| Enterococcus faecalis,a facultative bacterium,could invade and colonize in the host intestine and oral cavity.E.faecalis can quickly adapt to the environments between different oxygen contents including aerobic(18% oxygen content),micro aerobic(5%),hypoxic(6～12%)and anaerobic environments.It is the main pathogen that causes refractory periapical periodontitis.Taking the pathogen E.faecalis YN771 as the material,this thesis systematically analyzed the physiological,biochemical and environmental response of YN771,and discussed the adaptive regulation mechanism of YN771 under different oxygen content(anaerobic,micro aerobic,hypoxic and aerobic).The expression difference of virulence factors and the key factors involved in metabolic regulation were analyzed in this study.Firstly,the effects of different oxygen content on the physical and chemical characteristics of YN771 were studied.The results showed that the growth rate and cell morphology of YN771 were determined by oxygen content.The biomass was the largest under aerobic condition and the smallest under micro aerobic conditions.SEM showed that cell morphology changed significantly under Micro oxygen,and extracellular mucus could not be observed.The fatty acids in cell membrane were also significantly affected by oxygen content,and the ratio of C18:1 and c16:1 increased with the increase of oxygen content.The content of unsaturated fatty acids was the lowest under hypoxia,while the amount of saturated fatty acids was the highest under anaerobic conditions.These indicated that YN771 responded to the change of oxygen concentration through adjusting the composition and content of fatty acids in the membrane.Next,the transcriptome and metabolomics methods were used to study the transcriptional and metabolic characteristics of YN771 under different oxygen content.The results showed that at the transcriptome level,there were the least differences under anaerobic,hypoxic and micro aerobic conditions;However,there were 897 differentially expressed genes(DEG)between aerobic and anaerobic conditions.It was also found that the genes gsh AB and cys E involved in glutathione synthesis and thiometabolism were significantly up-regulated;The genes fab F and mur C involved in the synthesis of fatty acids and peptidoglycans were significantly down regulated in aerobic conditions,indicating that YN771 could resist ROS through the regulation of glutathione and sulfur metabolism.In addition,there were significant differences in the transcription of virulence factor Ebp、Gel E(gelatinase)and Spr E(serine protease)under different oxygen content.It was found that the metabolic pathways of YN771 were significantly different between aerobic and anaerobic conditions.The study showed Under hypoxia,gene nifj encoding pyruvate ferredoxin was up-regulated while one of the genes with lpd gene function(GKQ55＿05175)was significantly downregulated.Under aerobic conditions,glutamate racemase gene rac E was significantly down regulated,while fumarate reductase gene frd A and gene lpd were significantly up regulated,indicating that YN771 may start aerobic respiration and its metabolic capacity may be strengthened under 18% oxygen.Through LC-MC methods,we found there were 175 significantly different metabolites between anaerobic and aerobic conditions.The genes of different metabolites and the transcriptome DEGs could be clustered into 12 main related metabolic pathways.L-glutamate was a common differential metabolite in eight related metabolic pathways,such as glutathione metabolism.And L-glutamate can be produced by L-glutamate dehydrogenase α-Ketoglutarate,thus entering the TCA cycle.At the same time,L-glutamic acid can also produce D-glutamic acid through racemization,and D-glutamic acid is an important part for the synthesis of cell wall peptidoglycan.It preliminarily showed that the metabolic regulation of L-glutamae under different oxygen conditions was one of the important key link for YN771 response to oxygen,and the content of metabolite L-glutamate may play a key role in the metabolic regulation of YN771 in the response to oxygen.L-serine was a significantly different metabolite in four related corresponding pathways,such as thiometabolism,suggesting that L-serine also plays an important role in the response of YN771 to oxygen.Some important virulence factors and metabolic regulatory genes involved in oxygen response of YN771 were verified by RT-q PCR.The results showed that the expression of gsh AB,a bifunctional gene of glutathione metabolism,was significantly up-regulated in aerobic conditions and significantly down regulated in micro oxygen and hypoxia;The expression of virulence factor ebp A and gelatinase gel E were the highest in anaerobic and aerobic conditions,respectively,and were significantly down regulated in hypoxia;Under aerobic conditions,the key gene fab H of fatty acid synthesis,the key gene lpd involved in pyruvate metabolism and the fumarate reductase gene frd A involved in TCA cycle were significantly up-regulated,while the expression of glutamate racemase rac E was the lowest.The transcriptional regulation modes of key genes involved in YN771 oxygen response were consistent with the results of transcriptome analysis,which confirmed that oxygen content was an important factor affecting the expression of YN771 virulence factors,and further verified the oxygen response mechanism of YN771.Finally,according to the difference of metabolites of YN771 between anaerobic and aerobic conditions,we prepared biobased carbon dots(CDs)under anaerobic and aerobic conditions,and discussed the similarities and differences in element composition and fluorescence characteristics between them.The research shows that anaerobic AYN771-CDs has stronger fluorescence.The viability of Hela,BEAS-2B and other cells incubated with AYN771-CDs derived from anaerobic condition were more than 80%,indicating the CDs with high biocompatibility.In the biological imaging experiment,it was found that anaerobic CDs could fluorescently stain plant and animal cells,and could also reach mouse heart and other organs with blood circulation in mouse,and could be excreted in a short time.It was preliminarily shown that anaerobic CDs has special application potential in bioimaging in vitro and in vivo compared with aerobic CDs.In this study,the adaptive mechanism of YN771 under different oxygen conditions was studied.It was found that oxygen not only affected the physical and chemical characteristics of YN771,but also one of the factors affecting the expression of virulence factors.The host environment of E.faecalis is complex and harsh with the oxygen concentration alterring constantly.The analysis of the metabolic regulation,as well as the key enzymes and virulence factors involved in the metabolic regulation of YN771 under different oxygen concentrations,may help us better understand the pathogenic mechanism of E.faecalis invading and infecting the host.A number of the regulated genes might potentially be useful candidates for development of new therapeutic approaches for treatment of E.faecalis infections. |
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