Gérard Klein

Specific host genes required for the killing of Klebsiella bacteria by phagocytes

The 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.

The Arrestin Fold: Variations on a Theme

Endocytosis of ligand-activated plasma membrane receptors has been shown to contribute to the regulation of their downstream signaling. β-arrestins interact with the phosphorylated tail of activated receptors and act as scaffolds for the recruitment of adaptor proteins and clathrin, that constitute the machinery used for receptor endocytosis. Visual- and β-arrestins have a two-lobe, immunoglobulin-like, β-strand sandwich structure. The recent resolution of the crystal structure of VPS26, one of the retromer subunits, unexpectedly evidences an arrestin fold in this protein, which is otherwise unrelated to arrestins. From a functional point of view, VPS26 is involved in the retrograde transport of the mannose 6-P receptor from the endosomes to the trans-Golgi network. In addition to the group of genuine arrestins and Vps26, mammalian cells harbor a vast repertoire of proteins that are related to arrestins on the basis of their PFAM Nter and Cter arrestin- domains, which are named Arrestin Domain- Containing proteins (ADCs). The biological role of ADC proteins is still poorly understood. The three subfamilies have been merged into an arrestin-related protein clan. This paper provides an overall analysis of arrestin clan proteins. The structures and functions of members of the subfamilies are reviewed in mammals and model organisms such as Drosophila, Caenorhabditis, Saccharomyces and Dictyostelium.

Interaction of trichorzianines A and B with model membranes and with the amoeba Dictyostelium

Trichorzianines A (TA) and B (TB) are microheterogeneous mixtures of antibiotic nonadecapeptides of the peptaibol class which interact with lipidic membranes and modify their permeability properties. The TB differ from the TA by replacement of the Gln-18 by a Glu, giving rise to a C-terminal negative charge at neutral pH. The role of this charge on the trichorzianine-lipid interaction was investigated with model membranes by fluorescence spectroscopy and the results were correlated with the biological activity toward the amoeba Dictyostelium discoideum. The interaction of the acidic trichorzianine TB IIIc (Glu-18) with phospholipid bilayers and the subsequent induced permeability were weaker than that exhibited by the uncharged TA IIIc (Gln-18) and MeTB IIIc (TB IIIc monomethyl ester). The unfavourable effect of the negative charge in TB IIIc was strongly enhanced by incorporation of cholesterol in the bilayer. Similarly, TA IIIc as well as MeTB IIIc induced growth inhibition and lysis of the amoeba Dictyostelium at four times lower concentrations than TB IIIc. The results suggested that the interaction of trichorzianines with the phospholipid bilayer and the subsequent modifications of permeability were involved in the inhibitory properties and cell lysis induced by trichorzianines toward Dictyostelium.

Kinetics of fluid-phase pinocytosis in Dictyostelium discoideum amoebae

Kinetics of pinocytosis in Dictyostelium discoideum were investigated over an extended period of time (up to 6 hours) using fluorescein isothiocyanate (FITC)-dextran as a fluid-phase marker. FITC-dextran added to the medium accumulated rapidly inside the cells with a rate of influx equivalent to 9 microns3 of fluid/cell x min. After a period of about 90 min of uptake, the intracellular FITC-dextran level reached a plateau which corresponded to a strict balance between pinocytosis and exocytosis as shown both by efflux measurements and pulse experiments with (3H) dextran. At equilibrium, the amount of internalized marker reached a value equivalent to 790 microns3 of fluid taken up per amoeba, i.e. a volume paradoxically higher than the total aqueous space of the cell (520 microns3 ). FITC-dextran was thus markedly concentrated intracellularly. The endocytic compartment in which the intracellular FITC-dextran was concentrated could be completely washed out when FITC-dextran was removed from the external medium.

Identification of inositol hexaphosphate in 31P-NMR spectra of Dictyostelium discoideum amoebae. Relevance to intracellular pH determination.

A sugar phosphomonoester, myo-inositol hexakisphosphate (phytic acid), has been identified as a major phosphorylated metabolite in Dictyostelium discoideum amoeba. Its intracellular concentration was estimated to be 0.7 mM. The identification was made in perchloric acid extracts on the basis of 31P-NMR chemical shift values and their variations with pH, by addition of authentic compound and by hydrolysis with wheat phytase. Perchloric acid extracts were prepared so as to avoid losses of insoluble salts of polyphosphorylated compounds with divalent cations. The glycolytic intermediate, 3-phosphoglycerate accumulated intracellularly in amoebae incubated in the presence of fluoride. The pH sensitive NMR signal of 3-phosphoglycerate was used to monitor cytosolic pH and a value of pH 7.4 was found in aerobic Dictyostelium amoebae.

A necrotic cell death model in a protist.

While necrotic cell death is attracting considerable interest, its molecular bases are still poorly understood. Investigations in simple biological models, taken for instance outside the animal kingdom, may benefit from less interference from other cell death mechanisms and from better experimental accessibility, while providing phylogenetic information. Can necrotic cell death occur outside the animal kingdom? In the protist Dictyostelium, developmental stimuli induced in an autophagy mutant a stereotyped sequence of events characteristic of necrotic cell death. This sequence included swift mitochondrial uncoupling with mitochondrial 2′,7′-dichlorofluorescein diacetate fluorescence, ATP depletion and increased oxygen consumption. This was followed by perinuclear clustering of dilated mitochondria. Rapid plasma membrane rupture then occurred, which was evidenced by time-lapse videos and quantified by FACS. Of additional interest, developmental stimuli and classical mitochondrial uncouplers triggered a similar sequence of events, and exogenous glucose delayed plasma membrane rupture in a nonglycolytic manner. The occurrence of necrotic cell death in the protist Dictyostelium (1) provides a very favorable model for further study of this type of cell death, and (2) strongly suggests that the mechanism underlying necrotic cell death was present in an ancestor common to the Amoebozoa protists and to animals and has been conserved in evolution.

Natural protein ATPase inhibitor from Candida utilis mitochondria binding properties of the radiolabeled inhibitor

The energy-transducing ATPases of mitochondria, chloroplasts and bacteria are associated with an endogenous protein inhibitor [1 -7 ] . These naturally occurring inhibitors (IFl) are low molecular weight and trypsin-sensitive proteins. They are either part of the ATPase molecule in chloroplast and bacterial ATPases [5-7] or a distinct entity in mitochondrial ATPase [8]. They strongly mask the hydrolytic ATPase activity of soluble and membrane-bound ATPase. Studies by Ernster et al. [9,10] and Van de Stadt et al. [11,12] have underlined the regulatory function of IF~ in oxidative phosphorylation. IF1 is believed to control the backflow of energy from ATP to ATP-driven reactions. A similar regulatory mechanism has been proposed for chloroplasts [ 13]. These conclusions were drawn from experiments where the interaction between the natural ATPase inhibitor and the ATPase was measured in terms of inhibition of the ATPase activity, assuming that inhibition of ATPase reflects the binding of IFx to ATPase. IF~ can be purified from Candida utilis mitochondria, allowing the preparation of radioactivity labeled IF~ by growing Candida utilis in amedium supplemented with a radioactive amino acid [3]. This

Necrotic cell death: From reversible mitochondrial uncoupling to irreversible lysosomal permeabilization

Dictyostelium atg1- mutant cells provide an experimentally and genetically favorable model to study necrotic cell death (NCD) with no interference from apoptosis or autophagy. In such cells subjected to starvation and cAMP, induction by the differentiation-inducing factor DIF or by classical uncouplers led within minutes to mitochondrial uncoupling, which causally initiated NCD. We now report that (1) in this model, NCD included a mitochondrial-lysosomal cascade of events, (2) mitochondrial uncoupling and therefore initial stages of death showed reversibility for a surprisingly long time, (3) subsequent lysosomal permeabilization could be demonstrated using Lysosensor blue, acridin orange, Texas red-dextran and cathepsin B substrate, (4) this lysosomal permeabilization was irreversible, and (5) the presence of the uncoupler was required to maintain mitochondrial lesions but also to induce lysosomal lesions, suggesting that signaling from mitochondria to lysosomes must be sustained by the continuous presence of the uncoupler. These results further characterized the NCD pathway in this priviledged model, contributed to a definition of NCD at the lysosomal level, and suggested that in mammalian NCD even late reversibility attempts by removal of the inducer may be of therapeutic interest.

Intracellular pH control in Dictyostelium discoideum: A 31P-NMR analysis

Phosphorus metabolites and intracellular pH have been examined in the slime mold Dictyostelium discoideum by non-destructive 31P-NMR measurements. In a spectrum from a suspension of aerobic amoebae, the major peaks are inorganic phosphate, nucleotide di- and triphosphates. In the corresponding perchloric acid extract, resonances originating from purine and pyrimidine nucleotides are resolved. Adenine nucleotides are the most abundant components, but the other nucleotides are present in significant amounts. In a spectrum from intact spores in a dormant state, only inorganic phosphate and polyphosphates are detected and nucleotides are no longer present in large amounts. Of particular importance is the ability to observe separately in aerobic amoebae the resonance of inorganic phosphate localized in two different cell compartments: the cytosol and the mitochondria. The cytosolic pH and mitochondrial pH have been measured as 6.7 and 7.7, respectively, on the basis of intracellular inorganic phosphate chemical shifts. They are essentially unaffected over a large range of external pH and they are not modified transiently or permanently during the initiation of the developmental program of the organism. A weak acid, such as propionate, which modifies the progression of differentiation by favoring prestalk cells, perturbs intracellular pH gradients by selectively decreasing mitochondrial pH without any effect on cytosolic pH.

FYVE-Dependent Endosomal Targeting of an Arrestin-Related Protein in Amoeba

Background Visual and β-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins. Methodology and Principal Findings A detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation. Significance This work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.