In the aquatic environment, Legionella pneumophila (Lp) proliferates within amoeba but upon transmission to humans it causes pneumonia with high morbidity and mortality. Within amoeba and human macrophages, Lp proliferates within the Legionella-containing vacuole (LCV), which evades lysosomal fusion and is remodeled by the endoplasmic reticulum (ER). Biogenesis of the LCV is controlled by the Dot/Icm type IV secretion system, which injects into the host cell ~300 protein effectors that modulate a myriad of cellular processes to enable intracellular proliferation of Lp. Very few of this large battery o Lp effectors, such as AnkB and AnkH, are required for the intracellular infection. To attain biological function in the host cell, many injected bacterial effectors require post- translational modification by various host machineries. Post-translational modification of eukaryotic proteins by Asparagine (Asn) hydroxylation impacts their protein-protein interaction, and has been widely studied during hypoxic conditions in inflammatory foci. It is not known whether any injected bacterial effector is post-translationally modified by the host asparagine hydroxylase, FIH. The Asn hydroxylation motif has been identified within many human Ankyrin repeats-containing proteins (Ank) that have been shown to be modified by FIH. Our bioinformatics preliminary data have identified an Asn hydroxylation motif within the ankyrin repeats of the AnkB and AnkH effectors of Lp, and we show that both effectors are modified by the host FIH Asparagine hydroxylase. We show that the host FIH is rapidly recruited to the LCV membrane, where AnkH and AnkB are exclusively localized. Our in silico genomic analyses have identified the Asn hydroxylation motif in eight other translocated effectors of Lp. Importantly, RNAi-mediated knockdown or chemical inhibition of FIH abolishes intracellular proliferation of Lp and promotes fusion of the LCV to the lysosomes. Therefore, our hypothesis is: host-mediated Asn hydroxylation of Lp effectors by FIH is required for their functions in biogenesis of the LCV and intracellular proliferation of Lp. To test our hypothesis, our specific aims are to characterize th following: I) Asn hydroxylation of AnkH and its role in the intracellular infection; II) Asn hydroxylation of AnkB and its role in the intracellular infection; and III) Asn hydroxylation of oter Lp effectors and its role in biogenesis of the LCV. Upon completion of our proposed studies, the biological function and host targets of AnkH will be known, and the ubiquitination substrates of AnkB will be identified. The role of host FIH-mediated Asn hydroxylation in the biological function of AnkH and AnkB will be uncovered. This is significant, since both effectors are two of very few of the battery of ~300 Lp effectors to be required for intra-vacuolar proliferation. We wil report on our novel discovery of the acquisition of the FIH complex by a pathogen-containing vacuole and its role in modification of several Lp effectors. We will uncover the role of FIH-mediated Asn hydroxylation of several Lp effectors in lysosomal evasion by the LCV. Our proposed studies on Lp-primary hMDMs interaction and specific manipulation of the host post- translational modification by FIH are innovative with broad high impact and significance not only for Lp but for other pathogens. This is evident from our discovery of Asn hydroxylation of the YopM effector of Yersinia, and potential Asn hydroxylation of the IpaH4.5 effector of Shigella, and the EsxF effector of Mycobacterium.