Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance-associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo-lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth-phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V-H þ ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein-vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein-Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild-type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP-dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.

SummaryMembrane phosphatidylinositides recruit cytosolic proteins to regulate phagocytosis, macropinocytosis and endolysosomal vesicle maturation. Here, we describe effects of inactivation of PI3K, PTEN or PLC on Escherichia coli and Legionella pneumophila uptake by the professional phagocyte Dictyostelium discoideum. We show that L. pneumophila is engulfed by macropinocytosis, a process that is partially sensitive to PI3K inactivation, unlike phagocytosis of E. coli. Both processes are blocked by PLC inhibition. Whereas E. coli is rapidly digested, Legionella proliferates intracellularly. Proliferation is blocked by constitutively expressing Nramp1, an endolysosomal iron transporter that confers resistance against invasive bacteria. Inactivation of PI3K, but not PTEN or PLC, enhances Legionella infection and suppresses the protective effect of Nramp1 overexpression. PI3K activity is restricted to early infection and is not mediated by effects on the actin cytoskeleton; rather L. pneumophila, in contrast to E. coli, subverts phosphoinositide-sensitive fusion of Legionella-containing macropinosomes with acidic vesicles, without affecting Nramp1 recruitment. A model is presented to explain how Legionella escapes fusion with acidic vesicles and Nramp1-induced resistance to pathogens.