Structural And Functional Study Of TCS WalKR In Staphylococcus Aureus Cell Division

Staphylococcus aureus(S.aureus),a conditional pathogen,can cause many diseases in the human body.S.aureus has developed resistance to many antibiotics that currently exist.Therefore,the need for the discovery of new antibiotic drug targets is quite urgent.Two-component systems(TCSs),which are ubiquitous in bacteria,are important tools for bacteria to deal with various changing environments.TCSs only exist in bacteria,archaea,and a few lower organisms,but are lacking in higher mammalian cells.Therefore,TCSs as a hot antibiotic research target may provide us new ideas.WalKR two-component system mainly exists in Gram-positive bacteria with low G+C content,such as S.aureus and B.subtilis.Among these pathogens,WalKR is highly conserved,and multiple studies have shown that WalKR is a master regulatory TCS due to the reason that it controls essential life activities such as cell wall metabolism and cell survival.In this thesis,we used a variety of microscopy techniques(fluorescence microscope,3D-SIM,EM,AFM),protein analysis technology,two-hybrid system method and structural biology methods to study two roles of WalKR in cell wall homeostasis and cell division,respectively.In the study of cell wall homeostasis,we found that WalKR-depleted cells caused cell death due to the reduction of hydrolases,and the treatment of two cell wall targeted antibiotics,vancomycin and methicillin,could significantly reduce cell death.The expression rate of hydrolases controlled by the WalKR system in the homeostasis of the cell wall needs to be strictly regulated.The imbalance of peptidoglycan hydrolysis and synthesis will lead to abnormal cell division.By reducing the rate of cell wall synthesis in WalKR depleted cells,the cell wall division process can reach a new balance to maintain bacterial life.The revelation of these phenomena provides new evidence to support the importance of cell wall homeostasis.In the study of the role of WalKR in the mechanism of cell division,we found that the WalK protein can interact with the core division protein DivlC,and the interaction regions are located in the cPAS domain of WalK and the intracellular region of DivlC,respectively.We then used X-ray crystal diffraction method and NMR method to analyze the structure of the complex protein.In the X-ray crystal diffraction experiment,we used three complex crystallization methods and we successfully obtained crystals with good diffraction resolution.However,the processed diffraction electron density map only has cPAS structure,the density of DivlC 1-33 was not found.In the NMR experiment,the chemical shift titration experiment of cPAS and DivlC 1-33 provided strong support for the interaction between the two proteins.However,in the triple resonance experiment,the doublelabeled cPAS protein was more prone to precipitation,and we did not succeed in getting the structure of the complex.In summary,our research on WalKR in S.aureus will lay the foundation for the study of cell division mechanism and new antibiotic targets.