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Legionella pneumophila is a Gram-negative bacterial pathogen that can cause a severe form of pneumonia called Legionnaires disease.A major virulence determinant of L.pneumophila is the type IVB secretion system (T4SSB),which is used to inject more than 300 effector proteins into the host cell,modulating various cellular processes.One effector,WipA,was the first tyrosine protein phosphatase found in L.pneumophila with no clear host substrates;moreover,the role of tyrosine phosphorylation during L.pneumophila infection and the signal pathways responsible for it are relatively unknown.Here,liquid chromatography-tandem mass spectrometry for label-free quantitative phosphoproteomics was applied to systematically investigate the global tyrosine phosphorylation events in Legionella-infected pulmonary alveolar macrophages.Quantitative analysis of the phosphoproteomics data revealed an increase in the tyrosine phosphorylation levels of 44 sites,and a decrease of 36 sites in wild type Lp02 infected macrophages compared to LpO2-△wipA mutant infected macrophages.We uncover an early function of WipA,showing that it associates with host actin assembly.The majority of phosphosites were actin assembly signaling pathway associated proteins,including tyrosine kinase signaling regulator Nck1,tyrosine kinase (Lyn and Yes),nuclear promote factor NWASP,actin related protein Arp3,ADP ribosylation factor (Arf) 6 and Capping protein.Furthermore,western blotting further confirmed the decrease in several tyrosine phosphosites phosphorylation levels during infection.Additionally,we found that WipA inhibited host actin polymerization.These findings suggested that the WipA-mediated tyrosine phosphorylation pathways may play important roles in the regulation of actin assembly in host and may help us elucidate the pathogenic mechanisms of L.pnuemophila infection.