Legionella Eukaryotic-Like Type IV Substrates Interfere with Organelle Trafficking

Abstract

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.

Genome Dynamics in Legionella: The Basis of Versatility and Adaptation to Intracellular Replication

“…Therefore, many groups started functional studies focusing on these proteins and within a short period of time the importance of the eukaryotic-like proteins for the virulence of Legionella was shown (de Felipe et al 2008;Nora et al 2009;Hubber and Roy 2010). The main secretion system of Legionella, essential for intracellular growth, is the type IVB Dot/Icm secretion system (Marra and Shuman 1992;Berger and Isberg 1993).…”

“…The main secretion system of Legionella, essential for intracellular growth, is the type IVB Dot/Icm secretion system (Marra and Shuman 1992;Berger and Isberg 1993). Using many dif-ferent methods it has been shown that at least 275 L. pneumophila proteins are secreted by this system (Campodonico et al 2005;de Felipe et al 2005;Shohdy et al 2005;de Felipe et al 2008;Burstein et al 2009;Heidtman et al 2009;Zhu et al 2011), which represents nearly 10% of the protein-coding genes predicted in the L. pneumophila genomes. At least 75 of these are eukaryotic-like proteins or carry eukaryotic domains and, for certain of these, it has also been shown experimentally that they interfere with host-cell signaling pathways ( Table 2).…”

Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii

“…Our efforts with these strategies have led to the retrieval of 57 C. burnetii T4SS substrate candidates. Using independent translocation assays based on the Cya (18) or the β-lactamase (TEM1)-mediated FRET on CCF4-AM (6,19), we demonstrated that 32 of these proteins are translocated into host cells by the L. pneumophila Dot/Icm system.…”

“…Various strategies have led to the identification of more than 150 protein substrates for the L. pneumophila T4SS (5-7). These proteins are predicted to modulate various host processes, including apoptosis, ubiquitination (8)(9)(10), lipid metabolism, and membrane trafficking (6,(11)(12)(13). By combining bioinformatics tools with the use of Legionella as a surrogate host to measure protein translocation, 11 Coxiella proteins containing the ankyrin repeat motif have been identified as substrates of its Dot/ Icm transporter (11,14).…”

Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector ofLegionella pneumophila

“…Within both evolutionarily distant host cells, L. pneumophila evades endocytic fusion and intercepts ER to Golgi vesicle traffic to remodel its phagosome into an ER-derived vacuole (Kagan and Roy, 2002;Molmeret et al, 2005;Shin and Roy, 2008;Isberg et al, 2009). The Dot/Icm type IV secretion system (Segal et al, 1998;Vogel et al, 1998) injects into the host cell a cadre of 200 effectors to modulate a myriad of cellular processes to reprogram the host cell into a proliferation niche (de Felipe et al, 2008;Shin and Roy, 2008;Isberg et al, 2009). The Ankyrin B (AnkB) effector is injected into the host cell by the Dot/Icm system upon bacterial attachment to the plasma membrane and exploits an evolutionarily conserved eukaryotic machinery within mammalian and protozoan cells (Price et al, 2009).…”