Streptococcus Sanguinis Pox-mediated H₂O₂ Production Effect On Programmed Cell Death And Competence Development

Objective:Streptococcus sanguinis?S.sanguinis?is the normal oral flora of human,also one of the most common agents of infective endocarditis?IE?in the oral.Under normal conditions,S.sanguinis is fixed in dental plaque,and it is also thought to be a benign microbe in the oral environment.However,when introduced into the bloodstream through the tiny wound and cause bacteremia,S.sanguinis proved to cause IE.Despite improvements in medical and application of antibiotics to a certain extent to prevent IE,its mortality remains at 12⁴5%with antibiotic resistance and no effective vaccine.Base on the studies of S.sanguinis,study on the deeply pathogenic mechanism of S.sanguinis in the field is still have a great sense.Whether in dental plaque or on heart valves,S.sanguinis need to form biofilm.Biofilm formation plays a crucial role in adapting to the environment and invading the host.It is commonly known that in oral streptococci the pox?encoding the pyruvate oxidase?is responsible for endogenous hydrogen peroxide?H₂O₂?generation under aerobic conditions.As the second messenger,endogenous H₂O₂ could transmit extracellular stress information into the cell to affect extracellular polysaccharides?EPS?production and lead to programmed cell death?PCD?.And it also could help biofilm formation and reshape the bacterial genome to generate antibiotic resistant strains.Moreover,as a stress response,competence development could induce biofilm formation,increase bacterial adaptability in hostile conditions,and be conducive to aggressivity and antibiotic resistance.Also,it has been found that pox is involved in Streptococcus pneumoniae competence development.These results prompt us to decipher that endogenous H₂O₂ plays a key role in effector of pathogenicity,survivability and antibiotic resistance in S.sanguinis.Additionally,there are highly conservative global transcriptional regulatory factors Spx in Gram-positive bacteria,and Spx CXXC motif is the Spx function structure and is extremely sensitive to oxides.S.sanguinis site-directed mutagenesis strains have a defect in competence development,and it shows that the Spx CXXC motif could affect bacteria competence development.The objective of this study is to investigate whether S.sanguinis Pox-mediated endogenous H₂O₂ plays a role in EPS poduction and biofilm formation,regulation of PCD and competence development,and whether the Spx A2 CXXC motif is involved in S.sanguinis competence development,and to investigate the pathogenic mechanism,to provide the preliminary guidance of whether the H₂O₂ involves in SpxA2 CXXC motif redox and potential prevent and cure methods in present study.Methods:S.sanguinis SK36?WT?and its pox knockout mutant??pox mutant?were chosen as subjects.Under aerobic conditions,Prussian Blue?PB?agar plate was used to identify the production of H₂O₂ in WT and the?pox mutant,and viability of?pox mutant was decided by colony-forming unit?CFU?and growth curve.Congo red agar plate was used to determine the production of EPS.Biofilm formation in WT and?pox mutant was measured using crystal violet microtiter assay to stain the cells cultured in 96-well plates.Then to investigate whether Pox-mediated endogenous H₂O₂ induced cell death in S.sanguinis exhibited typical biochemical hallmarks of PCD,such as membrane potential,DNA ultrastructure and exposure of phosphatidylserine?PS?on the outer membrane,fluorescence-activated cell sorting?FACS?analysis was used.Meanwhile,real-time RT-PCR was used to analyze related DNA damage repair genes expression in WT and?pox mutant.Finally,this study used gene knockout technology to build spxA2 knockout mutant??spxA2 muatnt?,pox and spxA2 double genes knockout mutant??pox?spxA2mutant?and site-directed mutagenesis mutants[spxA2?C10A?and spxA2?C13A?].The competence development of WT and these mutants were compared by transformation efficiency assay and real-time RT-PCR.In present study,the?pox mutant was treated with 1mM H₂O₂ as one control group to investigate whether endogenous H₂O₂ has the effect on competence development and PCD.Results:1.?pox mutant built successful and its decreased H₂O₂ production.Under aerobic conditions,the?pox mutant was observed diminished H₂O₂ production compared with the WT by PB agar plate.2.Increased cell viability and EPS poduction,and reduced biofilm formation in the?pox mutant.The CFU counts of?pox mutant were higher than that in WT?P<0.05?.The cell viability also examined by growth curve showed that the?pox mutant later than WT reached the stationary phase.Meanwhile,the?pox mutant number of cells in the stationary phase was higher than that in the WT.The Congo red agar plate showed that?pox mutant colony color has changed into black for more EPS production compared with the WT.The phenotype of the cells cultured in 96-well plates and the result of crystal violet microtiter assay showed that?pox mutant biofilm formation reduced.3.The Pox-mediated endogenous H₂O₂ induces bacterial PCD.FACS results showed that the?pox mutant had a reduction in hydroxyl radical OH-formation compared to the WT?P<0.05?.Furthermore,we found typical biochemical hallmarks of PCD in the?pox mutant were significantly different from that in WT,such as decreased membrane potential depolarization,DNA fragmentation,chromosome condensation,and PS exposure on the outer membrane?P<0.05?.However,the biochemical hallmarks of PCD levels in 1 mM H₂O₂-treated?pox mutant went back to the WT levels.4.Enhanced related DNA damage repair genes expression in the?pox mutant.Compared with the WT,these DNA damage repair genes?comEA,recA,dnaC,dinG and pcrA?elevated expression in the?pox mutant by real-time RT-PCR?P<0.05?.5.Increased transformation efficiency and com gene expression in?pox mutant.In transformation efficiency assay,the?pox mutant transformation efficiency increased about 161.31%compared with WT?P<0.05?.The results from real-time RT-PCR showed that?pox mutant had a 2-to 8-fold increase in the expression levels of com genes?comD,comE,comX and comYA?compared with the WT?P<0.05?.However,the expression of com gene was noticeably reduced in the 1mM H₂O₂-treated?pox mutant?P<0.05?.6.Enhanced H₂O₂ production,decreased transformation efficiency and com gene expression in?spxA2 mutant.PB agar plate showed that?spxA2 mutant H₂O₂ production is slightly higher than that of WT,and?spxA2 mutant pox expression is higher than that in WT?P<0.05?.In the transformation efficiency assay,the transformation efficiency of?spxA2 mutant was significantly decreased compared with WT?P<0.05?.Also,the com gene expreaasion levels in the?spxA2 mutant were lower than that in WT by real-time RT-PCR?P<0.05?.7.Decreased H₂O₂ production,increased transformation efficiency and com gene expression in?pox?spxA2 mutant.The?pox?spxA2 mutant showed nearly none H₂O₂production on PB agar plate.Moreover,the?pox?spxA2 mutant had more transformants than WT?P<0.05?.The com gene expression in?pox?spxA2 mutant was significantly higher than that in WT?P<0.05?.8.Decreased transformation efficiency and com gene expression in spxA2?C10A?and spx A2?C13A?.In the transformation efficiency assay,spx A2?C10A?and spx A2?C13A?behaved like the?spxA2 mutant,also had a lower transformation efficiency than WT?P<0.05?.In addition,the real-time RT-PCR results showed that the level of com gene expression in spx A2?C10A?and spx A2?C13A?seemed like that in?spxA2 mutant,but significantly lower than that in WT?P<0.05?.Conclusion:1.The absence of pox could affect the viability of S.sanguinis and the formation of biofilm.2.S.sanguinis Pox-mediated H₂O₂ production could induce PCD,and affect competence development.It is an important factor for bacterial evolution,environmental adaptability and pathogenic ability.3.The effect of the global transcriptional regulatory factor Spx on S.sanguinis competence development was dependent on the CXXC motif;also Spx could affect endogenous H₂O₂ production.4.SpxA2 CXXC motif is sensitive to the oxides,while H₂O₂ as one of ROS may cause the changes of CXXC motif conformation by redox reaction,and affect the combination with the RNA polymerase?subunit to regulate the downstream com gene transcription.