The Construction Of Eno Gene Knockout Strain Of Fluoride-resistant Streptococcus Mutans Strain

Enolase,known as pyruvate hydrate synthase,is a highly expressed protein in many organisms,and a key enzyme in glucose metabolism,catalyzing 2-phosphoglycerate to produce phosphoenolpyruvate.Recent accumulation of evidence revealed that,in addition to its innate glycolytic function,enolase also plays an important role in several biological and pathophysiological processes.Enolase on the cell surface can bind to plasminogen and subsequently alter its configuration,making the plasminogen more easily activated to plasmin.Plasmin is a serum protein,once activated by enolase on the bacterial surface,triggers uncontrolled proteolysis leading to tissue degradation and runaway inflammation.The degradation of fibronectin by the enolase of periodontal pathogens such as Tannerella forsythia is associated with the loss of gingival attachment in periodontitis.Furthermore,the increase of the expression of enolase and the enhancement of the enzyme activity will help fluoride-resistant bacteria survive in the fluoride-containing environment,reducing the anti-caries effect of fluoride.As a moonlighting protein,enolase in the field of oral medicine research also need further in-depth study.Streptococcus mutans is the main pathogen of caries,and fluoride is a kind of widely used anti-caries agent.The long-term and large-scale application of fluoride has led to the selective growth of fluoride-resistant strains.The fluorine-resistant strains not only evolved enhanced fluoride tolerance but also gained stronger cariogenic ability.The genomics and proteomics of Streptococcus mutans fluoride-resistant strains have changed compared with their parental strains.Among them,eno gene has changed a lot in fluoride-resistant strains of Streptococcus mutans.It not only has a single base mutation,located at 1184373,but also the encoded protein expression is upregulated.The DNA base mutation was from cytosine to thymine,at the same time the encoded amino acid has changed from threonine to isoleucine.All these show that enolase plays a key role in the fluoride-resistant strains of streptococcus mutans.In this experiment,in order to study the function of eno gene in fluoride-resistance and cariogenecity,overlapping homologous recombination technology was used to construct homologous recombination fragments,and the sequence of resistance gene kanamycin was inserted into the open reading frame of the eno gene,so that the bacteria could not express functional enolase and the eno gene knockout strain was constructed.Then the successfully constructed homologous recombination DNA fragment was connected with pEASY-Blunt blunt-end vector,to facilitate preservation and amplification of the fragment.The amplified and purified fragments were introduced into the fluoride-resistant strain of Streptococcus mutans by electroporation,and the eno knockout strains of Streptococcus mutans fluoride-resistant strain was selected by kanamycin resistance and PCR identification.This experiment has successfully constructed eno gene knockout strain of fluoride-resistant strain of Streptococcus mutans.It was a useful attempt to study the function of the eno gene.The phenotypic differences between eno-knockout fluoride-resistant strains and non-knockout fluoride-resistant strains,transcriptomic sequencing,and the changes of eno's upstream and downstream gene expression could serve as future research directions.This experiment would lay the foundation to clarify the role of eno gene in bacterial pathogenesis and fluoride tolerance,the regulatory relationship between its upstream and downstream genes and mechanism of bacterial tolerance to environmental stress.