| At present,root canal therapy(RCT)is the effective method to cure pulpitis and periapical periodontitis.Because of bacterial infection in these diseases,the key to success of RCT is whether it can be thoroughly debrided and removed effectively the root canal infection.However,the anatomy of root canals varied dramatically and there are limitations of root canal preparation and root canal irrigation.It is difficult to completely kill the bacteria in the root canals.The residual bacteria have the ability to colonize and survive in the inner surface or dentin tubules of root canals,and when there is nutrition in the root canal,such as microleakage,these bacteria can proliferate in large quantities to cause a persistent infection again.The cases of chronic periapical diseases,which are difficult to cure,have been always encountered in clinical.After repeated treatments,the symptoms are still no improvement or still recurrent.These cases are usually referred to as refractory periapical periodontitis.E.faecalis is the main pathogens of refractory or secondary root canal infection and the biofilm formation is the main way to survive and grow in root canals after its adaptation to the environment.More importantly,E.faecalis could resist the human immune function and exert its pathogenicity through the biofilm in such micro-environment.Studies confirm that the biofilm of E.faecalis is up to 1,000 times more resistant to the planktonic E.faecalis.Furthermore,E.faecalis can be long-term survival in starvation,high alkali,hypoxia and other special environments,which adhere to the dentin surface to form a biofilm and result in root canal reinfection.Therefore,E.faecalis is one of the most important causes of refractory apical periodontitis,with the detection rate of up to 77% in root canals infected with refractory periapical periodontitis,whereas the detection rate only 7.5% in untreated infected root canals.Our research group have engaged in the etiology of refractory apical periodontitis for a long time and found that the clinical isolation of biofilm formation ability of E.faecalis is not the same,and what are the key molecules that regulate biofilm formation of E.faecalis? What is the molecular mechanism? How does it regulate the formation of biofilm? It has been a hot area and difficult point in the field of endodontics.With the high-throughput sequencing technology widely used in the study of microbial genomes,its research on the functions of metagenomic and genomic genomes in oral microorganisms has also been carried out.By high-throughput sequencing of clinical isolates of high biofilms formed by E.faecalis in this study,gene expression profiling results showed that the expression of nitrogen metabolism pathway compared with the control group were significantly different,which the gene expression of gltA encoded glutamate synthase was significantly up-regulated,suggesting that gltA gene and glutamate metabolic pathway in E.faecalis biofilm formation may play an important regulatory role.The aim of this study is to clarify the new mechanism of E.faecalis biofilm formation and improve our awareness of biofilm regulation,and to provide possible targets and pathways for the development of novel antibacterial biofilm drugs and inhibitors.The experiment is divided into the following four parts:PartⅠ Collection,isolation and identification of high biofilm formation of E.faecalis from refractory apical periodontitis in clinicalSelect 45 retreatment cases of refractory apical periodontitis in our department.Clinical sampling was performed under strict aseptic conditions.The samples were cultured in vitro in a laboratory using enterococci medium and bile aesculin medium,and the E.faecalis was identified by observing the color change.Then,strains identified as positive were identified using BBL CRYSTAL system was used to identify the bacterial biochemical reaction.Finally,the 16 SrRNA of bacteria was sequenced and compared with the standard bacteria in the gene bank by polymerase chain reaction(PCR)and DNA purification and recovery techniques.Clinically isolated E.faecalis was assayed for its biofilm formation ability using crystal violet staining.The results showed that in the collected 45 cases of clinical cases,14 cases were isolated and identified E.faecalis,the detection rate was 31.1%.Biofilm formation assay showed that there were 2 strains negative “–”,3 strains of “+”,5 strains of "++",4 strains of “+++”,and the rate of high biofilm formed strains was 28.6%.PartⅡ High-throughput sequencing analysis of differentially expressed genes of E.faecalis with high biofilm formation abilityFour strains of E.faecalis with high biofilm formation ability were isolated and purified.DNA library was extracted and sequenced using Illumina HiSeq2500 platform.Four bacterial transcriptome RNAs were extracted and sequenced using Illumina HiSeq2500 platform.Sequences of the original reads(double-ended sequence)quality assessment and filtering,the high-quality reads replies to the reference sequence,and then based on the results of the replies homogeneous distribution analysis,standard analysis of saturation analysis,as well as gene structure analysis,expression analysis,differential expression of gene function annotation and other advanced analysis.The results showed that compared with the standard strain of E.faecalis V583,four strains of high biofilm-forming clinical isolates had 152 genes down-regulated and 10 genes up-regulated,among which the expression of nitrogen metabolism pathway was significantly higher than that of the control group.The gltA gene encoding glutamate synthase was significantly up-regulated,which is an important molecule in glutamate metabolic pathway.These results suggest that gltA gene and glutamate metabolic pathway may play an important regulatory role in the biofilm formation of E.faecalis.PartⅢ The Study on the relationship between gltA gene and E.faecalis biofilm formationThe biofilm formation ability of 4 strains of high biofilm formed of E.faecalis after 24 h was observed by laser scanning confocal microscopy(CLSM).The number of viable cells in the biofilm and biofilm volume were analyzed by software.Then,RT-qPCR technique detected the expression level of gltA gene in 24 h.Finally,the relative expression level of Glutamate synthase(GOGAT)in 24 h was detected by the method of Label-free Quantitative Proteomic Analysis.CLSM showed that four strains of E.faecalis isolated from clinical specimens could form dense biofilm with a significantly higher viable cell count and biofilm volume at 24 h compared with control group E.faecalis V583(P<0.05).RT-qPCR showed that the expression of gltA gene decreased over time,and the expression of gltA gene in 4 high biofilm-forming E.faecalis at 6h and 12 h was significantly higher than that in control group(P<0.05);Label-free Quantitative Proteomic Analysis showed that the level of GOGAT at 6h was significantly higher than control group(P<0.05),while there was no significant difference at 12h(P>0.05),but none was detected at 24 h.The above results demonstrated that the high expression of gltA gene can enhance the biofilm formation ability of E.faecalis,and at the same time indicated that the gltA gene,encoding GOGAT,can regulate macromolecules,such as glutamic acid,required for metabolism in the early stage of biofilm formation(0 ~ 6h).When biofilm is initially formed(6 ~ 12h),the expression of gltA gene will be reduced by the negative feedback regulation in vivo,thus inhibiting the synthesis of GOGAT.PartⅣ Effects of up and down regulation of gltA gene on the biofilm formation of E.faecalis.The homologous recombination technique was used to transfer the pHS-AVC plasmid containing ampicillin resistance gene into the standard strain E.faecalis V583 to replace the gltA gene,and to successfully construct the gltA-deficient E.faecalis strain.The gltA gene was cloned into the plasmid pMg36 e containing the strong promoter p32 and electroporated into the standard strain E.faecalis V583.The gltA-highly expressed E.faecalis strain was successfully constructed.First,the growth curve and biofilm formation ability of two mutant strain and standard strain V583 were determined.Then CLSM was used to observe the biofilm formation ability of mutant strain and control group V583 after 24 h.The number of viable bacteria and biofilm volume in biofilm were calculated and analyzed.Finally,the expression level of genes related to biofilm formation in E.faecalis was detected by RT-qPCR.The results showed that the growth rate of gltA-highly expressed strain was higher than control group after entering the exponential phase.On the contrary,the growth rate of gltA-deficient strain was lower than control group entering the exponential phase,but there is no statistical difference between them(P>0.05).There was no significant difference in biofilm formation ability between the two mutant strains at 6h,but at 12 h and 24 h the biofilm formation capacity of the gltA-highly expressed strain was significantly higher than control group(P<0.05),and the gltA-deficient strain was significantly lower than control group(P<0.05).The CLSM images revealed that the gltA-deficient strain tends to grow slowly and relatively loose structures and poor thickness,whereas the gltA-highly expressed strain tends to aggregate into distinct clusters and relatively dense structures.The viable bacteria and biofilm volume of the gltA-deficient strain had reduced dramatically at 12 h and 24h(P<0.05),but the gltA-highly expressed strain had increased significantly at 12 h and 24h(P<0.05).The expression levels of gelE,fsrB,esp,ace,salB and bopD were significantly increased in the gltA-highly expressed strain(P<0.05),but significantly lower in gltA-deficient expressed strain(P<0.05).So we concluded that glutamate,which is synthesized by GOGAT encoded by gltA gene,is the backbone molecule of many macromolecular structures in E.faecalis,such as peptidoglycan.Peptidoglycan is also an important component of bacterial cell wall.Therefore,we think gltA gene may affect the formation of biofilm by affecting the surface structure of bacteria by affecting the synthesis of peptidoglycan in the cell wall of E.faecalis.The above results show that gltA,a gene related to glutamate metabolism,can affect the biofilm formation of E.faecalis and is a key molecule in regulating biofilm formation. |
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