Genetic Regulation Of Pyruvate Carboxylase Gene In Staphylococcus Aureus

Staphylococcus aureus,a gram-positive bacterium,is often parasitic on the surface of skin and mucous membrane and causes a variety of infections in many parts of the body.S.aureus is one of the major pathogens causing nosocomial and community-acquired infections and represents a serious burden on healthcare system.For the past few years,with the extensive use and abuse of antibiotics,the situation of bacterial drug resistance has become increasingly serious and the prevention and treatment of S.aureus infection has become more difficult.Methicillin-resistant S.aureus,recognized as a "superbug",is resistant to most penicillin antibiotics and has severely curbed the use of drugs in the clinic.Recent studies show that mutation of pyruvate carboxylase gene(pycA)in S.aureus results in markedly reduced bacterial virulence,suggesting that pyruvate carboxylase(Pyc A)may be a potential target for resistance to S.aureus infection.An object of this thesis is to screen the genes affecting the expression of pycA by transposon random insertion mutagenesis and explore the genetic regulation of pycA and its relationship with the virulence of S.aureus.Main results showed as follows:1.Using transposon random insertion mutagenesis,89 genes affecting the expression of pycA in S.aureus were screened.Among them,79 genes positively regulate pycA expression while 10 genes negatively regulate pycA expression.As pycA mutant exhibits aspartic acid(Asp)auxotrophy,phenotypic analysis on 79 strains with positive regulation gene of pycA mutation were performed.Transposon insertion mutations of aconitase(Cit B)gene and isocitrate dehydrogenase(Cit C)gene,which encode enzymes in the tricarboxylic acid cycle,caused Asp auxotrophy phenotype in S.aureus.Then gene deletion mutants of aconitase gene(citB)and isocitrate dehydrogenase gene(citC)were constructed by targeted gene knockout respectively.Similar to transposon insertion mutants,both gene deletion mutants((?)citB and (?)citC)exhibited Asp auxotrophy phenotype.Constitutive expression of pycA in (?)citB or (?)citC resulted in partial loss of Asp auxotrophy phenotype.Moreover,it was proved that citB or citC deletion mutation could significantly reduce the infectivity of S.aureus to G.mellonella larvae through functional complementarity experiments.2.Through experiments of bacterial physiology,biochemistry,growth curve analysis and G.mellonella larvae infection model,it was found that: 1)Compared with wild type strain,citric acid content was significantly increased in (?)citB and(?)citC.After citrate synthase gene citZ was further deleted on the basis of citB or citC deletion mutation,which means the strain couldn’t accumulate citric acid,double gene deletion mutant (?)citB(?)citZ or (?)citZC didn’t exhibit Asp auxotrophy phenotype and its virulence was similar to that of the wild type.2)Similarly,after further deletion of ccp E gene(encoding the transcriptional regulator Ccp E in response to citric acid)on the basis of citB or citC deletion mutation,the strain didn’t exhibit Asp auxotrophy phenotype while the pycA expression returned to wild type,and the virulence of double gene deletion mutant((?)citB(?)ccp E or(?)citC(?)ccp E)was higher than that of (?)citB or (?)citC.3)In vitro,purified Ccp E protein directly binded to pycA promoter.Based on the results,the following hypotheses are proposed: In S.aureus,the mutation of citB or citC causes the accumulation of citric acid,and citric acid activates Ccp E,which inhibits pycA expression and affects Asp synthesis,thereby reducing the virulence of strain.3.40 compounds which could cause Asp auxotrophy phenotype in S.aureus were obtained by disk diffusion method.And it was found that Cit C inhibitors DS-1001 B and BAY-1436032 could induce weaker Asp auxotrophy phenotype and reduce expression of pycA in wild type JE2.These results provide a basis for the study of small molecular compounds to inhibit the virulence of S.aureus.