Legionelladirected Phagosome Biogenesis

Phagocytosis in D. discoideum and macrophages is known to be regulated by a heterotrimeric G protein-linked signal transduction (Table 4.1). By using pharmacological inhibitors and Dictyostelium mutants it has been demonstrated that L. pneumophila uptake occurs by conventional phagocytosis which requires hetero-trimeric G proteins and the PLC pathway. Moreover, these experiments revealed that cytoplasmic calcium levels, the cytoskeleton proteins coronin, villidin and a-actinin/ filamin as well as the calcium-binding ER proteins calreticulin and calnexin significantly influence this process [17, 40]. The newly internalized phagosome is rapidly transported through the cell on microtubules (Figure 4.1). The decreased intracellular replication of L. pneumophila in a D. discoideum ratioA (rtoA~) mutant obviously results from a lowered efficiency of vesicle trafficking important for the integrity of the Legionella-containing compartment [35]. Of particular interest are the substrates of the Legionella Dot/Icm type IV secretion system [41]. SidM and LidA target the mammalian Rab1, a small GTPase regulating ER-to-Golgi traffic. RalF recruits and activates ADP-ribosylation factor 1 (Arf1), a small GTPase involved in retrograde vesicle transport from the Golgi apparatus to the ER [42, 43]. The recent analysis of the D. discoideum transcriptome upon infection with L. pneumophila revealed that by establishing its replicative niche Legionella not only interferes with bacterial degradation and intracellular vesicle transport and fusion but also profoundly influences and exploits the metabolism of its host. Furthermore, genes belonging to the Dictyostelium ubiquitination machinery are strongly upregulated after infection with L. pneumophila wild-type as compared to the dotA mutant [32]. Remarkably, the L. pneumophila genome analysis identified several homologs of eukaryotic genes and it has been speculated that the respective proteins may allow Legionella to communicate with eukaryotic cells [44].

Genetic and pharmacological evidence suggest that class I phosphatidylinositol 3-kinases (PI3Ks) are dispensable for phagocytosis of L. pneumophila but appear to play a major role for the establishment of the replicative vacuole [45]. L. pneumophila obviously subverts the host metabolism, favoring formation of specific phosphati-dylinositol forms in the Legionella-containing vacuole (LCV). Among the different phosphatidylinositols, PI(4)P is enriched in the Legionella-containing phagosome and anchors specifically SidC, one of the secreted protein substrates of the Legionella Dot/Icm type IV secretion system. In the absence of functional PI3K, SidC recruitment to Legionella-containing phagosomes is increased, suggesting that PI (4)P sites are enriched [45].

Exciting progress has been made by proteome studies and the analysis oflatex bead-containing Dictyostelium phagosomes which helped to define three maturation stages [19]. The first maturation stage is characterized by coronin and lysosomal glycoprotein (LmpB) acquisition. The second stage is characterized by the transfer of lysosomal enzymes. The third stage is characterized by quantitative retrieval of hydrolases from the phagolysosome and exocytosis of latex beads. So far, almost 200 phagosome proteins have been identified and ordered by their temporal appearance [20]. The phagosome proteins of Legionella-infected Dictyostelium cells also belong to a variety of functional categories, but specific alterations can be observed. Proteins involved in metabolism, protein biosynthesis as well as cytoskeleton and signal transduction make up the bulk of the Legionella phagosome proteome and agree well with the observed upregulation of their transcription [32]. The presence of elongation factors, ribosomal proteins, tRNA-synthetases as well as proteins typically associated with ER is consistent with the fact that the Legionella-containing phagosome intercepts secretory vesicles from ER exit sites and develops into a compartment that shares many features with the rER [46]. Unpublished results highlight the relevance of the ubiquitin-proteasome machinery (Shevchuk, Steinert et al., unpublished). This view is further supported by the observation that Legionella translocates an E3 ubiquitin ligase that has two U-boxes. U-box 1 is critical to the ubiquitin ligation and U-box 2 mediates interaction with Clk1 (Cdc2-likekinase 1) [47].

Since actin assembled by phagosomes can provide tracks for lysosomes to move vectorially toward them, it is an interesting finding that infection with the pathogenic L. pneumophila Corby infection causes degradation of actin on phagosomes (Shevchuk, Steinert et al., unpublished). The fact that phagosomes containing nonreplicative L. hackeliae do not display degraded actin indicates the importance of this phenomenon. Whether the cAMP-signaling system of D. discoideum plays a role in phagosome actin assembly and on phagosome maturation, as described for macrophages infected with mycobacteria, remains to be established [48].

The appearance of the actin-binding protein coronin on the phagosome raises another hypothesis. Coronin interacts with cytosolic NADPH oxidase components p40phox and p67phox and is known to be involved in their translocation to the phagosome [49]. Previous observations that Legionella replicates well in Dictyostelium mutants lacking coronin may thus be due to improper superoxide generation at the phagosome [30, 40]. Moreover, in neutrophils, phosphorylated p47phox protein has been shown to allow appropriate localization of PKC to coronin, leading to coronin phosphorylation. The phosphorylated coronin solubilizes, and the subsequent removal of the coronin coat facilitates phagolysosome fusion [49, 50, 51]. Since coronin as well as PKC inhibitors were observed on Legionella phagosomes we may have a first indication of how the Legionella-specific inhibition of the phagolysosome fusion is regulated.

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