 and the ?divIVA mutant were compared under the same conditions.Compared to the wild type strain 05ZYH33,the AdivIVA mutant growed obviously slow,and the colony was smaller.The mutant strain is more sensitive than wild type strain in hydrogen peroxide sensitivity experiments.Gram staining result showed that the ?divIVA mutant gathered into a cell mass,not tparallel to chain under in vitro conditions.Scanning electron microscope result showed that the cell division of mutant strain is abnormal,and cell division is disorder.Trasmission electron microscope result showed that the capsule structure of the mutant is more thinner and more looser than the wild strain.In vitro cell interaction experiments showed that the deletion of the divIVA gene in S.suis 2 Iead to increased adhesion to Hep-2 cells,and the ability of anti-phagocytosis for mouse macrophages and human polymorphonuclear neutrophil was reduced.BabL/c Mice model experiments showed that an inactivation of divIVA attenuated the virulence srain the virulence of the 05ZYH33.In conclusion,prokaryotic expression of DivIVA protein could be expression in prokaryotic system and showed a high affinity to rabbit polyclonal antibody,which layed a good foundation for the subsequent protein function research.The deletion of the divIVA gene indicated its importance in cell devision,maintenance of biological characteristic,and suggested its potent fuction in pathogenesis.