A systems biology analysis of the Drosophila phagosome.

Phagocytes have a critical function in remodelling tissues during embryogenesis and thereafter are central effectors of immune defence. During phagocytosis, particles are internalized into 'phagosomes', organelles from which immune processes such as microbial destruction and antigen presentation are initiated. Certain pathogens have evolved mechanisms to evade the immune system and persist undetected within phagocytes, and it is therefore evident that a detailed knowledge of this process is essential to an understanding of many aspects of innate and adaptive immunity. However, despite the crucial role of phagosomes in immunity, their components and organization are not fully defined. Here we present a systems biology analysis of phagosomes isolated from cells derived from the genetically tractable model organism Drosophila melanogaster and address the complex dynamic interactions between proteins within this organelle and their involvement in particle engulfment. Proteomic analysis identified 617 proteins potentially associated with Drosophila phagosomes; these were organized by protein-protein interactions to generate the 'phagosome interactome', a detailed protein-protein interaction network of this subcellular compartment. These networks predicted both the architecture of the phagosome and putative biomodules. The contribution of each protein and complex to bacterial internalization was tested by RNA-mediated interference and identified known components of the phagocytic machinery. In addition, the prediction and validation of regulators of phagocytosis such as the 'exocyst', a macromolecular complex required for exocytosis but not previously implicated in phagocytosis, validates this strategy. In generating this 'systems-based model', we show the power of applying this approach to the study of complex cellular processes and organelles and expect that this detailed model of the phagosome will provide a new framework for studying host-pathogen interactions and innate immunity.

Red cells from glutathione peroxidase-1-deficient mice have nearly normal defenses against exogenous peroxides.

The role of glutathione peroxidase in red cell anti-oxidant defense was examined using erythrocytes from mice with a genetically engineered disruption of the glutathione peroxidase-1 (GSHPx-1) gene. Because GSHPx-1 is the sole glutathione peroxidase in the erythrocyte, all red cell GSH peroxidase activity was eliminated. Oxidation of hemoglobin and membrane lipids, using the cis-parinaric acid assay, was determined during oxidant challenge from cumene hydroperoxide and H(2)O(2). No difference was detected between wild-type red cells and GSHPx-1-deficient cells, even at high H(2)O(2) exposures. Thus, GSHPx-1 appears to play little or no role in the defense of the erythrocyte against exposure to peroxide. Simultaneous exposure to an H(2)O(2) flux and the catalase inhibitor 3-amino-1,2,4-triazole supported this conclusion. Hemoglobin oxidation occurred only when catalase was depleted. Circulating erythrocytes from the GSHPx-1-deficient mice exhibited a slight reduction in membrane thiols, indicating that high exposure to peroxides might occur naturally in the circulation. (Blood. 2000;96:1985-1988)

Increased erythrocyte deformability in fetal erythropoiesis and in erythrocytes deficient in glucose-6-phosphate dehydrogenase and other glycolytic enzymes.

Erythrocyte deformability was determined in more than 500 clinical samples, and was found to be elevated in conditions in which fetal-like red cells are produced: aplastic anemia (3/3 cases), myelodysplastic syndromes, polycythemias, sickle cell anemia during treatment with hydroxyurea, paroxysmal nocturnal hemoglobinuria, and recovery from B12 deficiency. Elevated deformability was observed in neonatal erythrocytes, and during recovery from transient erythroblastopenia of childhood, when fetal-like red cells are known to be produced. Increased deformability appears to be a feature of fetal and fetal-like red cells. Forty-eight cases of enzymatically verified glucose-6-phosphate (G-6-PD) deficiency were also examined. Thirty out of 32 G-6-PD(A-) individuals, including both heterozygotes and hemizygotes, exhibited increased deformability during the steady state. In contrast, G-6-PD(Med) hemizygotes had normal deformability. Increased deformability was also found in G-6-PD(Huron) (n=3), G-6-PD(Wayne) (n=4), triose phosphate isomerase deficiency (n=2), and pyruvate kinase deficiency (n=2). An elevated osmoscan was found in more than 90% of female G-6-PD heterozygotes, affording a simple screening test for heterozygotes. Deformability remained high during hemolytic episodes, when older enzyme deficient cells are removed from the circulation. In four cases of G-6-PD deficiency with normal deformability, evidence for co-existing hereditary spherocytosis was found. The combination of conditions with opposing effects on deformability resulted in nearly normal deformability. Because increased red cell deformability is a feature of fetal erythrocytes, these results suggest that the red cells in many cases of glycolytic enzyme deficiency are fetal-like.

Oxidant damage to erythrocyte membrane in glucose-6-phosphate dehydrogenase deficiency: correlation with in vivo reduced glutathione concentration and membrane protein oxidation.

Chronic nonspherocytic hemolytic anemia has been observed in a recently described glucose-6-phosphate dehydrogenase (G6PD) variant, G6PDWayne. The mechanical properties of these erythrocytes and other G6PD variants were examined. The deformability of G6PD-deficient erythrocytes was normal, as determined by osmotic scan ektacytometry, and was not significantly affected by hemolytic crisis. In the common varieties of G6PD deficiency, the mechanical stability of the red blood cell (RBC) membrane was greater than normal, but G6PDWayne membranes were abnormally susceptible to shear-induced fragmentation. There was no evidence for a concurrent genetic defect in spectrin, because self-association constants and tryptic digests were normal. The fragility of G6PDWayne membranes appeared to be a consequence of oxidative damage to membrane thiol groups associated with a low glutathione (GSH) level in these RBCs. Associations among GSH level, thiol oxidation, and membrane instability were also found when a larger group of G6PD-deficient RBCs were examined. In normal erythrocytes, 1-chloro-2,4-dinitrobenzene was used to reduce GSH levels by 50%. Membrane thiol oxidation and membrane fragility both increased when these cells were kept at 4 degrees C for 3 to 5 days. Our findings suggest that chronic depletion of GSH leads to the destabilization of membrane skeleton through oxidation of membrane protein thiols.

Extensive colonization of the porcine colonic epithelium by a spirochete similar to Treponema innocens.

Specimens of colonic mucosa from two pigs with diarrhea were examined by light and electron microscopy. The epithelial surfaces of both pigs were extensively colonized by large spirochetes morphologically compatible with Treponema hyodysenteriae or Treponema innocens. The microorganisms were intimately attached end-on to the luminal cells. A weakly beta-hemolytic spirochete similar to T. innocens was isolated from the colon of one of the pigs.