The intracellular bacterial pathogen Legionella pneumophila (the causative agent of Legionnaires' Disease) remodels its phagosome to completely avoid fusion with lysosomal compartments and to intercept early secretory vesicles deriving from ER exit sites. Although L. pneumophila is a major cause of community and nosocomially-acquired pneumonia, the bacterium is believed to be an environmental organism that parasitizes multiple species of amoebozoans. A Type IVB secretion system known as the Icm-Dot complex is required for the translocation of effector proteins from L. pneumophila into host cells. These effector proteins are believed to modify many host cell trafficking pathways that are essential for L. pneumophila 's intracellular lifestyle. We believe that many genes that encode effector proteins were acquired via Inter-Kingdom Horizontal Gene Transfer, perhaps directly from host cells. We screened the genome of Legionella pneumophila for genes that have eukaryotic motifs and other hallmarks of horizontally acquired genes. We report the identification of 45 Legionella Eukaryotic-like Genes (leg), of which 33 encode substrates of the Icm-Dot complex. Further analysis reveals that many other species of pathogenic and endosymbiotic bacteria also contain large numbers of eukaryotic-like genes, suggesting that Inter-Kingdom Horizontal Gene Transfer is a major mechanism for the acquisition of effector molecules by bacteria. Since all of the leg genes tested are dispensable for intracellular growth in amoeba and macrophages, we believe significant functional redundancies arising from genome plasticity may exist. Ectopic expression of the leg genes in the model organism Saccharomyces cerevisiae reveals a set of genes that impair growth (legC5, legC8 and lepB) and another set of genes (legC3 and legC7) that interfere with endocytic maturation and trafficking of cargoes to the vacuole. Overexpression of legC3 in the social amoeba Dictyostelium discoideum induces the accumulation of endosomes-like structures containing undigested material. The use of bioinformatics screens combined with powerful heterologous expression systems offers attractive tools for the identification of determinants of infection as well as a functional analysis in eukaryotic systems.