Function Of DNA-binding Protein In Staphylococcus Aureus

Staphylococcus aureus(S.aureus)is a kind of gram-positive bacteria,which is an opportunistic pathogen and causes various kinds of infectious diseases,including skin infections,pneumonia,endocarditis and bacteremia.It has been reported that biofilms are involved in the survival and pathogenicity of S.aureus in vivo.S.aureus can form biofilm on various implants in the human body,including tympanic membranes,artificial heart valves,central venous catheters,and even contact lenses.During bacterial colonization and infection,biofilms can provide sufficient protection for bacteria through their own special structure.They not only can reduce the bactericidal action of host antimicrobial peptides but inhibit the phagocytic and cytotoxic effects of phagocytic cells.Moreover,biofilm can resist antibiotics so that antibiotics can not act on the bacteria effectively and lose the antibacterial effect.In addition,when developed to the mature stage,biofilms will dissociate and bacteria in biofilms can spread to new infection sites through the human circulatory system.Biofilm is a complex structure and microbial populations adhere to the surface of the matrix.The highly hydrated extracellular polymeric substances(EPS)envelop internal microbes.The EPS contains extracellular polysaccharides,extracellular proteins and extracellular DNA,among which the extracellular DNA serves as a structural component of the biofilm.DNABII family proteins include HU(histone-like protein)and IHF(host factor protein),which bind to DNAas dimers and condenses DNA upon binding.The DNABII proteins have a high affinity to pre-bent secondary structures of DNA.DNABII plays an important role in biofilm formation and maintaining the structure of living organisms.In vivo and in vitro studies have shown that antiserum targeting the DNABII family proteins can destroy the biofilm structure and release the entrapped bacteria in the biofilm,and further use antibacterial drugs to achieve therapeutic purposes.Phage display technology refers to infusion protein of Fab or scFv and geneIII on the surface of filamentous phage,a bacterial viruses that can infect and replicate within Escherichia coli,and DNA encoding the heavy chain(VH)and light chain(VL)are batchcloned into viral genome to creat fusion protein with phage coat proteins pIll or pVIII.After several rounds of “absorption-elution-amplification”,the specific antibody can be selected and enriched.The advantage of phage display is that it can couple phenotype and genotype to selecte antibodies with high specificity.DNABII family protein HU binds extracellular DNA in a sequence-independent manner,but there are few reports on the formation of biofilms in the S.aureus.Therefore,in our study,by expressing recombinant protein HU and preparing its monoclonal and polyclonal antibodies to explore the influence on the formation of biofilms in the S.aureus.ObjectivesBy expressing recombinant protein HU and preparing its monoclonal and polyclonal antibodies to explore the influence on the formation of biofilms in the S.aureus.Methods1)Expression of MethodsStaphylococcus aureus HU protein.We used E.coli expression system to recombinantly express HU protein and purify protein HU by Ni2+ affinity chromatograph.2)Preparation and Functional Preliminary Study of HU-specific Antibodies.We used recombinant HU protein to immunize rabbits and prepare the polyclonal antibody.At the same time,we used phage display technology to obtain a monoclonal antibody against HU protein through three rounds of screening,and further using a mammalian cell expression system to express and purify the antibody.ELISA and Western blot detected the binding ability of protein HU and the specific antibodies.3)Effect of HU-specific antibodies on the formation of S.aureus biofilms in vitro.Through in vitro biofilm formation assays,we used crystal violet staining and laser confocal microscopy assays to determine whether HU-specific antibodies could inhibit the formation of S.aureus biofilm.We further used PIA spot assay to examine the expression level of PIA after antibody treatment.4)Effect of HU-specific antibodies on biofilm formation in S.aureus.By constructing a catheter-implanted animal model,we used laser confocal microscopy to observe the formation of S.aureus biofilm in the implanted catheter.It was further examined whether the addition of HU-specific antibodies could inhibit the formation of S.aureus biofilms in implanted catheters in mice.Results1)The pET-28a-HU high-efficient expression vector was successfully constructed,and the purified recombinant protein HU with high staphylococcus aureus was obtained.2)Anti-HU polyclonal antibody(anti-HU)was prepared by immunizing rabbits with recombinant HU protein.At the same time,using the phage antibody library technology,a full human monoclonal antibody YG6 was obtained after three rounds of panning and screening,and YG6 was recombinantly expressed using a mammalian cell expression system.ELISA and Western blot results showed that both anti-HU and YG6 could specifically bind to HU.3)Anti-HU and YG6 can significantly inhibit the formation of Staphylococcus aureus biofilm in vitro,and have a certain concentration dependence.At the same time,anti-HU and YG6 can inhibit the expression of PIA.4)An animal model of biofilm formation wasconstructed.The Staphylococcus aureus 8325 strain containing the green fluorescent GFP plasmid successfully formed a biofilm in a mouse-implanted catheter,and the formed biofilm morphology was observed under a laser confocal microscope.At the same time,anti-HU and YG6 antibodies were able to inhibit the formation of S.aureus biofilm in implanted catheter in mice.conclusionsRecombinantly expressing the Staphylococcus aureus HU protein,preparing its corresponding polyclonal antibodies and screening the specific human antibodies.In vitro and vivo research results showed that anti-HU antibody could inhibit the formation of Staphylococcus aureus biofilm.The results suggested that HU protein was involved in the formation of S.aureus biofilm.Targeting HU protein may block the colonization and infection of S.aureus in vivo,providing a new theoretical basis and potential drug targets for S.aureus infection.