Isolation And Characterization Of An Avirulent Streptococcus Suis Serotype 2 Strain For Comparative Genomics Analysis

Streptococcus suis serotype 2(SS2) is an important zoonotic pathogen, which causing severe infections to swine industry or people in close contact with pork-derived products. Human infected with S. suis have been reported more than 40 years, but until now new cases of infections still appeared, and the number of reported human infections in Southeast Asia showed ascending tendency in recent years. Two notably outbreaks of SS2 infections have been reported in China(1998 in Jiangsu Province and 2005 in Sichuan Province), which resulted in a high mortality(62.7% ~ 81.3%), and caused global public consideration. Most patients during that two Chinese outbreaks exhibited serious streptococcal toxic shock syndrome(STSS), thus cannot reverse the course of the disease. Our research group paid close attention to the two emergency infections, and isolated two highly pathogenic SS2 strains(S. suis 98HAH12 and S. suis 05ZYH33), which may responsible for the prevalence of disease. We conducted comprehensive studies on biological property observation and genomic characteristic analysis, and characterized a specific pathogenicity island in a length of ~89 kb(89K PAI) in the highly virulent SS2 strains in these two outbreaks. Many gene components located on the 89 K PAI are closely related with bacterial pathogenicity, such as virulence-associated factor(VAF), two-component signal transduction system(TCSTS), and type IV secretion system(T4SS).20 VAFs have been identified in S. suis strains, most important VAFs are capsular polysaccharides(CPS) and suilysin(SLY). CPS could provide protective barrier for bacteria, and also participate in antiphagocytic ability of the host cell, while SLY induce high bacterial density and promote the bacteria trigger inflammatory response of the host. Other VAFs also take part in bacteria-host interaction, and facilitate the bacteria transmits the blood-brain-barrier of the host.However, the virulence differentiation of S. suis strains remains ambiguous, and the long-term objective of S. suis prophylaxis and treatment is to establish effective prevention methods. To investigate the genomic rearrangement and virulence differentiation of S. suis, and explore efficient vaccine, we isolated an avirulent SS2 strain from the epidemic region, and conducted a series of analyses on the biological characteristics identification, whole genome sequencing and comparative genomics analysis.In this dissertation, the following experiments are conducted:1. Identification of the biological characteristics of S. suis 05HAS68. To identify the biological properties of S. suis 05HAS68, we carried out Gram stain first. Hemolytic activity of the strain was measured by using the spectrophotometer method, and whole blood survival ability of the strain was enumerated by calculating surviving bacteria. Bacterial adhesive capacity to the Hep-2 cell was determined using the flow cytometer, and authenticate the biochemical reaction type through Bio Merieux VITEK 2 Systems. After that we confirmed that S. suis 05HAS68 belongs to SS2, and have higher adhesive capacity but lower hemolytic activity compared to 05ZYH33.2. Whole genome sequencing and annotation of S. suis 05HAS68. By using the next generation sequencing technique(Ion torrent PGM), we constructed two genomic sequencing library with a 4 kb Mate-pair library and a shotgun library. Sequence assembly was completed by Newbler v2.8 and CLC Genomics Workbench, and obtained a single circle genome, in a length of 2,176,073 bp. Each coding sequence was annotated by using the RAST annotation tool and aligning against BLAST, COG and KEGG database. The general characteristics of S. suis 05HAS68 genome were analyzed with DNAStar, CGView Server, t RNAScan-SE 1.21, and RNAmmer 1.2. These results indicated that S. suis 05HAS68 genome contains 2009 genes with a G+C content of 41.2%, 56 t RNA, and 12 r RNA.3. Comparative genomics analysis between S. suis 05HAS68 and highly virulent S. suis strains. To identify the VAF and TCSTS on S. suis 05HAS68 genome, VFDB and BLASTp algorithm were applied. Island Viewer, CRISPRfinder, and Prophage Finder tools were used to identified candidate genomic island(GI), CRISPR structure, and prophage structure on S. suis 05HAS68 genome. The ACT and Mauve program were used to investigate the pattern of large-scale genomic rearrangement between the genome of S. suis 05HAS68 and other S. suis strains. After that bioinformatics analyses, we identified a CRISPR structure, four prophage cluster, and a 113 K metabolic island on S. suis 05HAS68 genome. 1741 genes of S. suis 05HAS68 are shared with S. suis 05ZYH33 and S. suis 98HAH12, but important virulence-associated components are absent, such as SLY, MRP, EPF, Sal K/Sal R, Nis K/Nis R, Vir R/Vir S, and Rev S. Transposase and prophage genes mediated the large-scale genomic rearrangement between the genome of S. suis 05HAS68 and other S. suis strains, which exhibited an “X-shape” around the origins/terminus axis(ori/ter axis).4. Distribution of 89 K PAI in S. suis strains, and phylogenetic analyses of S. suis strains. Align the 89 K PAI DNA sequence of the S. suis and other Streptococcus strains. The genome of S. suis strains P1/7, A7, GZ1, S735, ST1 and ST3 has no similarity with the 89 K PAI sequence, and most of the 89 K PAI sequence is identified in the genome of S. suis SC84 and S. suis BM407. Meanwhile, several sequences were appeared in S. agalactiae, S. pneumoniae, and other S. suis strains. The phylogenetic analyses were carried out by using rpo B, 16 S r RNA, internal transcribed spacer gene(ITS), and core gene sequences, indicating that S. suis 05HAS68 occupied different evolutionary position with other SS2 strains.5. Evaluation the virulence of S. suis 05HAS68 and immune protection assay. The piglets were injected with S. suis 05HAS68, and highly virulent strains S. suis 05ZYH33 and S. suis 98HAH12 in a dose of 1 × 108 CFU/piglet, respectively. Results of experimental infections indicated that S. suis 05HAS68 is an avirulent strain. The piglets were injected with S. suis 05HAS68 or Δsal KR in a dose of 1 × 108 CFU/piglet, and gave the same dose for booster on day 14, then challenged with highly virulent strains S. suis 05ZYH33 or S. suis 98HAH12 in the same dose. The experimental results showed that piglets can be protected from the attack of highly virulent strains, which were pre-injected with S. suis 05HAS68, but piglets injected with Δsal KR cannot suffered the attack of S. suis 05ZYH33. Those results preliminary identified the protective effect of S. suis 05HAS68 to the infected animals.