Disruption of the vesicle budding from the ER repressed intracellular bacterial replication, several bacterial proteins have been shown to target molecules that regulate protein trafficking between the ER and the Golgi apparatus. For example, RalF activates the small GTPase Arf1 on the surface of LCVs whereas SidM/DrrA and LepB are guanine nucleotide exchange factor and GTPase activation protein for the Rasfamily small GTPase Rab1, respectively. In addition to vesicle trafficking, several other cellular processes are modulated by substrates of the Dot/Icm system. Mammalian cells harboring replicating L. pneumophila exhibits a strong resistance to exogenous cell death stimuli, probably by synergized effects resulted from NF-kB activation and the activities of some effectors such as SdhA and SidF, which have been shown to contribute to such resistance by different mechanisms. Two effectors, LegK1 and LnaB are able to activate NF-kB when ectopically expressed in mammalian cells, but whether such activation plays any role in modulating host cell death is unknown. Like other intravacuolar pathogens, L. pneumophila is able to maintain a neutral luminal pH in LCVs. Recently, the effector SidK has been shown to contribute to such regulation by antagonizing the activity of v-ATPase, the MK-0683 proton transfer machinery that controls organellar pH. The cytoplasmic face of LCVs is decorated by ubiquitinated proteins and the host proteasome function is important for intracellular bacterial replication. Not surprisingly, a number of Dot/Icm substrates are involved in protein ubiquitination. Finally, at least four proteins are capable of inhibiting host protein synthesis by inactivating the translation elongation factor eEF1A, which may contribute to the induction of host stress response and other unknown cellular processes. Given the large number of substrates, the diverse host functions modulated by these proteins and the possibility that translocation signals may be one of the parameters that control temporal translocation of these proteins, it is likely that previous screens have missed some substrates with undefined features. In this study we have greatly expanded the repertoire of Dot/Icm substrates by performing a comprehensive screen to test for translocation all the open reading frames larger than 300 base pairs annotated as hypothetical proteins in the genome L. pneumophila strain Philadelphia 1. Our efforts led to the identification of 70 novel Dot/Icm substrates. Furthermore, a protein translocated at a high efficiency does not share most of the features found in one or more groups of established substrates, indicating that the Dot/Icm system has a great flexibility in recognizing substrates. A total of 164 proteins that consistently promote TEM translational fusions in a Dot/Icm-dependent manner were obtained. Among these, 94 proteins had been reported as Dot/Icm substrates, further validating the reliability of our screen strategy.