Research Of The Important Pathogenic Gene Of ClpP、ClpX And SarZ In Staphyococcus Aureus

Staphylococcus aureus is an important pathogenic bacterium,,while clpPs clpX and SarZ are important genes regulation of virulence factors of Staphylococcus aureus,clpP and clpX are CLP proteolytic enzymes,they play an important role in the process of organism protein regulation of metabolic processes irreversible damage to proteins and removals.Additionally,SarZ protein belongs to the SarA protein family, is a dimer protein,it can through the combined with hemolysin gene promoter regulation the formation of alpha-hemolysin.MgrA,SarA and SarZ are SarA family protein,highly homologous, phosphorylation of MgrA,SarA and SarZ cysteine in proteins caused a conformational change of the protein,thereby regulating the Staphylococcus aureus virulence factors and bacterial resistance to vancomycin. Research and discovery based on above two aspects, carry out the following research work:1.The study and results of the clpP proteolytic enzyme substratesclpP and clpX by molecular cloning,using the principle of homologous recombination,gene knockout in Newman genome,discovered that the mutant hemolytic declined a lot compared with wild type.Through the gene function complementation experiment of pyj335::CLPP plasmid construction,found the hemolytic recovery.Observation of mutant phenotype in culture medium on the urea agar,found that urease activity increased a lot.By observing the2%milk board,found that the mutant extracellular protease activity has increased.Using the principle of homologous recombination,adding erm marker genes in plasmid construct,gene knockout clpX.Using bacterial two hybrid kit,explore clpP and clpX protein interactions.2. The characteristics and virulence studiesbetweenSarZ protein structure and DNA.In the C-and N-end of the SarZ wHTH area amino acid series of point mutations.The experimental results of EMS A showed that, the SarZ (C13E) and SarZ (C13S) mutations later can changethe binding ability of protein and DNA,SarZ (C13E) combined with DNA is weaker, the combination of SarZ (C13S) ability stronger. milk board is also the result of folding point mutation can alter protein on SarZ13C, resulting in the change ofprotein characteristicsand function.SarZ protein contains two kinds of new protein modification after translation, they are REDOX and cysteine (cys) phosphorylation modification. These two protein modification ways also exist in two family proteins SarA and MgrA.Through the study of the SarZ protein,in analogy to the SarA family protein MgrA,amino acid sequence alignment,MgrA C12E point mutation,to study the effect of protein structure change of its function.Research found that clpP and clpX proteins play an important role in the physiology and pathogenicity of Staphylococcus aureus,but it is not clear what is the real clpP protein substrates.Get clpP and clpX and the double deletion mutant clpP and clpX,use the means of mass spectrometry analysis of protein substrates. On staphylococcus aureus,another is SarA family protein-SarZ protein research,further study of cysteine phosphorylation in protein post-translational modifications.