Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.The bacterium Pseudomonas aeruginosa is an important causative agent of nosocomial infections, including severe pneumonia (10) and bacteremia. This opportunistic pathogen also colonizes the lungs of cystic fibrosis patients and leads to progressive lung damage, respiratory failure, and eventually death (3, 12). The seriousness of P. aeruginosa infections is further exacerbated by the rapid selection of antibiotic-resistant strains following antibiotic treatment (14).Studies in mammalian hosts have shown that quorum sensing is important for the virulence of P. aeruginosa (28,37,42). Secreted components essential for Pseudomonas virulence, such as proteases, rhamnolipids, pyocyanin, and exotoxin A, are under the control of two quorum-sensing systems, las and rhl ( Fig. 1) (31, 43). When the bacterial cell density reaches a certain threshold, the accumulation in the medium of signaling autoinducer molecules (3-oxo-C12-homoserine lactone [HSL] and C4-HSL) induces the las and rhl pathways, respectively, leading to transcription of virulence genes. Both systems involve a transcriptional regulator (LasR and RhlR, respectively) and an autoinducer synthase (LasI and RhlI, respectively). The las quorum-sensing system can also induce the transcription of rhlR and consequently activate, to some degree, the rhl quorum-sensing system (21).Strategies to develop innovative treatments against Pseudomonas infections rely on the elucidation of virulence and antibiotic resistance mechanisms. These studies involve the characterization of mutant strains and analysis of their virulence.However, the assessment of bacterial pathogenicity in mammalian hosts is time-consuming and expensive. Therefore, alternative yet equally relevant host systems would be extremely useful. Pseudomonas is remarkable in its ability to infect a number of alt...

To identify the molecular mechanisms involved in phagocytosis, we generated random insertion mutants of Dictyostelium discoideum and selected two mutants defective for phagocytosis. Both represented insertions in the same gene, named PHG1. This gene encodes a polytopic membrane protein with an N-terminal lumenal domain and nine potential transmembrane segments. Homologous genes can be identified in many species; however, their function is yet to be elucidated. Disruption of PHG1 caused a selective defect in phagocytosis of latex beads and Escherichia coli, but not Klebsiella aerogenes bacteria. This defect in phagocytosis was caused by a decrease in the adhesion of mutant cells to phagocytosed particles. These results indicate that the Phg1 protein is involved in the adhesion of Dictyostelium to various substrates, a crucial event of phagocytosis and demonstrate the usefulness of a genetic approach to dissect the molecular events involved in the phagocytic process.Phagocytosis is the process by which cells internalize large particles (typically Ͼ1 m diameter), such as bacteria or cell debris. In higher eucaryotes, phagocytic cells are essential players of the host defense against invading pathogens and tissue remodeling (for review see Ref. 1). Phagocytosis involves adhesion of the phagocytic cell to the particle, and reorganization of the actin cytoskeleton to allow engulfment. A number of receptors required for the recognition of particles to be phagocytosed have been identified in mammalian cells, e.g. Fc receptors involved in the phagocytosis of opsonized particles (2). These receptors presumably transduce a local activation signal upon recognition of their ligand, leading to reorganization of the actin cytoskeleton. Protein kinases such as Syk (3) as well as GTP-binding proteins of the Rho family (4) have been implicated in the transduction of the activation signal.The cellular slime mold Dictyostelium discoideum has been used previously as a model organism to study phagocytosis (5,6). Vegetative Dictyostelium amoebae multiply as single cells feeding phagocytically on bacteria. The mechanisms involved in phagocytosis by Dictyostelium cells are very similar to those used by mammalian phagocytes, and involve notably the actin cytoskeleton and RacF1, a member of the Rho family of GTPbinding proteins (7). The receptors responsible for phagocytosis have not yet been identified. However, previous studies suggest that there are at least two receptors, one a lectin and the other one a "nonspecific" receptor, which accounts for the phagocytosis of most hydrophilic particles in HL5 medium (5).Here we describe the identification of a new gene implicated in phagocytosis in Dictyostelium. The phenotype of the phg1 mutants suggests a role for Phg1p in adhesion to phagocytic substrates. EXPERIMENTAL PROCEDURESCell Culture and Internalization Assays-Wild-type cells used in this study are DH1-10 cells, a subclone of DH1 cells. They were grown at 21°C in HL5 medium (8) and subcultured twice a week. Cells were typically not a...

Glycosylphosphatidylinositol (GPI) anchors are added onto newly synthesized proteins in the ER. Thereby a putative transamidase removes a C‐terminal peptide and attaches the truncated protein to the free amino group of the preformed GPI. The yeast mutant gpi8–1 is deficient in this addition of GPIs to proteins. GPI8 encodes for an essential 47 kDa type I membrane glycoprotein residing on the luminal side of the ER membrane. GPI8 shows significant homology to a novel family of vacuolar plant endopeptidases one of which is supposed to catalyse a transamidation step in the maturation of concanavalin A and acts as a transamidase in vitro. Humans have a gene which is highly homologous to GPI8 and can functionally replace it.

Gpi7 was isolated by screening for mutants defective in the surface expression of glycosylphosphatidylinositol (GPI) proteins. Gpi7 mutants are deficient in YJL062w, herein named GPI7. GPI7 is not essential, but its deletion renders cells hypersensitive to Calcofluor White, indicating cell wall fragility. Several aspects of GPI biosynthesis are disturbed in ⌬gpi7. The extent of anchor remodeling, i.e. replacement of the primary lipid moiety of GPI anchors by ceramide, is significantly reduced, and the transport of GPI proteins to the Golgi is delayed. Gpi7p is a highly glycosylated integral membrane protein with 9 -11 predicted transmembrane domains in the C-terminal part and a large, hydrophilic N-terminal ectodomain. The bulk of Gpi7p is located at the plasma membrane, but a small amount is found in the endoplasmic reticulum. GPI7 has homologues in Saccharomyces cerevisiae, Caenorhabditis elegans, and man, but the precise biochemical function of this protein family is unknown. Based on the analysis of M4, an abnormal GPI lipid accumulating in gpi7, we propose that Gpi7p adds a side chain onto the GPI core structure. Indeed, when compared with complete GPI lipids, M4 lacks a previously unrecognized phosphodiesterlinked side chain, possibly an ethanolamine phosphate. Gpi7p contains significant homology with phosphodiesterases suggesting that Gpi7p itself is the transferase adding a side chain to the ␣1,6-linked mannose of the GPI core structure. Glycosylphosphatidylinositol (GPI)1 -anchored proteins represent a subclass of surface proteins found in virtually all eukaryotic organisms (1). The genome of Saccharomyces cerevisiae contains more than 70 open reading frames (ORFs) encoding for proteins that, as judged from the deduced primary sequence, can be predicted to be modified by the attachment of a GPI anchor (2, 3). In about 25 of them, the presence of an anchor has been confirmed biochemically. A majority of them lose part of the anchor and become covalently attached to the ␤1,6-glucans of the cell wall (4 -6). A minority of GPI proteins retain the GPI anchor in an intact form and stay at the plasma membrane (PM). For the biosynthesis of GPI anchors, phosphatidylinositol (PI) is modified by the stepwise addition of sugars and ethanolamine phosphate (EtN-P), thus forming a complete precursor lipid (CP) which subsequently is transferred en bloc by a transamidase onto newly synthesized proteins in the ER (7,8). The identification of genes involved in the biosynthesis of the CP and its subsequent attachment to proteins has been possible through the complementation of mammalian and yeast gpi Ϫ mutants, i.e. mutants being deficient in GPI anchoring of membrane proteins (7, 9 -20). In our laboratory, a series of recessive gpi Ϫ mutants (gpi4 to gpi10) has been obtained by screening for yeast mutants that are unable to display the GPI-anchored ␣-agglutinin (Sag1p) at the outer surface of the cell wall, although the synthesis and secretion of soluble proteins is normal (21,22).Here we report on the characterization of...

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SummaryThe amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes ( PHG1 and KIL1 ) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae . Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.

Phosphatidic acid is the intermediate, from which all glycerophospholipids are synthesized. In yeast, it is generated from lysophosphatidic acid, which is acylated by Slc1p, an sn-2-specific, acyl-coenzyme A-dependent 1-acylglycerol-3-phosphate O-acyltransferase. Deletion of SLC1 is not lethal and does not eliminate all microsomal 1-acylglycerol-3-phosphate O-acyltransferase activity, suggesting that an additional enzyme may exist. Here we show that SLC4 (Yor175c), a gene of hitherto unknown function, encodes a second 1-acyl-sn-glycerol-3-phosphate acyltransferase. SLC4 harbors a membrane-bound O-acyltransferase motif and down-regulation of SLC4 strongly reduces 1-acyl-sn-glycerol-3-phosphate acyltransferase activity in microsomes from slc1⌬ cells. The simultaneous deletion of SLC1 and SLC4 is lethal. Mass spectrometric analysis of lipids from slc1⌬ and slc4⌬ cells demonstrates that in vivo Slc1p and Slc4p generate almost the same glycerophospholipid profile. Microsomes from slc1⌬ and slc4⌬ cells incubated with [ 14 C]oleoyl-coenzyme A in the absence of lysophosphatidic acid and without CTP still incorporate the label into glycerophospholipids, indicating that Slc1p and Slc4p can also use endogenous lysoglycerophospholipids as substrates. However, the lipid profiles generated by microsomes from slc1⌬ and slc4⌬ cells are different, and this suggests that Slc1p and Slc4p have a different substrate specificity or have access to different lyso-glycerophospholipid substrates because of a different subcellular location. Indeed, affinity-purified Slc1p displays Mg 2؉ -dependent acyltransferase activity not only toward lysophosphatidic acid but also lyso forms of phosphatidylserine and phosphatidylinositol. Thus, Slc1p and Slc4p may not only be active as 1-acylglycerol-3-phosphate O-acyltransferases but also be involved in fatty acid exchange at the sn-2-position of mature glycerophospholipids.

The study of free-living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin b chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well-characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin b chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.