Rab5-associated Vacuoles Play a Unique Role in Phagocytosis of the Enteric Protozoan Parasite Entamoeba histolytica
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m403987200
ISSN1083-351X
AutoresYumiko Saito‐Nakano, Tomoyoshi Yasuda, Kumiko Nakada‐Tsukui, Matthias Leippe, Tomoyoshi Nozaki,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoIn mammals, Rab5 and Rab7 play a specific and coordinated role in a sequential process during phagosome maturation. Here, we report that Rab5 and Rab7 in the enteric protozoan parasite Entamoeba histolytica, EhRab5 and EhRab7A, are involved in steps that are distinct from those known for mammals. EhRab5 and EhRab7A were localized to independent small vesicular structures at steady state. Priming with red blood cells induced the formation of large vacuoles associated with both EhRab5 and EhRab7A (“prephagosomal vacuoles (PPV)”) in the amoeba within an incubation period of 5–10 min. PPV emerged de novo physically and distinct from phagosomes. PPV were gradually acidified and matured by fusion with lysosomes containing a digestive hydrolase, cysteine proteinase, and a membrane-permeabilizing peptide amoebapore. After EhRab5 dissociated from PPV, 5–10 min later, the EhRab7A-PPV fused with phagosomes, and EhRab7A finally dissociated from the phagosomes. Immunoelectron and light micrographs showed that PPV contained small vesicle-like structures containing fluid-phase markers and amoebapores, which were not evenly distributed within PPV, suggesting that the mechanism was similar to multivesicular body formation in PPV generation. In contrast to Rab5 from other organisms, EhRab5 was involved exclusively in phagocytosis, but not in endocytosis. Overexpression of wild-type EhRab5 enhanced phagocytosis and the transport of amoebapore to phagosomes. Conversely, expression of an EhRab5Q67L GTP form mutant impaired the formation of PPV and phagocytosis. Altogether, we propose that the amoebic Rab5 plays an important role in the formation of unique vacuoles, which is essential for engulfment of erythrocytes and important for packaging of lysosomal hydrolases, prior to the targeting to phagosomes. In mammals, Rab5 and Rab7 play a specific and coordinated role in a sequential process during phagosome maturation. Here, we report that Rab5 and Rab7 in the enteric protozoan parasite Entamoeba histolytica, EhRab5 and EhRab7A, are involved in steps that are distinct from those known for mammals. EhRab5 and EhRab7A were localized to independent small vesicular structures at steady state. Priming with red blood cells induced the formation of large vacuoles associated with both EhRab5 and EhRab7A (“prephagosomal vacuoles (PPV)”) in the amoeba within an incubation period of 5–10 min. PPV emerged de novo physically and distinct from phagosomes. PPV were gradually acidified and matured by fusion with lysosomes containing a digestive hydrolase, cysteine proteinase, and a membrane-permeabilizing peptide amoebapore. After EhRab5 dissociated from PPV, 5–10 min later, the EhRab7A-PPV fused with phagosomes, and EhRab7A finally dissociated from the phagosomes. Immunoelectron and light micrographs showed that PPV contained small vesicle-like structures containing fluid-phase markers and amoebapores, which were not evenly distributed within PPV, suggesting that the mechanism was similar to multivesicular body formation in PPV generation. In contrast to Rab5 from other organisms, EhRab5 was involved exclusively in phagocytosis, but not in endocytosis. Overexpression of wild-type EhRab5 enhanced phagocytosis and the transport of amoebapore to phagosomes. Conversely, expression of an EhRab5Q67L GTP form mutant impaired the formation of PPV and phagocytosis. Altogether, we propose that the amoebic Rab5 plays an important role in the formation of unique vacuoles, which is essential for engulfment of erythrocytes and important for packaging of lysosomal hydrolases, prior to the targeting to phagosomes. Phagocytosis is a critically important element of host defense against invading pathogens in higher organisms and its molecular mechanism in professional phagocytes, e.g. macrophage, has been extensively studied at the molecular level (1Tjelle T.E. Lovdal T. Berg T. Bioessays. 2000; 22: 255-263Crossref PubMed Scopus (138) Google Scholar, 2Greenberg S. Grinstein S. Curr. Opin. Immunol. 2002; 14: 136-145Crossref PubMed Scopus (439) Google Scholar). A number of steps including cell surface binding to ligands and the activation of a signaling pathway leading to F-actin polymerization have been identified as essential for phagocytosis. In addition, membrane trafficking plays an important role in the controlled maturation of phagosomes. The maturation is accompanied by sequential fusion with the endocytic compartment to form a phagolysosome, and is orchestrated by small GTPase, Rab proteins, which act as molecular switches regulating the fusion of vesicles with target membranes through the conformational change between active (GTP-bound) and inactive (GDP-bound) forms (3Novick P. Zerial M. Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (666) Google Scholar). It has been reported that Rab5 and Rab7 play an important role in the maturation of phagosomes in macrophages (4Desjardins M. Celis J.E. van Meer G. Dieplinger H. Jahraus A. Griffiths G. Huber L.A. J. Biol. Chem. 1994; 269: 32194-32200Abstract Full Text PDF PubMed Google Scholar). Rab5 was initially shown to be localized to early endosomes and the plasma membrane, and involved in endocytosis and the endosome fusion (5Stenmark H. Parton R.G. Steele-Mortimer O. Lutcke A. Gruenberg J. Zerial M. 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It has also been recently demonstrated that a novel effector protein, RILP, is recruited to the phagosomal membrane by Rab7, which promotes fusion between phagosomes and lysosomes (17Harrison R.E. Bucci C. Vieira O.V. Schroer T.A. Grinstein S. Mol. Cell. Biol. 2003; 23: 6494-6506Crossref PubMed Scopus (343) Google Scholar). Besides professional phagocytes from higher eukaryotes, some unicellular organisms such as Dictyostelium discoideum and Entamoeba histolytica show an inherent ability of phagocytosis. E. histolytica, an enteric protozoan parasite that causes an estimated 50 million cases of amebiasis: amebic colitis, dysentery, and extraintestinal abscesses (18Petri Jr., W.A. Curr. Opin. Microbiol. 2002; 5: 443-447Crossref PubMed Scopus (42) Google Scholar), and 40,000–100,000 deaths annually (19W.H.O./PAHO/UNESCO Epidemiol. Bull. 1997; 18: 13-14Google Scholar), colonizes the human gut and engulfs foreign cells including microorganisms and host cells. Phagocytosis has been implicated to be closely associated with the pathogenesis of the amoeba because phagocytosis-deficient amoeba mutants were shown to be avirulent (20Orozco E. Guarneros G. Martinez-Palomo A. Sanchez T. J. Exp. Med. 1983; 158: 1511-1521Crossref PubMed Scopus (181) Google Scholar). Although a number of amoebic molecules involved in attachment, phagocytosis, and degradation of microorganisms and host cells have been identified including galactose/N-acetylgalactosamine (Gal/GalNAc)-inhibitable lectin (21Vines R.R. Ramakrishnan G. Rogers J.B. Lockhart L.A. Mann B.J. Petri Jr., W.A. Mol. Biol. Cell. 1998; 9: 2069-2079Crossref PubMed Scopus (126) Google Scholar, 22Cheng X.J. Tsukamoto H. Kaneda Y. Tachibana H. Parasitol. Res. 1998; 84: 632-639Crossref PubMed Scopus (46) Google Scholar), cytoskeletal proteins and their associated regulatory molecules (23Voigt H. Olivo J.C. Sansonetti P. Guillen N. J. Cell Sci. 1999; 112: 1191-1201PubMed Google Scholar, 24Guillen N. 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We and other groups (28Saito-Nakano Y. Nakazawa M. Shigeta Y. Takeuchi T. Nozaki T. Mol. Biochem. Parasitol. 2001; 116: 219-222Crossref PubMed Scopus (25) Google Scholar, 29Rodriguez M.A. Garcia-Perez R.M. Garcia-Rivera G. Lopez-Reyes I. Mendoza L. Ortiz-Navarrete V. Orozco E. Mol. Biochem. Parasitol. 2000; 108: 199-206Crossref PubMed Scopus (42) Google Scholar, 30Temesvari L.A. Harris E.N. Stanley Jr., S.L. Cardelli J.A. Mol. Biochem. Parasitol. 1999; 103: 225-241Crossref PubMed Scopus (48) Google Scholar, 31Juarez P. Sanchez-Lopez R. Stock R.P. Olvera A. Ramos M.A. Alagon A. Mol. Biochem. Parasitol. 2001; 116: 223-228Crossref PubMed Scopus (23) Google Scholar) have reported about 20 EhRab genes. An additional 50 putative Rab genes showing significant homology to Rab from other organisms were found in the E. histolytica genome data base (data not shown, www.tigr.org). A few EhRab proteins have been shown to participate in phagocytosis. EhRabB was shown to be located on the plasma membrane and phagocytic mouths in the early phase (up to 5 min) of phagocytosis (29Rodriguez M.A. Garcia-Perez R.M. Garcia-Rivera G. Lopez-Reyes I. Mendoza L. Ortiz-Navarrete V. Orozco E. Mol. Biochem. Parasitol. 2000; 108: 199-206Crossref PubMed Scopus (42) Google Scholar). Putative EhRab7 and EhRab11 proteins were reported to be abundant in the endosome fraction labeled with iron-dextran, similar to their putative homologues from mammals (30Temesvari L.A. Harris E.N. Stanley Jr., S.L. Cardelli J.A. Mol. Biochem. Parasitol. 1999; 103: 225-241Crossref PubMed Scopus (48) Google Scholar). To dissect the molecular mechanism of Rab proteins involved in the phagosome biogenesis in Entamoeba, we characterized, in the present study, two amebic Rab proteins, EhRab5 and EhRab7A, that show significant homology to mammalian and yeast counterparts. The amoebic Rab5 homologue has several unique characteristics that are dissimilar to those of the mammalian and yeast Rab5. First, EhRab5 is primarily involved in phagocytosis, not endocytosis. Second, in contrast to mammalian Rab5, which is immediately recruited to phagosomes after the engulfment of bacteria or beads, EhRab5 is not recruited directly to phagosomes, but colocalizes with EhRab7A, forming prephagosomal vacuoles (PPV) prior to fusion with phagosomes. Third, EhRab5 is required for the formation of PPV and efficient engulfment of red blood cells. Fourth, EhRab5 plays an important role in the transport of the major membrane-permeabilizing peptide amoebapore. Therefore, in conjunction with EhRab7A, EhRab5 plays a key role in the biogenesis of phagosomes by regulating the formation of PPV and transport of membranolytic and hydrolytic factors during phagocytosis in this parasite. Organism and Culture—E. histolytica trophozoites of HM-I:IMSS cl 6 (32Diamond L.S. Mattern C.F. Bartgis I.L. J. Virol. 1972; 9: 326-341Crossref PubMed Google Scholar) were cultured axenically in BI-S-33 medium at 35 °C as described previously (33Diamond L.S. Harlow D.R. Cunnick C.C. Trans. R. Soc. Trop. Med. Hyg. 1978; 72: 431-432Abstract Full Text PDF PubMed Scopus (1574) Google Scholar). Isolation of EhRab5 and EhRab7A cDNAs—A full-length EhRab5 cDNA was obtained by a degenerate PCR approach, followed by 5′- and 3′-rapid amplification of cDNA ends as previously described (28Saito-Nakano Y. Nakazawa M. Shigeta Y. Takeuchi T. Nozaki T. Mol. Biochem. Parasitol. 2001; 116: 219-222Crossref PubMed Scopus (25) Google Scholar). A full-length EhRab7A gene was obtained by reverse transcriptase-PCR using oligonucleotide primers designed based on sequences previously reported (30Temesvari L.A. Harris E.N. Stanley Jr., S.L. Cardelli J.A. Mol. Biochem. Parasitol. 1999; 103: 225-241Crossref PubMed Scopus (48) Google Scholar, 34Tanaka T. Tanaka M. Mitsui Y. Biochem. Biophys. Res. Commun. 1997; 236: 611-615Crossref PubMed Scopus (24) Google Scholar). We identified at least eight genes showing significant homology to Rab7 from other species (data not shown). We designated the EhRab7 gene showing highest homology to mammalian and yeast Rab7 as EhRab7A in the present study and describe the characterization of other EhRab7 isotypes elsewhere. Plasmid Constructions to Produce Transgenic Amoeba Lines— EhRab5 and EhRab7A cDNA fragments were amplified by PCR using sense and antisense oligonucleotides containing appropriate restriction sites at the end. Three tandem repeats of hemaggulutinin (HA) or c-Myc tags, made of annealed complementary oligonucleotides, were inserted in the engineered NheI site, which was located at the fourth or second amino acid codon of EhRab5 or EhRab7A cDNA fragments, respectively (Fig. 1). An expression plasmid, pEhEx, contains the 5′-flanking region cysteine synthase gene (AB000266) containing a putative promoter (35Nozaki T. Asai T. Kobayashi S. Ikegami F. Noji M. Saito K. Takeuchi T. Mol. Biochem. Parasitol. 1998; 97: 33-44Crossref PubMed Scopus (83) Google Scholar), BglII and XhoI sites between cysteine synthase 5′- and 3′-flanking regions to insert a gene of interest, cysteine synthase 3′-flanking regions and neomycin resistance gene flanked by the 5′ and 3′ regions of actin gene, obtained from pA5′A3′NEO (36Hamann L. Buss H. Tannich E. Mol. Biochem. Parasitol. 1997; 84: 83-91Crossref PubMed Scopus (71) Google Scholar), for drug selection. The 3HA-EhRab5 cDNA fragment was inserted into the BglII-XhoI sites of pEhEx to produce pH5. For construction of a plasmid to co-express EhRab5 and EhRab7A (pH5-M7), a 1.7-kb fragment containing the 3Myc-EhRab7A protein-coding region flanked by cysteine synthase 5′ and 3′ regions was cloned into the SpeI site of pH5. EhRab5Q67L and EhRab5S22N mutants were constructed by PCR-mediated mutagenesis (37Landt O. Grunert H.P. Hahn U. Gene (Amst.). 1990; 96: 125-128Crossref PubMed Scopus (639) Google Scholar). Two EhRab5 mutants were fused with the 3-HA tag and cloned to pEhEx to produce pH5L or pH5N, respectively. Plasmids to co-express either EhRab5Q67L or EhRab5S22N and EhRab7A were constructed as described above (pH5L-M7 or pH5N-M7, respectively). A plasmid to express green fluorescent protein (GFP)-EhRab5 fusion protein in amoebae was constructed. GFP was amplified by PCR from GIR222 as a template (38Ramakrishnan G. Rogers J. Mann B.J. Petri Jr., W.A. Parasitol. Int. 2001; 50: 47-50Crossref PubMed Scopus (6) Google Scholar), and cloned into pKT-3M, which contained the cysteine synthase promoter, 3-Myc tag, and SmaI and XhoI restriction sites to produce pKT-MG. The EhRab5 protein coding region without the stop codon was ligated into SmaI-XhoI sites of pKT-MG to produce pKT-GFP5. Detailed information, e.g. nucleotide number based on sequences deposited in the data base and positions of inserted restriction sites and 3-HA or 3-Myc epitope, are also shown in Fig. 1B. Establishment of Epitope-tagged EhRab-expressing Amoeba Cell Lines—Wild-type trophozoites were transformed with plasmids by liposome-mediated transfection as previously described (39Nozaki T. Asai T. Sanchez L.B. Kobayashi S. Nakazawa M. Takeuchi T. J. Biol. Chem. 1999; 274: 32445-32452Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Transformants were initially selected in the presence of 3 μg/ml of Geneticin (Invitrogen). The Geneticin concentration was gradually increased to 6–20 μg/ml during the following 2 weeks before the transformants were subjected to analyses. Antibodies—Affinity purified anti-EhRab5 or anti-EhRab7A rabbit antibodies were commercially produced at Oriental Yeasts Co. (Tokyo, Japan) using recombinant amino-terminal glutathione S-transferase fusion proteins purified using glutathione-Sepharose 4B (Amersham Biosciences). Anti-HA 16B12 and anti-Myc 9E10 mouse monoclonal antibodies were purchased from Berkeley Antibody Co. (Berkeley, CA). Alexa Fluor anti-mouse and anti-rabbit IgG were obtained from Molecular Probes (Eugene, OR). Anti-amoebic CP2 and human band 3 rabbit antibodies were gifts from Iris Bruchhaus and Egbert Tannich (40Hellberg A. Leippe M. Bruchhaus I. Mol. Biochem. Parasitol. 2000; 105: 305-309Crossref PubMed Scopus (29) Google Scholar), and Yuichi Takakuwa (41Tomishige M. Sako Y. Kusumi A. J. Cell Biol. 1998; 142: 989-1000Crossref PubMed Scopus (167) Google Scholar), respectively. The production of anti-amoebapore A antibody was previously described (42Leippe M. Ebel S. Schoenberger O.L. Horstmann R.D. Muller-Eberhard H.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7659-7663Crossref PubMed Scopus (183) Google Scholar). Indirect Immunofluorescence—Amoeba transformants in a logarithmic growth phase were harvested and transferred to 8-mm round wells on glass slides and incubated for 30 min at 35 °C to let trophozoites attach to the glass surface. Gerbil red blood cells were added to each well at 107 cells/ml and incubated for 5–50 min at 35 °C. An indirect immunofluorescence assay was performed as follows. Amoebae were fixed with 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature. Ingested red blood cells were stained with diaminobenzidine (0.84 mm 3,3′-diaminobenzidine, 0.048% H2O2, and 50 mm Tris-HCl, pH 9.5) for 5 min (43Novikoff A.B. Novikoff P.M. Davis C. Quintana N. J. Histochem. Cytochem. 1972; 20: 1006-1023Crossref PubMed Scopus (185) Google Scholar). Cells were then permeabilized with 0.05% Triton X-100, PBS for 5 min. Samples were reacted with 16B12 (1:1000), 9E10 (1:400), anti-amoebapore A antibody (1: 1000), or affinity-purified anti-EhRab5, anti-EhRab7A, or CP2 antibody (1:200). In most experiments, we used a rabbit antibody raised against recombinant EhRab5, amoebapore, and CP, and anti-Myc mouse antibody to detect 3Myc-EhRab7A unless mentioned otherwise. The samples were then reacted with Alexa Fluor anti-mouse or anti-rabbit IgG (1:1000). The mouse monoclonal antibodies gave no background signal in the non-transformants because of nonspecific antibody binding under the conditions described above. For the staining of endosomal and lysosomal compartments, amoebae were pulsed with either 2 mg/ml FITC-dextran (Sigma) for 10 min or LysoTracker™ Red DND-99 (Molecular Probes) (1:500) for 12 h at 35 °C. Samples were examined on a Zeiss LSM510 confocal laser-scanning microscope. Images were further analyzed using LSM510 software. Time-lapse Microscopy—Amoeba transformants expressing GFP-EhRab5 were plated onto a 35-mm glass-bottom culture dish (D111100, Matsunami Glass Ind. Inc., Osaka, Japan) to settle amoebae at 30 °C. After the medium was removed, the glass chamber was enclosed by a glass coverslip. Time-lapse microscopy was performed with a Leica AS MDW system on a Leica DM IRE2 inverted microscope. Images of 18 slices (1.5 μm apart on the z-axis) were captured at 2.85-s intervals. This z-spacing was optimized to: 1) monitor the entire depth of amoebae from the top to the bottom, and 2) to accomplish fast capturing of a moving amoeba. Obtained raw images were further deconvoluted using Leica Deblur software. For each time point, images were three-dimensionally reconstituted and only a selected plane containing a PPV or a GFP-EhRab5-associated compartment was shown. Immunoelectron Microscopy—Immunoelectron microscopy was performed by pre-embedding labeling method (44Jaunin F. Burns K. Tschopp J. Martin T.E. Fakan S. Exp. Cell Res. 1998; 243: 67-75Crossref PubMed Scopus (36) Google Scholar). Amoebae were transferred to slide glass and incubated with red blood cells for 10 min as described above. Samples were prefixed with 3.7% paraformaldehyde, PBS for 20 min, and then incubated with 0.1 m glycine, PBS, and permeabilized with 0.1% Triton X-100. Samples were reacted with anti-amoebapore A (1:50), and subsequently with a goat anti-rabbit IgG conjugated with 5-nm gold (1:30). These cells were embedded into 2% soft agar, and further fixed with 0.1% OsO4, PBS for 30 min followed by dehydration, and embedded in Epon 812 (TAAB Laboratories Equipment LTD., UK). Ultrathin sections were made on an LKB-ultramicrotome (LKB-Produkter, Bromma, Sweden), and sections were stained with uranyl acetate and examined with a Hitachi-H-700 electron microscope. Measurement of FITC-dextran Uptake—Transformants were cultured in BI-S-33 medium containing 2 mg/ml of FITC-dextran for given periods at 35 °C. After the incubation, cells were washed three times with 6 ml of ice-cold PBS containing 2% glucose, and solubilized with 50 mm Tris-HCl, pH 7.0, containing 0.1% Nonidet P-40, and 10 μg/ml of trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64). Fluorescence emission at 520 nm was measured with excitation at 490 nm on a fluorescence spectrophotometer (VersaFluor Fluorometer, BioRad) and compared with standards of known concentrations. Identification of Entamoeba Homologues of Rab5 and Rab7 (EhRab5 and EhRab7A)—We isolated cDNAs coding for a putative homologue of Rab5 and Rab7, and designated them EhRab5 and EhRab7A, respectively. EhRab5 and EhRab7A showed 45 and 48% identity to mammalian Rab5 and Rab7, respectively. The effector region and α2 helix loop, which are important to the specificity of Rab proteins (45Stenmark H. Olkkonen V.M. Genome Biol. 2001; 2 (Reviews 3007)Crossref PubMed Google Scholar), were well conserved among mammalians, yeasts, and E. histolytica (Fig. 2). To examine whether the amoebic Rab5 and Rab7A play a role similar to that in other organisms, we attempted to rescue defects of a yeast Δypt51/Δvps21 mutant (46Singer-Kruger B. Stenmark H. Dusterhoft A. Philippsen P. Yoo J.S. Gallwitz D. Zerial M. J. Cell Biol. 1994; 125: 283-298Crossref PubMed Scopus (184) Google Scholar, 47Horazdovsky B.F. Busch G.R. Emr S.D. EMBO J. 1994; 13: 1297-1309Crossref PubMed Scopus (168) Google Scholar) and Δypt7 mutant (14Wichmann H. Hengst L. Gallwitz D. Cell. 1992; 71: 1131-1142Abstract Full Text PDF PubMed Scopus (203) Google Scholar) through ectopic expression of EhRab5 and EhRab7A, respectively. Overexpression of EhRab5 on a single-copy plasmid under the regulation of a GAL1 promoter did not complement either the fragmented vacuole morphology or a temperature-sensitive growth defect in Δypt51/Δvps21 cells (data not shown). Neither did overexpression of EhRab7A in the Δypt7 mutant rescue vacuole fragmentation (data not shown). These results indicate that amoebic Rab5 and Rab7A play a role distinct from that of yeast Ypt51p and Ypt7p. Dynamics of EhRab5 and EhRab7A during Phagocytosis and Identification of Unique PPV Associated with EhRab5 and EhRab7A—We examined the subcellular localization of EhRab5 and EhRab7A during phagocytosis of red blood cells. We constructed a stable transformant that constitutively expressed an 3HA-tagged EhRab5 and a 3Myc-tagged EhRab7A. EhRab5 and EhRab7A were estimated to be overexpressed by 3–5- and 1.5–2-fold, respectively, in the transformant when compared with wild-type cells by quantitation of immunoblots using an antibody raised against recombinant EhRab5 and EhRab7A (data not shown). Neither expression of the epitopetagged EhRab5 alone nor co-expression of both epitope-tagged EhRab5 and EhRab7A affected cell growth or morphology (see below and Fig. 8A). Immunofluorescence imaging using anti-EhRab5 and anti-Myc antibody, the latter of which reacts with 3Myc-tagged EhRab7A, showed that, at steady state (i.e. without red blood cells), EhRab5 and EhRab7A were localized to small non-overlapping vesicles throughout the cytoplasm (Fig. 3, A–D). The distribution of EhRab5 and EhRab7A dramatically changed upon incubation with red blood cells. After 5 min, large vacuoles (4.0 ± 0.9 μm in diameter) that colocalized with both EhRab5 and EhRab7A emerged (Fig. 3, E–H). At 10 min, EhRab5 began to dissociate from some of these vacuoles, whereas EhRab7A remained associated with them (Fig. 3, I–L). These EhRab5/EhRab7A-positive vacuoles also formed in the amoebae that did not ingest red blood cells (a trophozoite in Fig. 3, E–H, and a trophozoite on the right in Fig. 3, I–L). We designated these vacuoles PPV as this compartment emerged prior to fusion with phagosomes (see below). At 30 min, when the amoebae ingested an average of 3–4 red blood cells per cell, EhRab5/EhRab7A double-positive PPV disappeared and EhRab5 dispersed into the cytosol as seen at steady state. Approximately 40% of phagocytosed red blood cells were surrounded by EhRab7A (Fig. 3, N, P, and Q). EhRab5 was not localized to phagosomes containing red blood cells at any time point (Fig. 3, A, E, I, and M), which is in good contrast to the dynamics shown for mammalian Rab5 in macrophages, where phagosomes are simultaneously associated with both Rab5 and Rab7 (4Desjardins M. Celis J.E. van Meer G. Dieplinger H. Jahraus A. Griffiths G. Huber L.A. J. Biol. Chem. 1994; 269: 32194-32200Abstract Full Text PDF PubMed Google Scholar). To unequivocally demonstrate the dynamics of the maturation of PPV and phagosomes, we counted (i) EhRab5/EhRab7A double-positive PPV, (ii) EhRab7A single-positive PPV, (iii) EhRab7A positive phagosomes, and (iv) EhRab7A negative phagosomes (Fig. 3Q). The number of these vacuoles per cell changed during the course of phagocytosis. The number of EhRab5/EhRab7A double-positive PPV peaked at 5 min and gradually decreased after 10 min, whereas the number of EhRab7A single-positive PPV increased at 5–10 min, and remained elevated up to 30 min. The proportion of EhRab5/EhRab7A double-positive PPV among all PPV (i.e. i/(i + ii)) sharply decreased between 5 and 30 min (78, 37, and 8% at 5, 10, and 30 min, respectively). The number of phagosomes increased linearly during 30 min (0.8 per cell at 5 min to 5.3 per cell at 30 min). However, the proportion of EhRab7A-positive phagosomes among all phagosomes (i.e. iii/(iii + iv)) did not significantly change during the course (30–40%). These results support the following model: 1) upon interaction with red blood cells, EhRab5/EhRab7A double-positive PPV forms; 2) EhRab5 is dissociated from EhRab5/EhRab7A double-positive PPV; 3) EhRab7A is subsequently targeted to phagosomes; and 4) EhRab7A is finally dissociated from phagosomes. We also verified that PPV was not an artifactually misi
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