Cloning And Knockout Of Ace Genes In Enterococcus For The Study Of Bacterial Adhesion And Biofilm Formation

Abstract:Enterococci are a kind of Gram-positive cocci which are spherical or ovoid, catalase negative, no spore, aerobic or facultative anaerobic, often occur with a single, pairs or arranged in chains. As part of the normal microflora, It mainly exist in the gastrointestinal tract of humans and animals, and is widely distribute in the soil, water and so on, among them E.faecium and E. faecalis are the dominant bacteria. In the early, Enterococci are mainly used as leavens, fungicides and prebiotics in fodder or food. But with its antibiotic resistence is recognized, their pathogenicity has been taken more and more attention.Enterococcus faecalis mainly through adhesion, invasion and tissue damages to infect and make people and animals ill, among which adhesion is the first step and also the most important step. Though adhesion to cells and the extracellular matrix, Enterococcus faecalis can intrude into the body, and eventually lead to tissue injury. Among the numerous virulence factors in enterococci, adhersin of collagen from enterococcus faecalis is kind of adhesion which exist on the surface of cell, and plays an important role in the formation of bacterial biofilm. Through the research on ace virulence factors, hoping that can provide a theoretical basis on the the future rapid diagnosis of enterococcus faecalis and development of effective vaccines.According to ace gene sequence of enterococcus which published in the GenBank, the specific primers of ace were designed. Taking the enterococcus faecalis DNA as template, through PCR amplification a 1358bp target gene segment was obtained. After purification and connected with cloning vector pTG19-T, it was transformed into Escherichia coli DH5a. Then the plasmids were successfully identified by PCR amplification, restriction enzyme digestion and analyzing of their sequences. Three standard strains which preserved in our laboratory and 43 isolates were identified by PCR amplification for deteceing the carrier rate of ace gene.Consequently, by using the method of homologous recombination, the ace gene deletion mutant was constructed. Firstly, Restriction enzyme digestion of ace gene and plasmid pK18mobSacB, after PCR confirmation and purification it was connected with cloning vector pTG19-T, then transformed it into Escherichia coli DH5a. After this, the vector pK18-ace-kan with Kanamycin resistance gene marker was constructed. Then through PCR and digest confirmation, the recombinant plasmid was subjected to sequencing. If the results of DNA sequencing was right, after deionized it will be transformed into the E. faecalis wild bacteriaATCC29212. According to the homologous recombination, the mutant ATCC29212A Ace::kan was screened. After PCR confirmation, the recombinant plasmid was subjected to sequencing to confirm whether the mutant was success or not. The genomic DNA of the mutant and wild-type strain was extracted, and digested by EcoRI. With the Kanamycin resistance marker gene-labeled probe, whether the ace gene was knocked out was confirmed by Southern blot.Then, the primary biological characteristics of the the mutant and wild-type strain, such as the bacterial adhesion and biofilm formation, were analyzed.PCR results showed 33 Ace-positive strains were screened, therefore the carrier rate of ace gene reached at 71.7%. At the same time, homology of the isolates were compared, and the rate reached at 98.0%--100%, which was consistent with the value of 97.5% or greater than that.The results indicted that adhersin of collagen from enterococcus faecalis widely existed in Enterococci and also highly conserved.After the molecular cloning and transformation, PCR amplification, plasmid restriction map analysis, a ace gene mutant was screened, designated ATCC29212A Ace::kan. This mutant was further confirmed by Southern blot, ace gene was successfully replaced by the kan gene.The ability of bacterial adhesion and biofilm formation of the mutant strain was remarkably reduced in comparison with the wide-type strain. These data inferred that comparing with the wild-type strain, the pathogenicity of the mutant was probably slightly attenuated.In summary, the ace gene was prevalent among these Enterococcus faecalis isolated from pig farms in Henan Province. Most of these Enterococcus faecalis were pathogenic, inferring that the ace gene was important in the pathogenicity of these strains. By the homologous recombination method, the ace gene deletion mutant ATCC29212A Ace::kan was successfully constructed. The bacterial adhesion and biofilm formation tests of the mutant revealed and the wild-type strain show that the Biofilm forming ability of the mutant was significantly reduced and the Adhesion ability of mutant was mildly impaired after the deletion of ace gene, infering that compared with the wild-type strain, the pathogenicity of the mutant was probably attenuated. This study provided a new strategy to search for new antibacterial drug for prevention and control of Enterococcus faecalis.