SezAT And Plcr-the Role In The Highly Virulent Streptococcus Suis Serotype2

As an important zoonosis, Streptococcus suis serotype2can not only cause economicloss to the worldwide swine industry, but also threaten the public health security. Infectionsin humans used to be considered as sporadic, and the pathological manifestations usuallyrestricted to brain, heart, lung and joint, patients exhibit good prognosis. However, twolarge-scale outbreaks occurred during1998and2005respectively in China showedremarkably different infective characteristics compared with previous reports, among those,two were outstanding: one is that it’s the first time to find this pathogen can infect generalpopulation, the other is large proportion of the patient exhibited typical Streptococcal toxicshock syndrome (STSS), even though dosed these patients with antibiotics, thepathogenesis could not be reversed. A lot of them died of severe cytokines storm induced bythe SS2infection. Such situations indicated that, a highly pathogenic SS2species hasevolved in the epidemic area of our country.To investigate the Chinese highly virulent SS2strain’s strategy to favor substantialvirulence genes persistence in the genome without natural pressure and the pathogenic roleof newly acquired virulence-associated gene, in this dissertation we intend to conductrelevant experiments from two aspects:①Previously,our research group identified a Chinese highly pathogenic SS2strain-owned pathogenicity island and nominated it as89K PAI with the support of wholegenome sequencing, genome functional annotation and comparative genomics. Furtherstudy showed this specific89K PAI can excise spontaneously from the SS2chromosomethus forming a plasmid-like molecule able to transfer itself to89K PAI-free SS2strains. Inorder to explore the function of89K PAI in depth, we attempted to induce its excisionthrough the plasmid curing method, but without success. Making a further step to analyzethe architecture of89K PAI, a toxin-antitoxin system was found inside of it and we namedit SezAT. TA systems have been proved to be capable of stabilizing mobile genetic elements,such as plasmids, genomic islands and so on. To reveal SezAT has the ability of enhancing 89K PAI’s maintenance in SS2populations, we aim to destroy SezAT through homologousrecombination firstly and then adopt Cre/loxP site-specific recombination system, thepowerful tool of genomic manipulation, to mediate89K’s excision and obtain the89KPAI-excised strain with a serious screening methods. A control experiment which isconducted with the same assist of Cre/loxP system, except for the difference that in thecontrol one, SezAT is intact. These two experiments together prove that SezAT, as aputative stabilize component, indeed plays a crucial role in the genetic stabilization of89KPAI. As the89K PAI-excised strain is available, basic biological characteristics experimentis then conducted to estimate the phenotypic influence due to the89K PAI’s excision.②To further investigate the high pathogenicity of epidemic strain in China(98HAH12and05ZYH33), our research group previously utilize NimbleGen tiling arrays methodcombined with comparative genomics to systematically analyze18SS2strains, selected onthe basis of virulence and geographic origin and identified a few virulence factors onlypossessed by virulent strain. Among these virulence factors, the PlcR transcriptionalregulator interests us since it plays a very important role in regulating the virulence ofBacillus species and what’s more, the role of PlcR in the Streptococci has not been reportedyet. In order to explore PlcR’s role in the SS2infection.We employ the bioinformaticsanalysis to predict the function of it and then delete the gene with homologousrecombination thus evaluate its effect in the pathogenesis of SS2.In this dissertation, the following experiments are performed:1. construction of knockout mutant of SezAT system: design and construct geneknockout vector based on the principle of homologous recombination, adopt aspectinomycin resistance cassette(Spcr) flanked with loxP sites as select marker. Theknockout mutant was obtained by homologous recombination. Then the introduction of Crerecombinase can catalyze the deletion of Spcr, after the deletion,the plasmid lost under thecondition of37℃. This part of the experiment indicates the Cre/loxP system work well inthe SS2cells.2. the excision of89K PAI with SezAT system destroyed: design and construct theknockin vector based on the the principle of homologous recombination. The attL offsideloxP knockin vector was introduce into the sezTloxP+/Spc-cell thus obtaining a strain thathad a loxP site on the attL’s right hand side. Then the introduction of Cre recombinase can catalyze the excision of the left half of89K, after the excision,the plasmid lost under thecondition of37℃. Next the attR left side loxP knockin vector was introduce into the lefthalf of89K deficient mutant. A Cre recombianse mediated excise reaction was performed inorder to obtain the whole89K deficient mutant89K.3. the attempt to excise89K PAI with SezAT system intact: The attL offside loxPknockin vector was introduce into the wild-type SS2cell thus obtaining a strain that had aloxP site on the attL’s right hand side. Then the introduction of the attR left side loxPknockin vector aid us to obtain a89K PAI flanked with loxP sites genetically engineeredSS2. A Cre recombianse mediated excise reaction was performed but failed to obtain thewhole89K deficient mutant89K. This control experiment highlighted the key role ofSezAT system in stabilizing89K PAI.4. the analysis of basic biological characteristics of89K: compare the growthcurve, hemolytic activity and capsule form between wild-type strain and89K mutantstrain. The results showed there’s no significant influence after the excision of whole89KPAI.5. bioinformatics analysis of PlcR: sequences were analyzed with the tool of BLASTin NCBI. The domain was interpreted by Pfam and InterPro. These results together suggestthe05SSU0241and05SSU0242gene are the plcR genes in Streptococci, but the realfunction of transcriptional regulation remained to be testified.6. construction of plcR knockout mutant:design and construct the gene knockoutvector based on the the principle of homologous recombination. Strategy for plcR deletionis conducted with the allelic replacement with a spectinomycin resistance cassette(Spcr),multiple-PCR and sequence analysis were performed to identify the mutant strain named asplcR.7. role of plcR in the virulence of SS2: the BALB/c mice were challenged with thesame amount of SS2(107CFU/ml). The results showed that both group of the miceexhibited typical infection symptom, while the mice challenged with wild-type strainbehaviour much more serious than the mutant strain challenged mice. Besides, the mutantstrain challenged group has60%survival rates much higher than the wild-type strainchallenged one. The latter has30%survival as the experiment ended. A significantdifference was obtained between the wild-type challenged group and mutant challenged group.In brief, this dissertation draw some conclusions about these experiments describedabove:1.89K PAI-excised mutant89Kwas obtained by using Cre/loxP system combinedwith homologous recombination.2. SezAT plays a crucial role in the genetic stabilization of89K PAI.3. Knockout mutant plcR was generated by homologous recombination.4. Deletion of plcR can cause significant virulence reduction according to the result ofmice challenged experiment.