Mechanism Underlying The Recruitment Of Atlastin 1 To Legionella-containing Vacuole

Background:Legionella pneumophila is a Gram-negative bacterial pathogen that causes Legionnaires’disease.After entering host cells,L.pneumophila multiplies in a specific vacuole named Legionella-containing vacuole（LCV）,that initiates a trafficking pathway to evade endocytic maturation.Biogenesis of this replicative phagosome requires the Dot/Icm type IV secretion system,which translocates a large number of bacterial effectors into host cells.The host endoplasmic reticulum（ER）is thought to be a major membrane source for the expansion of the LCV.After its formation following phagocytosis,LCVs recruit vesicles originating from the ER and quickly（within 5 min）converts its membranes into membranes with features associated with those of the ER.Yet,the mechanism of this process has not yet been fully elucidated.In this study,we found that the ER large GTPase Atlastin is required for optimal intracellular growth of L.pneumophila in mammalian cells.Furthermore,members of the Sid E ubiquitin ligases catalyze Atlastin ubiquitination and is important for its association with the LCV.Thus,Atlastin is targeted by Sid Es and this process is important for intracellular growth of L.pneumophila in mammalian cells.Purpose:The purpose of this project was to investigate the role of Atlastin 1（ATL1）in the intracellular growth of L.pneumophila and the ubiquitination sites induced by Sde A on ATL1.Method:1.The COS-7 cells with the Atlastin gene（KO）knocked out was infected by L.pneumophila.At 24 hours and 48 hours after infection,growth of the bacterium was examined.2.Atlastin KO cells expressing ATL1 were infected by L.pneumophila strains,with wild-type,?sid Es and the dot A^-mutant defective in the Dot/Icm transporter..The?sid Es mutant was complemented with Sde A,Sde A_E/A,Sde B,Sde C or Sid E,and modification of ATL1 was assessed by changes in its molecular weight,which was validated by biochemical experiments with recombinant proteins.3.The modification sites on ATL1 were determined by mass spectrometric analysis.Result:1.The growth of L.pneumophila in host cells need Atlastin.2.Each member of the Sid Es family can modify ATL1 by phosphoribosyl ubiquitination.3.Ser22 of ATL1 is the site modified by Sid Es-induced phosphoribosyl ubiquitination.Conclusion:1.ATL family proteins play an important role in the replication of Legionella pneumophila in the host cell.2.Sde A targets the Ser22 residue of ATL1 for phosphoribosyl ubiquitination.