Study On The Biological Characteristics Of Enterococcus Faecalis

Enterococcus faecalis(E.faecalis) is common bacteria in human oral,is the most frequent Enterococcus species detected in human disease,especially in the re-infected root canals,it is the major bacterial species detected.The infection caused by E.faecalis result from the ability to form biofilms.Meanwhile,the pathogenicity of E.faecalis is associated with the types and functions of its surface proteins.Analysis of the mechanism which made E.faecalis escaping from several antibacterial methods during the root canal treatment(RCT),will possess clinical significance for the therapy and prevention of the failed RCT canals from the risk of repeat infection.The study is consisted of three parts:The first part is to induce a “viable but nonculturable”(VBNC)state in E.faecalis cells using three different protocols in vitro,and we investigated the physiological and biochemical characteristics of the VBNC cells,and the characteristics associated with its pathogenicity,including their morphology,metabolic profile,acid production and acid-proof ability,adherence properties and gene expression.As a result,E.faecalis may enter into the VBNC state when faced with several adverse conditions.The VBNC E.faecalis will resist to environmental change damage through changing morphology and reducing metabolic degree.Meanwhile,the VBNC E.faecalis cells maintain their acid production and adherence ability,and express part of genes associated with their pathogenicity.The VBNC E.faecalis may be able to resume cell division and promote disease transmission and persistence when permissive conditions are restored.The second part,using the method of “label-free” mass spectrometry,we identified proteins expressed significantly different between the planktonic E.faecalis and the biofilm cells.To analyze the difference of expression quantities and functions of the surface proteins between the two phases of E.faecalis,then explore the mechanism of infections caused by the biofilm phase E.faecalis.The results showed that,compared with the planktonic E.faecalis,there were 108 proteins detected significantly different expressed(difference fold ?2).Compared with the planktonic E.faecalis,73 proteins expressed up-regulated in the biofilm group of E.faecalis,and 35 proteins expressed down-regulated.71 identified proteins were predicted to be cytoplasmic,most of them were ribosomal proteins(Rbps)or elongation factor(EF).The other 37 identified proteins were predicted to be surface-located proteins.Of the 37 surface-associated proteins expressed differently by E.faecalis,5 proteins were anchored with the cell wall with LPx TG;17 proteins with lipo-anchor were predicted to be extracellular proteins,which act as bacterial extracellular solute-binding proteins(SBPs);the other 15 proteins were membrane proteins,in which there were 6 proteins with N-terminal anchor and 9 proteins with multi-transmembrane helices(TMHs).The third part,using the method of RNA-sequencing,we sequenced the biofilm E.faecalis in the total genome scale and discribed its transcript pathway map.The results showed that: Mapped Reads are 26911175 in biofilm E.faecalis,total trinity transcripts are 2552;1657 transcripts can be compared in the 4 reference gemomes;1913 transcripts are annotated to reference genes,the other 639 transcripts are sequence similar compared and received 387 transcripts with proteins in NR database;1763 transcripts are classified into different COGs,most focus on the information store and process,metabolism,cell procedure and signal peptide;KEGG analysis got 170 pathways,74 pathways are analyzed after exclude Eukaryotes,significantly enriched in energe and substance metabolism,replication and repair,transcript,signal transduction,glycan biosynthesis and metabolism.Our study results,to some extent,confirmed the possibility of the mechanism which E.faecalis resist adverse environment through entering into the VBNC state.The surface proteins of E.faecalis are associated with the cell wall synthesis and maintenance,bacterial adherence profile,virulence factors and signal transduction function.These surface-located proteins are also involved in the bacteria behavior,such as the transport of the solutions and the regulation process of the pheromone.The study provided a further supplement for the bacterial etiology doctrine of root canal infection from the theory point,and provided theoretical basis for controlling the source of the disease during the clinical RCT,and prevention from the recurrence.This study presents analysis of differentially expressed proteins and transcripts characteristics of the biofilm E.faecalis.Make the deep understanding of the E.faecalis surface proteins and their related functions possible.