Studies On Contribution Of Glutamine Synthetase GlnA And Its Regulator GlnR To The Virulence Of Streptococcus Suis Serotype 2

Streptococcus suis 2(S.suis 2) is an important zoonotic pathogen of pigs and humans,associated with meningitis,arthritis,endocarditis,septicemia and even sudden death.Glutarnine,one of the central molecules in nitrogen metabolism,is used as the nitrogen donor for many nitrogen-containing molecules in the cell.Recently studies indicated that several systems involved in nutrition and metabolism,especially glutamine metabolism,were very important for the virulence of various bacterial pathogens. However,so far,and the contribution of glutamine metabolism to virulence in S.suis 2 have not been studied.In the present work,S.suis 2 glnR,glnA and glnRA null mutant strains were constructed and characterized.Functional complementation of each mutant was constructed and characterized by PCR,RT-PCR.The impact of glnR,glnA or glnR/glnA deletion on the biological characteristics and virulence was evaluated.Using quantitative PCR,the transcription profiles of the wild type strain with the glnR,glnA or glnRA deficient mutant were compared to define the precise regulatory mechanism of glnR/glnA in the virulence of highly pathogenic S.suis 2.Below is more detailed information on this study:1.Construction of recombination suicide plasmidsThe upstream and downstream flanking regions of glnR or glnA were separately amplified from S.suis 2 wild-type strain SC19 genomic DNA respectively.Followed by digestion with the corresponding restriction enzymes,the PCR products were directly cloned into a pSET4s vector.The resultant plasmids,pSET4Sâ–³GlnR,pSET4Sâ–³GlnA, pSET4Sâ–³GlnRA were confirmed by DNA sequencing.2.Construction of the S.suis 2 glnR,glnA,glnR/A deletion mutantThe resultant plasmids,pSET4Sâ–³GlnR,pSET4Sâ–³GlnA,pSET4Sâ–³GlnRA were confirmed by DNA sequencing and transformed into SC 19 as described by Takamatsu et al.The resultant mutant strains were verified by PCR and RT-PCR amplification respectively,and direct DNA sequencing of the mutation sites using genomic DNA preparations of the mutant strains.For complementation analysis,a DNA fragment containing glnR,glnA,glnRA and its upstream promoter were amplified and cloned into the E.coli-S,suis shuttle vector pAT18.Then the recombinant plasmids were electrotransformed into the mutants to screen the complemented strains Câ–³glnR,Câ–³glnA or Câ–³glnRA. 3.Effect of glnR,glnA,glnRA deletion on the general biological characteristics of S.suis SC19The general biological characteristics of the wild type strain SC19 and theâ–³glnR,â–³glnA orâ–³glnRA mutant were compared under the same conditions.First,the colony and cell morphology,killing by PMNs and hemolytic activity were examined but,remarkably, no significant differences between the wild type and mutant strains could be ascertained. However,compared with WT,â–³glrd orâ–³glnRA mutant were found to displayed a significant decrease in adhesion to the HEp-2 cells4.Impact of theâ–³glnR,â–³glnA orâ–³glnRA mutant on the virulence of S.suis SC19To evaluate the impact of glnR/glnA deletion on the virulence of SC19,three sets of experimental infections were carried out in parallel.To study the effect of glnR,glnA deletion on the pathogenesis of S.suis 2,the virulence of the WT and glnR,glnA deletion mutant was assessed in a CD1 mouse infection model.Results of the trial showed that the LD₅₀ of the glnA deletion mutant strain was increased up to 2 log compared with the wild-type strain,indicating that glnA deletion significantly reduced the virulence of S. suis 2.5.Role of glnR,glnA in the virulence regulation of S.suis SC19To gain further insights into the network mediated by glnR,gird,quantitative PCR was applied to reveal the differential transcription profiles between the wild type strain SC19 with theâ–³glnR,â–³glnA mutant independently.In total,the absence ofâ–³glnR,â–³glnd led to changed expression of 18 genes,which can be roughly categorized into the following 3 groups:(â…°)Genes involved in glutamine transport;(â…±)Genes involved in carbohydrate and glutamine metabolism;(â…²)Genes associated with virulence of S.suis 2.