Legionella pneumophila, the causative agent of Legionnaires' disease, evades phago-lysosome fusion in mammalian and protozoan hosts to create a suitable niche for intracellular replication. To modulate vesicle trafficking pathways, L. pneumophila translocates effector proteins into eukaryotic cells through a Type IVB macro-molecular transport system called the Icm-Dot system. In this study, we employed a fluorescence-based translocation assay to show that 33 previously identified Legionella eukaryotic-like genes (leg) encode substrates of the Icm-Dot secretion system. To assess which of these proteins may contribute to the disruption of vesicle trafficking, we expressed each gene in yeast and looked for phenotypes related to vacuolar protein sorting. We found that LegC3-GFP and LegC7/YlfA-GFP caused the mis-secretion of CPY-Invertase, a fusion protein normally restricted to the yeast vacuole. We also found that LegC7/YlfA-GFP and its paralog LegC2/YlfB-GFP formed large structures around the yeast vacuole while LegC3-GFP localized to the plasma membrane and a fragmented vacuole. In mammalian cells, LegC2/YlfB-GFP and LegC7/YlfA-GFP were found within large structures that co-localized with anti-KDEL antibodies but excluded the lysosomal marker LAMP-1, similar to what is observed in Legionella-containing vacuoles. LegC3-GFP, in contrast, was observed as smaller structures which had no obvious co-localization with KDEL or LAMP-1. Finally, LegC3-GFP caused the accumulation of many endosome-like structures containing undigested material when expressed in the protozoan host Dictyostelium discoideum. Our results demonstrate that multiple Leg proteins are Icm/Dot-dependent substrates and that LegC3, LegC7/YlfA, and LegC2/YlfB may contribute to the intracellular trafficking of L. pneumophila by interfering with highly conserved pathways that modulate vesicle maturation.

Intracellular pathogens exploit host cell functions to create a replication niche inside eukaryotic cells. The causative agent of Legionnaires' disease, the ␥-proteobacterium Legionella pneumophila, resides and replicates within a modified vacuole of protozoan and mammalian cells. L. pneumophila translocates effector proteins into host cells through the Icm-Dot complex, a specialized type IVB secretion system that is required for intracellular growth. To find out if some effector proteins may have been acquired through interdomain horizontal gene transfer (HGT), we performed a bioinformatic screen that searched for eukaryotic motifs in all open reading frames of the L. pneumophila Philadelphia-1 genome. We found 44 uncharacterized genes with many distinct eukaryotic motifs. Most of these genes contain G؉C biases compared to other L. pneumophila genes, supporting the theory that they were acquired through HGT. Furthermore, we found that several of them are expressed and up-regulated in stationary phase in an RpoS-dependent manner. In addition, at least seven of these gene products are translocated into host cells via the Icm-Dot complex, confirming their role in the intracellular environment. Reminiscent of the case with most Icm-Dot substrates, most of the strains containing mutations in these genes grew comparably to the parent strain intracellularly. Our findings suggest that in L. pneumophila, interdomain HGT may have been a major mechanism for the acquisition of determinants of infection.The ␥-proteobacterium Legionella pneumophila is an opportunistic human pathogen that multiplies within alveolar macrophages and causes the nosocomial and community-acquired pneumonia known as Legionnaires' disease (18,25,48). Human disease occurs when aerosolized L. pneumophila is inhaled from man-made or natural freshwater reservoirs harboring the bacteria. L. pneumophila poses a significant worldwide public health problem, particularly for individuals with compromised immune systems (19,38,40). Eradication of the pathogen from freshwater, industrial settings has proven difficult, since L. pneumophila thrives in environments that exclude antibacterial agents, such as biofilms and the intracellular compartments of protozoa (8,46,47).In order to create a replicative niche inside eukaryotic cells suitable for replication, L. pneumophila is believed to modulate host cell functions by the delivery of effector proteins through a type IVB secretion system known as the Icm-Dot complex (52, 59). Effector proteins presumably regulate several pathways in the host, including up-regulation of phagocytosis (23), delay in phagosome-lysosome fusion (24), recruitment of ARF1 to the phagosome (43), acquisition of endoplasmic reticulum-derived vesicles (29), and nonlytic egress from the host cell (11).Several laboratories found Icm-Dot substrates through genetic screens and bioinformatic approaches (3,7,11,13,36,43,44,57). Most of the known effector proteins are not individually required for intracellular multiplication, since knocking out th...

Legionella pneumophila, the bacterial agent of legionnaires' disease, replicates intracellularly within a specialized vacuole of mammalian and protozoan host cells. Little is known about the specialized vacuole except that the Icm/Dot type IV secretion system is essential for its formation and maintenance. The Legionella genome database contains two open reading frames encoding polypeptides (LepA and LepB) with predicted coiled-coil regions and weak homology to SNAREs; these are delivered to host cells by an Icm/Dot-dependent mechanism. Analysis of mutant strains suggests that the Lep proteins may enable the Legionella to commandeer a protozoan exocytic pathway for dissemination of the pathogen.