Large-scale identification ofLegionella pneumophilaDot/Icm substrates that modulate host cell vesicle trafficking pathways

Abstract

SummaryThe bacterial pathogen Legionella pneumophila replicates in a specialized vacuole within host cells. Establishment of the replication vacuole depends on the Dot/Icm translocation system that delivers a large number of protein substrates into the host cell. The functions of most substrates are unknown. Here, we analysed a defined set of 127 confirmed or candidate Dot/Icm substrates for their effect on host cell processes using yeast as a model system. Expression of 79 candidates caused significant yeast growth defects, indicating that these proteins impact essential host cell pathways. Notably, a group of 21 candidates interfered with the trafficking of secretory proteins to the yeast vacuole. Three candidates that caused yeast secretory defects (SetA, Ceg19 and Ceg9) were investigated further. These proteins impinged upon vesicle trafficking at distinct stages and had signals that allowed translocation into host cells by the Dot/Icm system. Ectopically produced SetA, Ceg19 and Ceg9 localized to secretory organelles in mammalian cells, consistent with a role for these proteins in modulating host cell vesicle trafficking. Interestingly, the ability of SetA to cause yeast phenotypes was dependent upon a functional glycosyltransferase domain. We hypothesize that SetA may glycosylate a component of the host cell vesicle trafficking machinery during L. pneumophila infection.

Identification of Novel Coxiella burnetii Icm/Dot Effectors and Genetic Analysis of Their Involvement in Modulating a Mitogen-Activated Protein Kinase Pathway

“…It was previously shown, using effectors from various pathogenic bacteria, including C. burnetii and L. pneumophila, that ectopic expression of effectors sometimes results in inhibition of yeast growth (21,25,32,33,67,68). Yeast growth inhibition suggests that a conserved and essential eukaryotic process that is modulated by the effector in the host cell was also modulated in the yeast cell, resulting in inhibition of yeast cell growth (33,34,67,69). Examination of the 13 newly identified C. burnetii effectors (CBU0062, CBU0113, CBU0978, CBU1063, CBU1370, CBU1387, CBU1409, CBU1634a, CBU1676, CBU1794, CBU1818, CBU2007, and CBU2013) in yeast revealed that all of them were expressed (Fig.…”

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

“…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).…”

Characterization of Enzymes from Legionella pneumophila Involved in Reversible Adenylylation of Rab1 Protein

“…The bacterium Legionella pneumophila is a prominent example, and a large amount of research has been performed on the mechanisms and proteins involved in evasion of lysosomal degradation (10). After Legionella is taken up by the cell into a Legionella-containing vacuole (LCV), the bacterium injects more than 250 proteins into the host cell using a type IV secretion system termed DOT/ICM (defect in organelle trafficking/intracellular multiplication) (11,12). Several of these proteins have been observed to modify Rab1 function, i.e.…”

“…Amino acids 1-339 represent the adenylyltransferase domain, amino acids 340 -533 represent the GEF domain, and amino acids 534 -647 represent the phosphatidylinositol-4-phosphate binding domain (P4M) of DrrA, respectively. The catalytic motif GX 11 DXD of the adenylyltransferase domain of DrrA (Fig. 2B, indicated by a red arrow) is quite far from the GEF binding site of Rab1, and on the opposite side of DrrA (Fig.…”