Screening And Identification Of In Vivo-induced Genes Of Streptococcus Suis 2

Streptococcus suis 2, as a zoonosis, has drawn wide public concern due to two major breakouts in Jiangsu and Sichuan provinces in 1998 and 2005, respectively. Since the first patient of streptococcus infection in Denmark, over 200 cases were reported, spanned across more than 20 countries[1], with a mortality from 20% to 45%. In the events of 1998 and 2005, infectants were as many as 229, with a mortality reached 60% to 80%, presenting a even new clinical syndrome named Streptococcal toxic shock syndrome, STSS[2]. Such phenomenon may indicate an important mutation of S. suis 2, leading to high pathogenicity and in vivo survival. Nevertheless, the mechanism of interaction between streptococcus suis and host cells were not elucidated so far. Previous research on streptococcus suis discovered factors include capsular polysaccharide (CPS), suilysin (SLY), extracellular factor (EF), muramidase released protein (MRP) and adhesin[3-5] played pivotal roles in the pathogenisis. However, none of these could explain the unprecedented pathogenic capability of streptococcus suis in 1998 and 2005[6, 7].In vivo induced (ivi) gene[8] is a set of unique gene expressed by pathogenic organisms only after entering host. A vast number of researches demonstrated ivi gene often possessed close relationship with pathogen survival within the host, some may even be the key factors of pathogenic toxicity, which could serve as an excellent target for design and research of antibiotics, diagnostic antigen and vaccines. In vivo induced antigen technology (IVIAT) is the method developed to identify antigens expressed specifically in vivo during human infection, by using convalescent serum to remove in vitro antigen from organism expression library for ivi antigen, this technique maintain the advantage of independence from animal model, making it a key approach for ivi gene research. The current study was designed to screen genome library of Chinese streptococcus suis 2 (05ZYH33) by IVIAT to discover ivi gene for better illustrate the its pathogenesis.1. collection and preparation of streptococcus suis infected serum: a total of nine S. suis 2 convalescent serums were kindly provided by research group led by Prof. Jiaqi Tang. All patients were confirmed with classical clinical syndrome, and 8 effective serums were pooled with protease inhibitor. Aliquots of patients'convalescent serum were adsorbed by whole cell and ultrasound lysates from in vitro cultivated 05ZYH33 and BL21 (DE3) strain to produce adsorbed serum. 5-10μl serum left after each step were validated by ELISA (05ZYH33 and BL21 lysates as envelope antigen) for adsorption effect of serum. 5-10μl serum left after each step were validated by ELISA (05ZYH33 and BL21 lysates as envelope antigen) for adsorption effect of serum.2. establishment of genome library: pET-30 a/b/c vehicle expression system were adopted. Genome DNA of 05ZYH33 stain was extracted, with 0.5–3 kb DNA segments recycled after incomplete digestion with Sau3A I. Segments were inserted into pET-30 a/b/c expression vehicle system digested by its isocaudarner BamH I and dephosphorylated; the product were electro transformed into DH5αcompetent cells, and positive recombinant were screened by using kanamycin LB plates. Positive clones were randomly selected for double endonuclease identification by Xho I and Kpn I after plasmid extraction, the results demonstrate that most insertions were 0.5 ~ 1.5 kb, and total collection of positive recombinant clones were 3.20 X 10~4 approximately, higher than the requirement of a library, indicating the success of library establishment.3. screen of genome library by immunoblotting: genome library transformed organism fluid were evenly distributed on Kana-LB plates (300~500 colonies/plate), positive colonies were 10h 37℃cultured and printed on NC film. The NC films were transferred on LB plates with Kana and IPTG for induced culture 4 h, and 15 min over chloroform for protein from organism lysates. NC film was blocked with 10% nonfat skim milk overnight at 4°C. Each membrane was reacted with adsorbed convalescent sera and horseradish peroxidase labeled sheep anti human IgG for 1~2 h in RT. Those with apparent denser color than background were determined as positive clones in primary screen, a second screen was performed with empty pET-30 a/b/c vehicles as negative control for eliminating false positives. A total of 151 positive clones were determined, with 122 possible ivi genes.4. RT-PCR validation: total RNA of streptococcus suis 05ZYH33 strain were extracted by TRIzol in colonies cultured on Todd-Hewitt (TH) broth. RNA was then treated with RNase-Free DNaseI, the 122 ivi genes were amplified using specific primers and compared on positive controls. Results from gel electrophoresis demonstrated that, 6 genes were suspected ivi genes as they showed weak band; 19 genes were determined as ivi genes as there's no relevant band showing in vitro presentation.In conclusion, by using IVIAT, a total of 556 clones were primarily screened, and 151 left after two repeated screen, with 122 ivi suspected genes. Finally, we determined 25 ivi genes of streptococcus suis 2, functions of which including 5 for ABC transportation system, 2 for type IV secretion system, 1 for virulence, 3 for nucleic acid metabolism, 3 for cellular structure synthesis, 4 for key metabolism route, 1 for transposase enzyme, 1 for DNA translation, transcription and 5 for functinal unknown proteins. Functional analysis suggest taht there ivi genes may well related with in vivo survival and pathogenesis of organisms in host, some may serve and key candidates for toxicity or vaccination factors, requiring further research.