Structural Study Of Legionella Pneumophila Dot/Icm ? Secretion System Core Complex Component DotH And Functional Studies Of Bacterial Type ? Effectors In Yeast Model System

Dot/Icm IV secretion system(Dot/Icm T4BSS)plays a critical role in Legionella pneumophila infection by delivering numerous effector proteins into host cells,thus assisting bacterium in surviving,proliferating,and escaping from host cells.Dot/Icm T4BSS core complex consists of five proteins,DotC,DotD,DotH,DotG and DotF.It is supposed that lipoproteins DotC and DotD recruit periplasmic protein DotH to the outer membrane,thus assembling into an outer membrane complex.To gain structural insights into the outer membrane complex of Dot/Icm T4BSS,we expressed truncated DotH(residues 101-360)and DotH(residues 123-360)in E.coli.Through multiple steps of purification,including affinity purification,ion exchange chromatography and gel filtration,we obtained DotH protein with high purity and high uniformity.After crystallization screen and optimization,we successfully obtained crystals of DotH(101-360)that can be applied to X-ray diffraction.The results reported here provide successful methods for DotH protein purification,crystallization and optimization,and make contributions to further structural studies for DotH protein and the outer membrane complex of Dot/Icm T4BSS,Type ? secretion systems(T3SS)are found in many important bacterial pathogens,including Shigella flexneri,Salmonella Typhimurium,Bordetella pertussis,Vibrio cholera,and Enterohemorrhagic Escherichia coli,etc.Using T3SS,Bacterium directly inject numerous effector protein into host cells to manipulate or arrest cellular process.Therefore,identification and characterization of these type ? effectors is essential to understanding pathogenesis.Here,we established a yeast model system and expressed bacterial type ? effectors in yeast Saccharomyces cerevisiae.We discovered that ectopic expression of Chromobacterium violaceum effector CopA significantly inhibited yeast growth.Combined secondary structure prediction and conserved sequence analysis,we identified critical amino acids required for virulence in CopA.Meanwhile,we also observed that Vibrio cholera effector VopK and Enterohemorrhagic Escherichia coli effector EspV exhibit strong toxicity in yeast cells.However,ectopic expression of EspO and NleA from Enterohemorrhagic Escherichia coli,BteA from Bordetella bronchiseptica,and putative effector protein CP0014 from Shigella flexneri did not induce yeast growth arrest.In essence,we have investigated the functions of several bacterial type ? effectors in yeast eukaryotic model system,and our data provide valuable clues for further studies on the molecular mechanisms of these effectors.