Structural And Functional Investigation Of Legionella Effectors LegA3 And LegA15

Legionella pneumophila,an intracellular pathogen,is a principle infectious Gramnegative bacterium to cause an atypical lobar pneumonia,namely Legionaires’ disease.During infection,L.pneumophila hijacks host vesicular tranfficking as well as recruit host mitochondria,ribosomes and ER-derived vesicles to form Legionella-contained vesicule(LCV),where is satisfied with its replication and proliferation in host.L.pneumophila secretes approximately three hundred effectors into host cells through its T4SS,and both of pathogenicity and intracellular replication of L.pneumophila depend on its secreted effectors.Despite intensive studies in this field,structure and function of most of effectors remain unknown.In current thesis,we mainly focused on structural and functional investigations of Legionella effectors LegA3 and LegA15.LegA3 is one of seven core conserved effectors in Legionella species,and depletion of gene lega3 inhibits intracellular replication and proliferation of L.pneumophila in host.We first determined a crystal structure of LegA3 at～2.5 A resolution.Based on structural analysis,we found that LegA3 possesses a triad catalytic center "C324-H243-D258" as a ubiquitin ligase,which was supported by our in vitro biochemical ubiquitination assays.Mutantions of C324,H243 or D258 inhibited its activity.L.pneumophila carrying these mutations retarded its intracellular replication and proliferation in host cells.We further identified a host protein mixed lineage kinase domain-like protein MLKL by LC-MS/MS,which is degraded when coexpressed with LegA3,suggesting LegA3 likely contributes to the intracellular replication of L.pneumophila by blocking host necroptosis.LegA15,an ankyrin repeat containing effector,was found toxic to host cell.LegA15 was largely localized in endoplasmic reticulum(ER)and lipid droplets,causing dysfunction of edoplasmic reticulum and possibly mitochondria.We determined a crystal structure of LegA15 at～2.4 ? resolution.Through structural analyses,we found LegA15 binds to phosphatidic acid and a typical phosphate-binding loop(P-loop)in LegA15.Several pieces of biochemical evidences suggested that NAD+is potentially a LegA15 cofactor.Expression of LegA15 was highly correlated with intracellular ADP-ribosylation level.We further found that glycines G292 and G294 in the P-loop and lysine K367 and arginine R291 in positive charged pocket are essential to cytotoxicity of LegA15.In addtion,we found the C-terninal domain of LegA15 is reqiured for localization of the endoplasmic reticulum and closely releatedt to its cytotoxicity.Our results are not only a complementary of pathogenitc mechnism of L.pneumophila,but also provide a reference to the interpaly mechnism of pathogen and host.