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.

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.

Background: Phagocytosis plays a major role in the defense of higher organisms against microbial infection and provides also the basis for antigen processing in the immune response. Cells of the model organism Dictyostelium are professional phagocytes that exploit phagocytosis of bacteria as the preferred way to ingest food, besides killing pathogens. We have investigated Dictyostelium differential gene expression during phagocytosis of non-pathogenic bacteria, using DNA microarrays, in order to identify molecular functions and novel genes involved in phagocytosis.

Recently we reported that Dictyostelium cells ingest Legionella pneumophila by macropinocytosis, whereas other bacteria, such as Escherichia coli, Mycobacterium avium, Neisseria meningitidis or Salmonella typhimurium, are taken up by phagocytosis.1 In contrast to phagocytosis, macropinocytosis is partially inhibited by PI3K or PTEN inactivation, whereas both processes are sensitive to PLC inhibition. Independently from reduced uptake, L. pneumophila proliferates more efficiently in PI3K-null than in wild-type cells. PI3K inactivation also neutralizes resistance to infection conferred by constitutively expressing the endo-lysosomal iron transporter Nramp1. We have shown this to be due to altered recruitment of the V-H(+) ATPase, but not Nramp1, in the Legionella-containing vacuole (LCV) early during infection.1 As further evidence for impaired LCV acidification we examine here the effects of disrupting the small G protein RacH on Legionella infection.