| Staphylococcus aureus is a common Gram-positive pathogen.The bacterium is widely present in the environment,and is able to parasitize various types of hosts.Being parasitic in the human body,S.aureus will cause a series of diseases,ranging from skin and soft tissue infections to life-threatening diseases such as pneumonia,osteomyelitis,sepsis and infectious myocarditis.At present,antibiotics are often used in the treatment of S.aureus infection,but with the long-term use of antibiotics,multi-drug resistant strains such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Staphylococcus aureus have been clinically evolved,greatly improving the clinical treatment difficulty and cure cycle of S.aureus infection,and bring heavy pressure on public health.It is imperative to find new therapeutic targets and treatments for S.aureus infection.The pathogenicity of S.aureus is mainly related to the expression and release of a large number of virulence factors.Various virulence factors help Staphylococcus aureus to approach and adhere to the host surface,thereby infecting host cells.The expression of these virulence factors is regulated by a variety of effectors,and the two-component regulatory system is one of the most important regulatory factors.The two-component control system is considered to be an important signal transduction method for bacteria,lower eukaryotes,and plants that sense environmental changes and respond accordingly.The system generally consists of a signal-sensitive histidine kinase and a downstream effector.The working mode of this system is that after histidine kinase receives an environmental change stimuli signal,autophosphorylation occurs at a conserved histidine site,followed by delivery of a phosphorylation group to a downstream effector,thereby altering effect regulation.The binding affinity for a particular DNA substrate ultimately alters the transcription level of a specific gene.In S.aureus,two-component regulatory systems are considered to be very promising new therapeutic targets because they are directly involved in the control of various key life activities of S.aureus.ArlR/S is a member of the two-component regulation system of Staphylococcus aureus,in which ArlS is a histidine kinase with signal-sensing ability,and ArlR is its corresponding effector regulatory factor.At present,in vivo experimental studies have proved that the two-component regulation system can play a role in various physiological processes,including affecting the production of regulatory molecules RNAII,RNAIII and autolysin,and inhibiting the biofilm formation of S.aureus.Among these physiological functions,the recognition and regulation mechanism of ArlR protein on the global regulatory factor agr have been widely concerned,but the molecular mechanism is still unclear.In order to solve above-mentioned scientific problems,in the first part of this thesis,we have studied the recognition and regulation mechanism between ArlR protein and global regulatory factor agr.By recombinant expression in vitro,we obtained the full-length ArlR protein,the ArlRDBD protein(only containing the DNA binding domain),and the full-length ArlRD52E protein(mimicked phosphorylated state).The interaction between ArlR protein and its mutants and agr gene was studied by EMSA method.It was found that ArlRDBD and ArlRD52E are able to bind agr,while ArlR protein bind agr only when the concentration is quite high.Through the DNasel enzymatic footprinting method,we found two agr segments that can be specifically recognized by ArlR,which are 21 bp and 31 bp in length respectively.By FPA method,we determined three protein samples and these two specific recognition regions.Using molecular exclusion chromatography,dynamic light scattering and chemical cross-linking,we found that in solution,the aggregation state of ArlR protein is monomer,the phosphorylated ArlRD52E protein is dimer,and the ArlRDBD protein is mainly monomer..To elucidate the relationship between the above-mentioned changes in aggregation state in DNA binding capacity,we obtained a crystal structure of ArlRDBD protein with a resolution at 2.5 A.Through alignment analysis of the structure and a series of mutant experiments,we confirmed the DNA binding site of the ArlRDBD protein and its preference for the length of the DNA substrate.Combining the above results of structural biology and biochemistry experiments,we propose a possible mechanism by which ArlR protein regulates the global regulator agr.This work has an important role in understanding the mechanism by which phosphorylation modification in the two-component system regulates the DNA binding affinity of the protein.Pyrimidine dimers are a common form of DNA damage produced by DNA under ultraviolet radiation.Ultraviolet light can induce a nucleic acid strand to form a covalent linkage near the C=C double bond of an adjacent base.Under sunlight,a single human skin cell will undergo 50-100 times of the above-mentioned damage reaction per second,and some pyrimidine dimers that are not repaired in time will inhibit the function of DNA polymerase,thereby affecting the transcription or replication of the gene,causing mutations,chromosomal abnormalities and apoptosis,and even triggering the formation of melanoma.In response to the damage caused by ultraviolet radiation,organisms have evolved a variety of repair mechanisms.Light repair system is one of the important DNA damage repair pathways.In lower organisms,the photorepairase/cryptochrome family protein is responsible for the above-mentioned repair function.The family of proteins are photoreceptor proteins that promote the transfer of electrons between the FADH-and the site of damage by absorbing light energy and reverting the pyrimidine dimer to a normal base.The photoremediation enzyme typically combines two cofactors,the catalytic group FAD and the chromophoric group responsible for absorbing light.In the second part of this paper,through gene data analysis,we found a gene in Arthrospira platensis(gene number NIES39_D05560)that may encode a photo-repairing enzyme,which is confirmed by recombinant expression and enzyme activity detection.The gene-encoded Ap-phr protein repairs pyrimidine dimers in single-stranded and double-stranded DNA and is assigned to the CRY-DASH photolyase subfamily.By analyzing the crystal structure of the Ap-phr protein at 1.6 A resolution,we found that,in addition to the catalytic cofactor FAD,the protein also has a light-absorbing chromophore MTHF,which is significantly different in binding position and conformation.The above-mentioned differences may enhance the energy transfer rate between MTHF and FAD in Ap-phr protein,which may be one of the important reasons why CRY-DASH subfamily protein is capable of collecting accumulated light energy under low light conditions. |
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