Modulation of phagosome biogenesis by Legionella pneumophila creates an organelle permissive for intracellular growth

Abstract

†These authors contributed equally to this work undamental to the pathogenic lifestyle of several intracellular bacteria is their ability to modulate trafficking of the phagosomes in which they reside 1 . The bacterial pathogen Legionella pneumophila resides in a phagosome that restricts its fusion with host endosomes and lysosomes 2 . The intracellular fate of L. pneumophila is determined by a type-IV-related transport system encoded by 24 dot and icm virulence genes 3,4 . L. pneumophila dot and icm mutants reside in phagosomes that rapidly fuse with lysosomes, resulting in decreased intracellular survival and a severe intracellular growth defect 5,6 . Here we show that the primary function of the dot and icm products is to transmit a functionally cis-acting signal to the host macrophage that drives maturation of the L. pneumophila phagosome into a replicative vacuole. We find that L. pneumophila dot and icm mutants are able to grow intracellularly when the bacteria are residing in a phagosome established by L. pneumophila that retains its ability to alter phagosome trafficking. These results show that L. pneumophila tailors the phagosome into a specialized organelle permissive for bacterial growth through the specific actions of the dot/icm genes.We first determined whether macrophages infected with wildtype L. pneumophila are defective in delivering phagosomes containing killed yeast particles to lysosomes. We infected mouse bone-marrow-derived macrophages with wild-type L. pneumophila, and then fed the macrophages a suspension of heat-killed Saccharomyces cerevisiae. S. cerevisiae phagosomes rapidly acquired the lysosomal membrane protein LAMP-1 in macrophages preinfected with L. pneumophila (Fig. 1a). Phagosomes containing S. cerevisiae were uniformly LAMP-1-positive in macrophages that had been infected for 1 h or for as long as 8 h with virulent L. pneumophila (see Supplementary Information). The finding that trafficking of S. cerevisiae phagosomes was unaffected in macrophages containing virulent L. pneumophila indicates that the Dot/Icm transport apparatus does not generate a signal that accumulates and affects the trafficking of newly formed phagocytic compartments in the host cell.To further address whether there is a signal transmitted by virulent L. pneumophila that has the capacity to act in trans, we used a mixed inoculum consisting of a wild-type and a dotA mutant strain of L. pneumophila to infect bone-marrow-derived macrophages. If the wild-type bacteria were secreting a trans-acting factor, it could potentially restore intracellular growth to a dotA mutant that has lost the ability to alter phagosome trafficking. In these studies, we used a dotA mutant carrying a plasmid that produces green fluorescent protein (GFP). In macrophages that had ingested both wildtype and mutant bacteria, we did not detect replication of dotA mutants in phagosomes that were isolated from those containing wild-type L. pneumophila (Fig. 1b).Upon close inspection of all macrophages that had been infected with both wild...

Caveolae regulate the nanoscale organization of the plasma membrane to remotely control Ras signaling

“…Signaling through H-and K-Ras isoforms is differentially sensitive to cholesterol depletion (Roy et al, 1999;Prior et al, 2001;Prior et al, 2003a). Cells transformed by either K-Ras or H-Ras also show altered ganglioside levels (Suzuki et al, 1989) and oncogenic K-Ras activity can be regulated by the levels of PM PS van der Hoeven et al, 2013).…”

“…7 A), suggesting that Ras signaling in CAV1 / MEFs may be largely driven by K-Ras as both the protein level is elevated and nanoclustering is enhanced in cells with abrogated CAV1 levels. We have shown previously that K-Ras signal transmission is unaffected by cholesterol depletion, whereas signaling by other Ras isoforms is cholesterol sensitive (Roy et al, 1999(Roy et al, , 2005Prior et al, 2001). Therefore, we compared CAV1 +/+ and CAV1 / MEFs for activation of the MAP kinase pathway in response to serum stimulation under normal and cholesterol-depleted conditions.…”

“…Previous work has shown that expression of dominantnegative or mutant CAV1 proteins selectively abrogate H-Ras but not K-Ras signal transmission (Roy et al, 1999;Carozzi et al, 2002). In the present study we have examined the consequences of loss of caveolae on specific Ras signaling pathways using CAV1 / cells, cells with down-regulated CAV1 and Cavin1/ polymerase transcript release factor (an essential structural component of caveolae) or an acute loss of caveolae by modulating cellular osmotic pressure.…”

Plasma membrane regulates Ras signaling networks

“…H-Ras localizes to lipid rafts in a nucleotide dependent manner. [36][37][38] Inactive GDP-bound HRas is found in lipid rafts, 39,40 while H-Ras-GTP occupies the disordered membrane regions where it interacts with galectin-1. 41,42 N-Ras also partitions into lipid rafts, and membrane curvature plays an important role in N-Ras enrichment in the raft-like liquid ordered phases.…”

The mystery of oncogenicKRAS: Lessons from studying its wild-type counter part

“…We speculate that this specificity might be related to the differential membrane trafficking and/or localization of different Ras isoforms, which is mediated by their highly variable C-termini. [14][15][16][17][18][19] To our surprise, loss of WT Kras not only further promoted activation of WT Hras and Nras but also significantly enhanced oncogenic Kras-GTP levels. It is known that Ras G12D mutation affects the intrinsic GTPase activity of Ras proteins by preventing proper position of the Ras-GAP (GTPase activating protein) arginine finger within its catalytic site.…”