Structural And Functional Analysis Of Genomic Islands In Streptococcus Suis Serotype 2

Streptococcus suis is an important pathogen that causes meningitis, endocarditis, septicemia, arthritis, polyserositis, pneumonia and even sudden death. There are 35 known serotypes of S. suis: serotype 1-34 and 1/2. Most cases of S. suis disease are cased by strains belonging to the serotype 2. S. suis 2 is a major swine pathogen responsible for severe economic losses to the porcine industry world-wild. Moreover, S. suis 2 is an zoonotic agent that afflicts people in close contact with infected swine or pork-derived products. Two large-scale outbreaks of human S. suis 2 infections in China (25 cases with 14 deaths in Jiangsu in 1998, and 204 cases with 38 deaths in Sichuan in 2005), which featured streptococcal toxic shock syndrome (STSS), have presented a challenge to public health and have sparked new interest in the zoonotic potential of this organism.Whole genome sequencing of several S. suis 2 strains world-wide was undertaken, but knowledge on virulence factors of S. suis 2 is still limited and pathogenesis of infection is not fully understood. Since, different S. suis 2 isolates have different virulence, we can get new view on pathogenesis by comparative genomics assay. Pathogenicity islands (PAIs), one kind of distinct genetic element, are considered as a subgroup of genomic islands (GIs) which may be acquired by horizontal gene transfer (HGT). Currently, it is accepted that PAIs may contribute to virulence in a wide range of pathogenic bacteria such as E. coli, S. aureus, Enterococcus faecalis and S. pneumoniae. The availability of genomics, a robust tool in the research fields of bacterial genomic plasticity or pan-genome, molecular variation or evolution, will make it possible to identify and characterize novel PAIs that may be linked to the pathogenesis or virulence mechanism caused by S. suis 2.In this work, in silico genome-wide search of published sequences of S. suis 2 virulent strains, we observed some regions with abnormal GC content and often flanked by transposable elements (transposase, site-specific recombinase and phage integrase). In our analysis, there are 14 putative genomic islands (GI1-GI13, and 89K) in S. suis 2 Chinese virulent strain SC84 genome. These putative genomic islands, potentially acquired by horizontal gene transfer, may play a critical role in virulent distinction between different S. suis 2 isolates. Then we analyzed the preferential distribution of these GIs in pathogenic and non-pathogenic S. suis 2 isolates, and investigated the potentiality of these GIs to be PAIs. Comparative genomics accompanied by PCR and sequencing assay, we identified three candidate PAIs (GI4, GI8 and GI12), which are resided in S. suis 2 virulent strains, but not in some avirulent isolates. However, the other GIs (GI1-GI3, GI5-GI7, GI9-GI11, and GI13) have genetic stability and conservatism in all analyzed S. suis 2 strains (no matter virulent and avirulent strains), those GIs may not have direct association to virulence.We investigated the genetic constitution and function of candidate pathogenicity island GI12. The novel PAI candidate contains a gene cluster, with three class C sortase gene (named srtC3, SrtC4 and SrtC5 in finished sequencing project of strain SC84, or named srtD, srtC and srtB in strain P1/7) and four putative pilin genes. The gene cluster was then named srtC345 pilus cluster in our work, and found to be homologous to the S. pneumoniae rlrA pilus cluster. As the important virulence factor, the pneumococcal pili encoded by rlrA islet are not only to participate in adherence and virulence but also stimulate the host inflammatory response.We analyzed the function of putative pilus island GI12 in vivo and in vitro. Human laryngeal epithelial cell line Hep-2 was used to determine the difference of different S. suis 2 strains in the cellular adhesion ability. The results of cell adherence assays showed that: 1) The cellular adhesion ability of the virulent strain ZY458 with GI12 island was about ten times higher than avirulent strain B22. 2) Introduction of the GI12 island into avirulent strain B22 enhanced adherence to epithelial cells, about four times higher than original strain. 3) Inactivation the srtC5 gene within GI12 island of virulent strain ZY458 displayed significant reduction in adherence to epithelial cells. The in vitro cell adherence assays indicated the important role of GI12 island in cellular adhesion, implying the contribution of pilins to this process.To further investigate the role of GI12 island in the pathogenesis of S. suis 2, we constructed a srtC5 gene deletion mutant (?srtC5) and, compared its virulence in vivo with that of WT (S. suis 2 Chinese virulent strain ZY458). The results of rabbits infection experiment showed that the srtC5 deletion mutant (?srtC5) attenuated the full virulence of its wild-type strain. During the experiment, All six rabbits infected by intraperitoneal injection with WT strain, ZY458, developed most of the typical disease symptoms of S. suis 2 infection and died within 5 days post-infection. Postmortem examination showed pathological changes in multiple organs and WT bacteria could be recovered from all of the examined tissue specimens. In contrast, the six rabbits infected with the ? srtC5 mutant, only one rabbits died on day 2 post-infection, and the other five survived until the end of the experiment (15 days after infection). Collectively, we concluded that the pathogenicity island GI12 encoding a putative pilus gene cluster is involved in adherence to host epithelial cells and systemic virulence.