Rab5-associated Vacuoles Play a Unique Role in Phagocytosis of ...

Rab5-associated Vacuoles Play a Unique Role in Phagocytosis of theEnteric Protozoan Parasite Entamoeba histolytica*

Received for publication, April 4, 2004, and in revised form, August 27, 2004Published, JBC Papers in Press, September 3, 2004, DOI 10.1074/jbc.M403987200

Yumiko Saito-Nakano‡, Tomoyoshi Yasuda‡, Kumiko Nakada-Tsukui‡, Matthias Leippe§,and Tomoyoshi Nozaki‡¶�

From the ‡Department of Parasitology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku,Tokyo 162-8640, Japan, the §Zoologisches Institut der Christian-Albrechts-Universitat Kiel, Olshausenstrasse 40,24118 Kiel, Germany, and the ¶Precursory Research for Embryonic Science and Technology, Japan Science andTechnology Agency, 2-20-5 Akebono-cho, Tachikawa, Tokyo 190-0012, Japan

In mammals, Rab5 and Rab7 play a specific and coor-dinated role in a sequential process during phagosomematuration. Here, we report that Rab5 and Rab7 in theenteric protozoan parasite Entamoeba histolytica,EhRab5 and EhRab7A, are involved in steps that aredistinct from those known for mammals. EhRab5 andEhRab7A were localized to independent small vesicularstructures at steady state. Priming with red blood cellsinduced the formation of large vacuoles associated withboth EhRab5 and EhRab7A (“prephagosomal vacuoles(PPV)”) in the amoeba within an incubation period of5–10 min. PPV emerged de novo physically and distinctfrom phagosomes. PPV were gradually acidified and ma-tured by fusion with lysosomes containing a digestivehydrolase, cysteine proteinase, and a membrane-perme-abilizing peptide amoebapore. After EhRab5 dissociatedfrom PPV, 5–10 min later, the EhRab7A-PPV fused withphagosomes, and EhRab7A finally dissociated from thephagosomes. Immunoelectron and light micrographsshowed that PPV contained small vesicle-like structurescontaining fluid-phase markers and amoebapores,which were not evenly distributed within PPV, suggest-ing that the mechanism was similar to multivesicularbody formation in PPV generation. In contrast to Rab5from other organisms, EhRab5 was involved exclusivelyin phagocytosis, but not in endocytosis. Overexpressionof wild-type EhRab5 enhanced phagocytosis and thetransport of amoebapore to phagosomes. Conversely, ex-pression of an EhRab5Q67L GTP form mutant impairedthe formation of PPV and phagocytosis. Altogether, wepropose that the amoebic Rab5 plays an important rolein the formation of unique vacuoles, which is essential

for engulfment of erythrocytes and important for pack-aging of lysosomal hydrolases, prior to the targeting tophagosomes.

Phagocytosis is a critically important element of host defenseagainst invading pathogens in higher organisms and its molec-ular mechanism in professional phagocytes, e.g. macrophage,has been extensively studied at the molecular level (1, 2). Anumber of steps including cell surface binding to ligands andthe activation of a signaling pathway leading to F-actin polym-erization have been identified as essential for phagocytosis. Inaddition, membrane trafficking plays an important role in thecontrolled maturation of phagosomes. The maturation is ac-companied by sequential fusion with the endocytic compart-ment to form a phagolysosome, and is orchestrated by smallGTPase, Rab proteins, which act as molecular switches regu-lating the fusion of vesicles with target membranes through theconformational change between active (GTP-bound) and inac-tive (GDP-bound) forms (3). It has been reported that Rab5 andRab7 play an important role in the maturation of phagosomesin macrophages (4).

Rab5 was initially shown to be localized to early endosomesand the plasma membrane, and involved in endocytosis and theendosome fusion (5, 6). Rab5 was also observed on nascentphagosomes, and has been implicated to play an important rolein the fusion between phagosomes and early endosomes (7–9).Expression of the GTP form Rab5Q67L mutant or down-regu-lation of wild-type Rab5 by antisense oligonucleotides per-turbed the regulated fusion between phagosomes and endo-somes, and resulted in the formation of giant phagosomes inthe former case, and reduced activity for killing of ingestedbacteria because of the inhibition of phagosome maturation inthe latter case (8, 9). In addition to Rab5 per se, some of theRab5 effectors that were implicated in endosome fusion, e.g.EEA1 and phosphatidylinositol 3-kinase (Vps34) (10, 11), alsohave been identified on the phagosome membrane, suggestingthat phosphoinositide metabolism is important for phagosomematuration as seen in the endocytic pathway (12, 13). Rab7 hasbeen implicated in late endosomal membrane trafficking in theendocytic pathway (14), and also in the late stage of phagosomematuration (4, 13). Although a specific role for Rab7 duringphagocytosis has not yet been well demonstrated, some intra-cellular microorganisms have been reported to be capable ofblocking the maturation and acidification of phagosomes byinterfering with Rab7 (15, 16). It has also been recently dem-onstrated that a novel effector protein, RILP, is recruited to thephagosomal membrane by Rab7, which promotes fusion be-tween phagosomes and lysosomes (17).

* This work was supported in part by a grant for Precursory Researchfor Embryonic Science and Technology (PRESTO), Japan Science andTechnology Agency, Grant-in-aid for Scientific Research 15790219 (toY. S.-N.) and 15019120 and 15590378 (to T. N.) from the Ministry ofEducation, Culture, Sports, Science and Technology of Japan, a grantfor Research on Emerging and Re-emerging Infectious Diseases fromthe Ministry of Health, Labor, and Welfare, a grant for the Project toPromote Development of Anti-AIDS Pharmaceuticals from the JapanHealth Sciences Foundation (to T. N.), and a grant from the DeutscheForschungsgemeinschaft (to M. L.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AB054582(EhRab5) and AB054583 (EhRab7A).

� To whom all correspondence should be addressed: Dept. of Parasi-tology, National Institute of Infectious Diseases, 1-23-1 Toyama,Shinjuku-ku, Tokyo 162-8640, Japan. Tel.: 81-3-5285-1111 (ext. 2733),Fax: 81-3-5285-1173; E-mail: [email protected] and [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 47, Issue of November 19, pp. 49497–49507, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 49497

Besides professional phagocytes from higher eukaryotes,some unicellular organisms such as Dictyostelium discoideumand Entamoeba histolytica show an inherent ability of phago-cytosis. E. histolytica, an enteric protozoan parasite that causesan estimated 50 million cases of amebiasis: amebic colitis,dysentery, and extraintestinal abscesses (18), and 40,000–100,000 deaths annually (19), colonizes the human gut andengulfs foreign cells including microorganisms and host cells.Phagocytosis has been implicated to be closely associated withthe pathogenesis of the amoeba because phagocytosis-deficientamoeba mutants were shown to be avirulent (20). Although anumber of amoebic molecules involved in attachment, phago-cytosis, and degradation of microorganisms and host cells havebeen identified including galactose/N-acetylgalactosamine(Gal/GalNAc)-inhibitable lectin (21, 22), cytoskeletal proteinsand their associated regulatory molecules (23–25), cysteineproteinases (CP),1 and pore-forming peptides (i.e. amoeba-pores) (26, 27), the molecular mechanism of phagocytosis inthis parasite remains largely unknown.

We presumed that Rab proteins also play an essential andcentral role in the regulation of phagocytosis and endocytosis inE. histolytica. We and other groups (28–31) have reportedabout 20 EhRab genes. An additional 50 putative Rab genesshowing significant homology to Rab from other organismswere found in the E. histolytica genome data base (data notshown, www.tigr.org). A few EhRab proteins have been shownto participate in phagocytosis. EhRabB was shown to be locatedon the plasma membrane and phagocytic mouths in the earlyphase (up to 5 min) of phagocytosis (29). Putative EhRab7 andEhRab11 proteins were reported to be abundant in the endo-some fraction labeled with iron-dextran, similar to their puta-tive homologues from mammals (30). To dissect the molecularmechanism of Rab proteins involved in the phagosome biogen-esis in Entamoeba, we characterized, in the present study, twoamebic Rab proteins, EhRab5 and EhRab7A, that show signif-icant homology to mammalian and yeast counterparts. Theamoebic Rab5 homologue has several unique characteristicsthat are dissimilar to those of the mammalian and yeast Rab5.First, EhRab5 is primarily involved in phagocytosis, not endo-cytosis. Second, in contrast to mammalian Rab5, which is im-mediately recruited to phagosomes after the engulfment ofbacteria or beads, EhRab5 is not recruited directly to phago-somes, but colocalizes with EhRab7A, forming prephagosomalvacuoles (PPV) prior to fusion with phagosomes. Third,EhRab5 is required for the formation of PPV and efficientengulfment of red blood cells. Fourth, EhRab5 plays an impor-tant role in the transport of the major membrane-permeabiliz-ing peptide amoebapore. Therefore, in conjunction withEhRab7A, EhRab5 plays a key role in the biogenesis of phago-somes by regulating the formation of PPV and transport ofmembranolytic and hydrolytic factors during phagocytosis inthis parasite.

EXPERIMENTAL PROCEDURES

Organism and Culture—E. histolytica trophozoites of HM-I:IMSS cl6 (32) were cultured axenically in BI-S-33 medium at 35 °C as describedpreviously (33).

Isolation of EhRab5 and EhRab7A cDNAs—A full-length EhRab5cDNA was obtained by a degenerate PCR approach, followed by 5�- and3�-rapid amplification of cDNA ends as previously described (28). Afull-length EhRab7A gene was obtained by reverse transcriptase-PCRusing oligonucleotide primers designed based on sequences previouslyreported (30, 34). We identified at least eight genes showing significant

homology to Rab7 from other species (data not shown). We designatedthe EhRab7 gene showing highest homology to mammalian and yeastRab7 as EhRab7A in the present study and describe the characteriza-tion of other EhRab7 isotypes elsewhere.

Plasmid Constructions to Produce Transgenic Amoeba Lines—EhRab5 and EhRab7A cDNA fragments were amplified by PCR usingsense and antisense oligonucleotides containing appropriate restrictionsites at the end. Three tandem repeats of hemaggulutinin (HA) or c-Myctags, made of annealed complementary oligonucleotides, were insertedin the engineered NheI site, which was located at the fourth or secondamino acid codon of EhRab5 or EhRab7A cDNA fragments, respectively(Fig. 1). An expression plasmid, pEhEx, contains the 5�-flanking regioncysteine synthase gene (AB000266) containing a putative promoter(35), BglII and XhoI sites between cysteine synthase 5�- and 3�-flankingregions to insert a gene of interest, cysteine synthase 3�-flanking re-gions and neomycin resistance gene flanked by the 5� and 3� regions ofactin gene, obtained from pA5�A3�NEO (36), for drug selection. The3HA-EhRab5 cDNA fragment was inserted into the BglII-XhoI sites ofpEhEx to produce pH5. For construction of a plasmid to co-expressEhRab5 and EhRab7A (pH5-M7), a 1.7-kb fragment containing the3Myc-EhRab7A protein-coding region flanked by cysteine synthase 5�and 3� regions was cloned into the SpeI site of pH5. EhRab5Q67L andEhRab5S22N mutants were constructed by PCR-mediated mutagenesis(37). Two EhRab5 mutants were fused with the 3-HA tag and cloned topEhEx to produce pH5L or pH5N, respectively. Plasmids to co-expresseither EhRab5Q67L or EhRab5S22N and EhRab7A were constructedas described above (pH5L-M7 or pH5N-M7, respectively). A plasmid toexpress green fluorescent protein (GFP)-EhRab5 fusion protein inamoebae was constructed. GFP was amplified by PCR from GIR222 asa template (38), and cloned into pKT-3M, which contained the cysteinesynthase promoter, 3-Myc tag, and SmaI and XhoI restriction sites toproduce pKT-MG. The EhRab5 protein coding region without the stopcodon was ligated into SmaI-XhoI sites of pKT-MG to produce pKT-GFP5. Detailed information, e.g. nucleotide number based on sequencesdeposited in the data base and positions of inserted restriction sites and3-HA or 3-Myc epitope, are also shown in Fig. 1B.

Establishment of Epitope-tagged EhRab-expressing Amoeba CellLines—Wild-type trophozoites were transformed with plasmids by lipo-some-mediated transfection as previously described (39). Transfor-mants were initially selected in the presence of 3 �g/ml of Geneticin(Invitrogen). The Geneticin concentration was gradually increased to6–20 �g/ml during the following 2 weeks before the transformants weresubjected to analyses.

Antibodies—Affinity purified anti-EhRab5 or anti-EhRab7A rabbitantibodies were commercially produced at Oriental Yeasts Co. (Tokyo,Japan) using recombinant amino-terminal glutathione S-transferasefusion proteins purified using glutathione-Sepharose 4B (AmershamBiosciences). Anti-HA 16B12 and anti-Myc 9E10 mouse monoclonalantibodies were purchased from Berkeley Antibody Co. (Berkeley, CA).Alexa Fluor anti-mouse and anti-rabbit IgG were obtained from Molec-ular Probes (Eugene, OR). Anti-amoebic CP2 and human band 3 rabbitantibodies were gifts from Iris Bruchhaus and Egbert Tannich (40), andYuichi Takakuwa (41), respectively. The production of anti-amoebaporeA antibody was previously described (42).

Indirect Immunofluorescence—Amoeba transformants in a logarith-mic growth phase were harvested and transferred to 8-mm round wellson glass slides and incubated for 30 min at 35 °C to let trophozoitesattach to the glass surface. Gerbil red blood cells were added to eachwell at 107 cells/ml and incubated for 5–50 min at 35 °C. An indirectimmunofluorescence assay was performed as follows. Amoebae werefixed with 3.7% paraformaldehyde in phosphate-buffered saline (PBS)for 10 min at room temperature. Ingested red blood cells were stainedwith diaminobenzidine (0.84 mM 3,3�-diaminobenzidine, 0.048% H2O2,and 50 mM Tris-HCl, pH 9.5) for 5 min (43). Cells were then permeabi-lized with 0.05% Triton X-100, PBS for 5 min. Samples were reactedwith 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 againstrecombinant EhRab5, amoebapore, and CP, and anti-Myc mouse anti-body to detect 3Myc-EhRab7A unless mentioned otherwise. The sam-ples were then reacted with Alexa Fluor anti-mouse or anti-rabbit IgG(1:1000). The mouse monoclonal antibodies gave no background signalin the non-transformants because of nonspecific antibody binding underthe conditions described above. For the staining of endosomal andlysosomal compartments, amoebae were pulsed with either 2 mg/mlFITC-dextran (Sigma) for 10 min or LysoTrackerTM Red DND-99 (Mo-lecular Probes) (1:500) for 12 h at 35 °C. Samples were examined on a

1 The abbreviations used are: CP, cysteine proteinase; FITC, fluores-cein isothiocyanate; PPV, prephagosomal vacuole; GFP, green fluores-cent protein; PBS, phosphate-buffered saline; HA, hemagglutinin;EhRab5, Entamoeba histolytica Rab5; EhRab7A, Entamoeba histolyticaRab7A.

Entamoeba Rab5 in Phagocytosis49498

Zeiss LSM510 confocal laser-scanning microscope. Images were furtheranalyzed 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 aglass coverslip. Time-lapse microscopy was performed with a Leica ASMDW system on a Leica DM IRE2 inverted microscope. Images of 18slices (1.5 �m apart on the z-axis) were captured at 2.85-s intervals.This z-spacing was optimized to: 1) monitor the entire depth of amoebaefrom the top to the bottom, and 2) to accomplish fast capturing of amoving amoeba. Obtained raw images were further deconvoluted usingLeica Deblur software. For each time point, images were three-dimen-sionally reconstituted and only a selected plane containing a PPV or aGFP-EhRab5-associated compartment was shown.

Immunoelectron Microscopy—Immunoelectron microscopy was per-

formed by pre-embedding labeling method (44). Amoebae were trans-ferred to slide glass and incubated with red blood cells for 10 min asdescribed above. Samples were prefixed with 3.7% paraformaldehyde,PBS for 20 min, and then incubated with 0.1 M glycine, PBS, andpermeabilized with 0.1% Triton X-100. Samples were reacted withanti-amoebapore A (1:50), and subsequently with a goat anti-rabbit IgGconjugated 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 bydehydration, and embedded in Epon 812 (TAAB Laboratories Equip-ment LTD., UK). Ultrathin sections were made on an LKB-ultramic-rotome (LKB-Produkter, Bromma, Sweden), and sections were stainedwith uranyl acetate and examined with a Hitachi-H-700 electronmicroscope.

Measurement of FITC-dextran Uptake—Transformants were cul-tured in BI-S-33 medium containing 2 mg/ml of FITC-dextran for givenperiods at 35 °C. After the incubation, cells were washed three times

FIG. 1. Plasmids used to express epitope-tagged EhRab5 and EhRab7A in E. histolytica. A, construction and schematic representationof the plasmids. All plasmids shown are derivatives of pBluescript KS II�. CS5�, CS3�, Act5�, Act3�, or Neo, 5� upstream or 3� downstream fromthe cysteine synthase gene, 5� upstream or 3� downstream from the actin gene, or the neomycin resistance gene, respectively. Only representativeconstructs to express wild-type EhRab5 and EhRab7 and GFP are shown. B, nucleotide and amino acid sequences of selected regions of theexpression cassette for EhRab5, EhRab7A, and GFP are shown. Nucleotide (nt) number of genes deposited under accession numbers (inparentheses) is shown. Amino acid sequences are shown above nucleotide sequences. (A S), (ASSKKKPL), or (P G) depict inserted amino acidsbecause of engineered restriction sites shown below the nucleotide sequences. An asterisk (*) depicts the stop codon. Restriction sites areunderlined. EhRab5, EhRab7A, and GFP open reading frame are italicized.

Entamoeba Rab5 in Phagocytosis 49499

with 6 ml of ice-cold PBS containing 2% glucose, and solubilized with 50mM Tris-HCl, pH 7.0, containing 0.1% Nonidet P-40, and 10 �g/ml oftrans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64). Fluo-rescence emission at 520 nm was measured with excitation at 490 nmon a fluorescence spectrophotometer (VersaFluor Fluorometer, Bio-Rad) and compared with standards of known concentrations.

RESULTS

Identification of Entamoeba Homologues of Rab5 and Rab7(EhRab5 and EhRab7A)—We isolated cDNAs coding for a pu-tative homologue of Rab5 and Rab7, and designated themEhRab5 and EhRab7A, respectively. EhRab5 and EhRab7Ashowed 45 and 48% identity to mammalian Rab5 and Rab7,respectively. The effector region and �2 helix loop, which areimportant to the specificity of Rab proteins (45), were wellconserved among mammalians, yeasts, and E. histolytica(Fig. 2).

To examine whether the amoebic Rab5 and Rab7A play arole similar to that in other organisms, we attempted to rescuedefects of a yeast �ypt51/�vps21 mutant (46, 47) and �ypt7mutant (14) through ectopic expression of EhRab5 andEhRab7A, respectively. Overexpression of EhRab5 on a single-copy plasmid under the regulation of a GAL1 promoter did notcomplement either the fragmented vacuole morphology or a

temperature-sensitive growth defect in �ypt51/�vps21 cells(data not shown). Neither did overexpression of EhRab7A inthe �ypt7 mutant rescue vacuole fragmentation (data notshown). These results indicate that amoebic Rab5 and Rab7Aplay a role distinct from that of yeast Ypt51p and Ypt7p.

Dynamics of EhRab5 and EhRab7A during Phagocytosis andIdentification of Unique PPV Associated with EhRab5 andEhRab7A—We examined the subcellular localization ofEhRab5 and EhRab7A during phagocytosis of red blood cells.We constructed a stable transformant that constitutively ex-pressed an 3HA-tagged EhRab5 and a 3Myc-tagged EhRab7A.EhRab5 and EhRab7A were estimated to be overexpressed by3–5- and 1.5–2-fold, respectively, in the transformant whencompared with wild-type cells by quantitation of immunoblotsusing an antibody raised against recombinant EhRab5 andEhRab7A (data not shown). Neither expression of the epitope-tagged EhRab5 alone nor co-expression of both epitope-taggedEhRab5 and EhRab7A affected cell growth or morphology (seebelow and Fig. 8A).

Immunofluorescence imaging using anti-EhRab5 and anti-Myc antibody, the latter of which reacts with 3Myc-taggedEhRab7A, showed that, at steady state (i.e. without red bloodcells), EhRab5 and EhRab7A were localized to small non-over-lapping vesicles throughout the cytoplasm (Fig. 3, A–D). Thedistribution of EhRab5 and EhRab7A dramatically changedupon incubation with red blood cells. After 5 min, large vacu-oles (4.0 � 0.9 �m in diameter) that colocalized with bothEhRab5 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 theamoebae that did not ingest red blood cells (a trophozoite inFig. 3, E–H, and a trophozoite on the right in Fig. 3, I–L). Wedesignated these vacuoles PPV as this compartment emergedprior to fusion with phagosomes (see below). At 30 min, whenthe amoebae ingested an average of 3–4 red blood cells per cell,EhRab5/EhRab7A double-positive PPV disappeared andEhRab5 dispersed into the cytosol as seen at steady state.Approximately 40% of phagocytosed red blood cells were sur-rounded by EhRab7A (Fig. 3, N, P, and Q). EhRab5 was notlocalized to phagosomes containing red blood cells at any timepoint (Fig. 3, A, E, I, and M), which is in good contrast to thedynamics shown for mammalian Rab5 in macrophages, wherephagosomes are simultaneously associated with both Rab5 andRab7 (4).

To unequivocally demonstrate the dynamics of the matura-tion of PPV and phagosomes, we counted (i) EhRab5/EhRab7Adouble-positive PPV, (ii) EhRab7A single-positive PPV, (iii)EhRab7A positive phagosomes, and (iv) EhRab7A negativephagosomes (Fig. 3Q). The number of these vacuoles per cellchanged during the course of phagocytosis. The number ofEhRab5/EhRab7A double-positive PPV peaked at 5 min andgradually decreased after 10 min, whereas the number ofEhRab7A single-positive PPV increased at 5–10 min, and re-mained 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 in-creased linearly during 30 min (0.8 per cell at 5 min to 5.3 percell at 30 min). However, the proportion of EhRab7A-positivephagosomes among all phagosomes (i.e. iii/(iii � iv)) did notsignificantly change during the course (30–40%). These resultssupport the following model: 1) upon interaction with red bloodcells, EhRab5/EhRab7A double-positive PPV forms; 2) EhRab5is dissociated from EhRab5/EhRab7A double-positive PPV; 3)

FIG. 2. Comparison of amino acid sequences of Rab5 and Rab7from E. histolytica, human, and yeast. A, sequence alignment ofEhRab5, Homo sapiens Rab5a, and Saccharomyces cerevisiae Ypt51p.B, sequence alignment of EhRab7A, H. sapiens Rab7, and S. cerevisiaeYpt7p. Sequences were aligned by DNASIS (Hitachi Software Engi-neering Co.). Amino acid residues conserved among at least two speciesare shown in reverse type. The GTP-binding consensus sequences, theeffector region, and the �2 helix are depicted by gray bars, black bars,and double lines, respectively, below the sequences. Computer-gener-ated gaps are shown as dashes.


EhRab7A is subsequently targeted to phagosomes; and 4)EhRab7A is finally dissociated from phagosomes.

We also verified that PPV was not an artifactually misiden-tified phagosome, i.e. the phagosome that contains debris of redblood cells, but is not stained by diaminobenzidine because ofloss of its content. To exclude this possibility, we used anantibody raised against a major component of the membranecytoskeleton of red blood cells, band 3 (48). The anti-band 3antibody clearly reacted with red blood cells in phagosomes

(Fig. 3V, red, arrow), whereas none of the EhRab5-positive PPVwas reacted with the antibody (green, arrowheads). In addition,localization of red blood cells by diaminobenzidine staining oranti-band 3 antibody agreed very well (Fig. 3, V and W). Theseresults clearly showed that PPV are distinct from phagosomes.The PPV formation was not a secondary defect caused by ex-pression of epitope-tagged EhRab5 and EhRab7A because itwas also observed in wild-type amoebae at a comparable fre-quency, as detected by the antibodies raised against recombi-

FIG. 3. Subcellular localization ofEhRab5 and EhRab7A changed dur-ing red blood cell phagocytosis. A–P,subcellular localization of EhRab5 andEhRab7A was examined by immuno-fluorescence assay using the amoebatransformant co-expressing 3HA-taggedEhRab5 and 3Myc-tagged EhRab7A inthe absence of red blood cells (A–D), orafter 5 (E–H), 10 (I–L), and 30 min (M–P)incubation with red blood cells. Localiza-tion of EhRab5 and EhRab7A was exam-ined with anti-EhRab5 antibody (green;A, E, I, and M) and anti-Myc monoclonalantibody (red; B, F, J, and N), respec-tively. Merged images of EhRab5 andEhRab7A (C, G, K, and O) and phase-contrast images under transmission light(D, H, L, and P) are also shown. Largearrowheads show EhRab5/EhRab7A-containing PPV (E–K). Small arrowheads(N–P) show EhRab7A-positive phago-somes. Thick arrows (J, K, N, O, and P)indicate EhRab7A-PPV, not associatedwith EhRab5. A thin arrow (L) indicatesan engulfed red blood cell associated withneither EhRab5 nor EhRab7A. Q, quanti-tative analysis of EhRab5 and EhRab7Alocalization to PPV and phagosomes dur-ing erythrophagocytosis. The number ofEhRab5/EhRab7A double-positive PPV(open bars; also marked as 5/7A-PPV),EhRab7A single-positive PPV (gray bars;7A-PPV), EhRab7A-positive phagosomes(hatched bars; 7A-phagosome), andEhRab7A-negative phagosomes (filledbars; phagosome) per cell is shown at 5,10, and 30 min after the addition of redblood cells. R–U, subcellular localizationof EhRab7A was examined by immunoflu-orescence assay using wild-type amoebaeand anti-EhRab7A antibody in the ab-sence of red blood cells (R and S) or aftera 10-min (T and U) incubation with redblood cells. Panels S and U show phaseimages of panels R and T, respectively.Arrowheads in T and U depict PPV. V andW, three-dimensional sections of theamoeba containing red blood cell showingthe presence of red blood cells in phago-somes, but not in PPV. Localization ofPPV and red blood cells was examinedwith anti-EhRab5 antibody (green, arrow-heads) and anti-band 3 antibody (arrows,red), respectively. Among 17 z-sections(1-�m intervals) obtained with confocallaser scanning microscopy, only one rep-resentative xy section, together with se-lected xz (green line), and yz (red line)sections, are shown. W shows a phase im-age of V.


nant EhRab5 and EhRab7A (Fig. 3, R-U; data of EhRab5 notshown).

EhRab5 Is Not Associated with Endosomes or Lysosomes butExhibits Cross-talk with These Compartments during Matura-tion of PPV—To see whether EhRab5 is associated with endo-somes, we examined colocalization of an endocytosed fluid-phasemarker, FITC-dextran, and EhRab5. Amoebae were either incu-bated with FITC-dextran for 10 min to label the early endosomesor incubated with FITC-dextran for 10 min and further chasedwithout FITC-dextran for 45 min to label the late endosomes, andthen subjected to immunofluorescence assay using anti-HA an-tibody that recognizes EhRab5 (Fig. 4A). Endocytosed FITC-dextran and EhRab5 did not colocalize at either 10-min pulse(Fig. 4A) or at the 10-min pulse followed by a 45-min chase (datanot shown). These findings imply that the EhRab5-positive com-partment is neither early nor late endosomes.

When amoebae were simultaneously incubated with redblood cells and FITC-dextran for 10 min, 30% of PPV containedendocytosed FITC-dextran (Fig. 4B). When the amoebae werepulsed with FITC-dextran for 10 min and chased for 45 min tolabel the late endosomes, and further incubated with red bloodcells for 10 min, the extent of colocalization of FITC-dextranand PPV was comparable (26%) (data not shown). These resultssuggest that PPV fuse with both early and late endosomesduring maturation.

To assess where and how PPV are formed during phagocy-tosis, we examined the dynamics of EhRab5 using the amoebatransformant expressing GFP-EhRab5 under time-lapse mi-croscopy. Images of 18 planes of the z-section with 1.5-�mintervals to cover from the top to the bottom of the cell wererecorded at 2.85-s intervals. This allowed us to evaluate thedetailed dynamism of PPV formation. After a few minutes ofcoincubation with red blood cells, an EhRab5-positive vacuolesuddenly emerged in less than 20 s. Neither plasma membraneinvagination nor ruffling were observed during this period,suggesting that PPV forms de novo (Fig. 5).

We also excluded a possibility that PPVs are micropinosomesor phagosomes. First, the fact that only a minor proportion ofPPV contained FITC-dextran at 10 min (Fig. 4B) suggests thatPPVs are not formed by invagination of the plasma membrane-

like macropinosomes, which form by the closure of membraneruffles and contain a fluid-phase marker (49, 50). Second, PPVis formed in a range of 10 s (Fig. 5), much faster than macropi-nosomes or phagosomes (49, 51). Membrane closure of macropi-nosomes and phagosomes was previously shown to occur in 1and 5 min, respectively. Third, the major Gal/GalNAc lectin onthe plasma membrane was abundantly demonstrated in pha-gosomes by proteomic analysis of phagosome proteins duringthe course of phagosome maturation (from 0 min to 2 h afteringestion)2 but was not demonstrated on PPV by immunofluo-rescence study using a specific monoclonal antibody againstheavy or intermediate lectin subunits (data not shown). Theseresults strongly argue against two possibilities: 1) PPV origi-nates from the plasma membrane, and 2) PPV is a remnant ofphagosomes.

Acidification of phagosomes has been shown to occur byfusion with late endosomes and lysosomes in mammalian cells(52). We examined by using LysoTracker Red, a membrane-diffusible probe accumulated in acidic organelles (53), whetherthe PPV and phagosomes of the amoeba are acidified duringmaturation. Amoebae were pulsed with LysoTracker and thensubjected to immunofluorescence assay. At steady state, nei-ther EhRab5 (Fig. 6A, left) nor EhRab7A (Fig. 6A, right),probed with anti-HA or anti-Myc antibody, respectively, colo-calized with LysoTracker. After a 5–10-min incubation withred blood cells when EhRab7A-positive PPV were formed, only20–30% of PPV contained LysoTracker, suggesting that PPVwere only partially acidified in the early stage (Fig. 6, B, upperpanels, and C, data at 5 min not shown). After 30–40 min, alarge proportion (50–70%) of PPV became acidified (Fig. 6, B,lower panels, and C).

PPV Are Involved in the Transport of Amoebapore to Phago-somes—We then examined which cargo proteins were trans-ported via PPV. Among several hydrolases and membrane-permeabilizing factors involved in the degradation ofinternalized host cells and microorganisms, e.g. CP (26), amoe-bapores (27), lysozyme (54), and phospholipases (55), we testedwhether amoebapore A and CP2 were transported to phago-somes via PPV. Immunostaining of amoebapore and CP2 usingspecific antisera showed similar patterns to those obtainedwith LysoTracker in the absence of red blood cells (Fig. 7A, 0

2 M. Okada, C. D. Huston, B. J. Mann, W. A. Petri, Jr., K. Kita, andT. Nozaki, submitted for publication.

FIG. 4. Immunofluorescent micrographs showing cross-talkbetween early endosomes and PPV. A, the amoebae were pulsedwith 2 mg/ml FITC-dextran (green) for 10 min, washed with PBS, andthen subjected to immunofluorescence assay using anti-HA antibody toprobe 3HA-tagged EhRab5 (red). Yellow arrowheads indicate endocy-tosed FITC-dextran. B, the amoebae were pulsed with FITC-dextran inthe presence of red blood cells for 10 min, and then subjected to immu-nofluorescence assay. A yellow or white arrow indicates an EhRab5-associated PPV that contains or does not contain endocytosed FITC-dextran, respectively. A yellow arrowhead depicts the endocytosedFITC-dextran that is not associated with EhRab5 in the cytoplasm.Bars, 10 �m.

FIG. 5. Time-lapse micrographs of an amoeba expressing GFP-EhRab5, showing de novo formation of PPV. Amoebae were mixedwith red blood cells, and then images of a stack of 18 sections along thez-axis (every 1.5 �m) were immediately recorded every 2.85 s. Fromeach time point, a representative section showing EhRab5-associatedvesicle or vacuole during the course of PPV formation was chosen toshow the de novo generation of PPV at a site indicated by the arrow-heads. Times in seconds are also shown. Bars, 10 �m.


min), suggesting that both amoebapore and CP2 were con-tained in the lysosomes at steady state. The subcellular local-ization of both amoebapore and LysoTracker changed duringerythrophagocytosis. At 10 min, 80% of acidified EhRab7A-

positive PPV were associated with amoebapore (Fig. 7, A, 10min, and C). At 30 min, all acidified EhRab7A-positive PPVremained amoebapore-positive (Fig. 7C, 30 min). In contrast,amoebapore and LysoTracker did not perfectly overlap on pha-gosomes at 30 min; all combinations of amoebapore and Lyso-Tracker positive or negative phagosomes were seen (Fig. 7A, 30min). As the number of total phagosomes increased duringincubation with red blood cells, the number of amoebapore- orLysoTracker-positive phagosomes increased in parallel (Fig.7D). However, the number of EhRab7A-associated phagosomesdid not increase after 30 min; the percentage of EhRab7A-positive phagosomes transiently increased at 20 min and thendecreased (i.e. 20, 31, 25, and 19% at 10, 20, 30, and 50 min),consistent with the notion that EhRab7A was dissociated fromphagosomes at this stage. The kinetics of CP2 was indistin-guishable from that of amoebapore (data not shown). Theseresults indicate that amoebapore and CP2 were transportedfrom lysosomes to phagosomes via PPV.

We noticed that amoebapore was concentrated in the periph-eral part of PPV, and not evenly distributed in the vacuole (e.g.Fig. 7A, 10 min). An immunoelectron micrograph using ananti-amoebapore A antibody further documented detailed lo-calization of amoebapore in the PPV (Fig. 7B). At the 10-minaddition of red blood cells, gold particles were detected on anamorphous structure that partially occupies the lumen. Fur-thermore, the amoebapore-containing vacuole included mem-brane structures (Fig. 7B, arrow). The concentrated localiza-tion of amoebapore within PPV was similar to that observed forendocytosed FITC-dextran (Fig. 4B).

Expression of EhRab5 Wild Type or Mutants Influences CellGrowth, Ingestion of Red Blood Cells, and Amoebapore Trans-port to Phagosomes but Not Endocytosis—To further examinethe specific role of EhRab5 and PPV, we introduced a consti-tutively active GTP form (EhRab5Q67L) or an inactive GDPform (EhRab5S22N) mutant of EhRab5 into wild-type amoeba.Introduction of neither wild-type EhRab5 nor EhRab5S22Naffected the amoeba growth compared with the vector controlindependent of coexpression of EhRab7A (Fig. 8A). In contrast,expression of EhRab5Q67L unexpectedly caused a severegrowth defect. This is the first case of a growth defect caused bythe expression of a mutant Rab5.

We also studied the effects of expression of wild-type andmutant EhRab5 on phagocytosis of red blood cells. The numberof red blood cells engulfed by the amoebae at 10, 20, or 30 minwas counted (Fig. 8B). Expression of wild-type EhRab5 accel-erated engulfment of red blood cells by 1.4–2.2-fold, whereasexpression of either the EhRab5Q67L or EhRab5S22N mutantinhibited the efficiency of phagocytosis by 50–70% comparedwith the control transformant.

Next, we assessed whether expression of EhRab5 wild-typeor mutants influences the transport of cargo proteins, e.g.amoebapore, to phagosomes. Efficiency of the amoebaporetransport was evaluated by calculating percentages of phago-cytosed red blood cells that colocalized with amoebapore (Fig.8C). In the control transformant cells, 67.0 � 7.5% of engulfedred blood cells colocalized with amoebapore at 30 min of incu-bation, whereas 87 � 2.3% of the ingested red blood cellscolocalized with amoebapore in wild-type EhRab5-expressingcells (p � 0.01). In contrast, the expression of EhRab5Q67Lreduced efficiency of the amoebapore transport to 45 � 3.0%(p � 0.05), whereas no significant change was observed in theEhRab5S22N-expressing transformant (58 � 2.8%, p � 0.1).These data indicate that overexpression of wild-type EhRab5 orthe EhRab5Q67L mutant increased or interfered with theamoebapore transport to phagosomes, respectively. Neitherfluid-phase nor receptor-medicated endocytosis (56), as indi-

FIG. 6. Acidification of EhRab5/EhRab7A-associated PPV andphagosomes. A, localization of EhRab5 (green) and LysoTracker (red)(left panel), and EhRab7A (green) and LysoTracker (red) (right panel) inthe absence of red blood cells. Amoebae were pulsed with LysoTrackerovernight and subjected to immunofluorescence assay with anti-HA (forEhRab5) or anti-Myc (for EhRab7A) monoclonal antibody. B, localiza-tion of EhRab7A (green) and LysoTracker (red) at 5 and 30 min of redblood cell incubation. White arrowheads (5 and 30 min) indicateEhRab7A-positive PPV that do not contain LysoTracker. Yellow arrow-heads (30 min) indicate EhRab7A-positive PPV containing Lyso-Tracker. White arrows indicate phagosomes that contain LysoTrackerand are also associated with EhRab7A. Yellow arrows indicate phago-somes containing LysoTracker, but not associated with EhRab7A. Bars,10 �m. C, quantitative analysis of PPV acidification. The number ofLysoTracker-associated (filled bars) or non-associated PPV (open bars)per amoeba is shown together with the percentages of the acidified PPV(circles and a line). Error bars represent S.D. of three independentexperiments.


cated by FITC-dextran (Fig. 8D) or lactoferrin (data not shown)internalization, respectively, was influenced by expression ofwild-type or mutant EhRab5 up to 3 h. Together with the lackof colocalization of EhRab5 and FITC-dextran shown above(Fig. 4A), these results also support the premise that EhRab5 isunlikely involved in endocytosis. In addition, the efflux of theinternalized fluid-phase marker was not affected in EhRab5mutants (data not shown).

EhRab5 Plays an Essential Role in the Formation of PPV—Subcellular localization of EhRab5 mutants was examined to

assess possible reasons for a defect in growth, phagocytosis,and amoebapore transport to phagosomes. Confocal immuno-fluorescence micrographs showed that both EhRab5Q67L andEhRab5S22N, probed with anti-EhRab5 antibody, were local-ized to small vesicular-like structures that resemble those ob-served for wild-type EhRab5 and EhRab7A at steady state(data not shown). After a 10-min incubation with red bloodcells, in contrast to wild-type EhRab5, EhRab5Q67L- orEhRab5S22N-associated vacuoles were not observed; their lo-calization appeared to be identical to that at steady state (Fig.

FIG. 7. Amoebapore A (AP-A) andcysteine proteinase 2 (CP2) weretransported to the red blood cell-con-taining phagosomes via PPV. A, sub-cellular localization of EhRab7A (green),LysoTracker (red), and CP2 or AP-A(blue) in the absence (0 min) or presenceof red blood cells (l0 and 30 min). Whitearrowheads indicate EhRab7A-positivePPV that contain neither LysoTrackernor AP-A. Yellow arrowheads indicatePPV containing both LysoTracker andAP-A. White arrows indicate phagosomesassociated with AP-A, but not with Lyso-Tracker. Yellow arrows indicate phago-somes that contain both LysoTracker andAP-A. Bars, 10 �m. B, an immunoelectronmicrograph using the amoebapore anti-body after a 10-min incubation of redblood cells. Localization of amoebapore in-dicated by 5-nm of gold particles (arrowheads) was partially localized in the vac-uole of 2.5 �m in diameter. Luminal mem-brane structure found in the vacuole wasindicated with an arrow. Bar, 1 �m. C,colocalization of LysoTracker and AP-Aon PPV. The percentages of PPV that con-tained both LysoTracker and AP-A (filledbars), or LysoTracker only (gray bars) at10 and 30 min of red blood cell incubationare shown. D, quantitative analysis ofphagosome maturation. The number oftotal phagosomes, and the AP-A-, Lyso-Tracker-, or EhRab7A-associated phago-somes is shown at 10–50 min of red bloodcell incubation.


8E). These results, together with the data shown above, sug-gest that EhRab5 is essential for the formation of PPV, whichis required for the efficient phagocytosis and transport of thedegradative proteins to phagosomes.

DISCUSSION

Although EhRab5 showed about 50% identity to the mam-malian and yeast counterparts and the putative effector do-main is very similar between E. histolytica and other organ-isms, the function of the amoebic Rab5 appears to be divergentfrom that of the mammalian and yeast homologues. First,whereas the mammalian and yeast Rab5/Ypt51p play a role inendocytosis, EhRab5 is involved exclusively in phagocytosis,but not in endocytosis. This has been shown by the absence ofcolocalization of EhRab5 and the endocytosed FITC-dextran(Fig. 4), and also by a lack of augmented uptake of the endo-cytosis marker by expression of wild-type or the dominantactive EhRab5 mutant (Fig. 8D). Second, the localization of

EhRab5 and its association to 3–5-�m translucent PPV, whichhas not been described in other organisms, are unique to thisorganism. In the mammalian cells, Rab5 is localized to theearly endosomes, and early endosomes directly fuse with pri-mary phagosomes during phagocytosis (4, 7), whereas EhRab5is not localized to phagosomes at any stages of phagocytosis inthe amoeba (Fig. 3). Instead, EhRab5 is localized, in conjunc-tion with EhRab7A, to PPV before these vacuoles fuse withphagosomes containing red blood cells. Third, in contrast tomammals, where similar phenotypes were observed in trans-formants expressing wild-type and GTP-mutant Rab5 and op-posite phenotypes were observed in the GDP-mutant Rab5-expressing transformants (5), expression of EhRab5 GTP orGDP mutant showed a similar defect in erythrophagocytosisand PPV formation (Fig. 8). This may indicate that require-ment of GTP hydrolysis by Rab5 for membrane fusion maydiffer between the amoeba and other organisms. Fourth,

FIG. 8. Effects of expression of EhRab5 wild-type and mutants. A, population doubling times of the transformants. E. histolyticatransformants expressing EhRab5 (Rab5), EhRab5/EhRab7A (Rab5 Rab7A), EhRab5S22N/EhRab7A (S22N Rab7A), EhRab5Q67L/EhRab7A(Q67L Rab7A), and the control transformant with a mock vector (vector) were cultured in the presence of 20 �g/ml Geneticin. Doubling times werecalculated from three independent experiments performed in triplicate. B, overexpression of EhRab5 wild-type and mutants influenced byphagocytosis of red blood cells. E. histolytica transformants overexpressing EhRab5/EhRab7A, EhRab5S22N/EhRab7A, or EhRab5Q67L/EhRab7Aand the control transformant were incubated with red blood cells for the indicated times. Phagocytosed red blood cells in these transformants werecounted under a microscope. C, the transport of AP-A to red blood cell-containing phagosomes was affected by the overexpression of EhRab5wild-type and EhRab5Q67L. The efficiency of AP-A transport was evaluated by calculating percentages of phagocytosed red blood cells colocalizingwith AP-A. Two hundred phagosomes containing red blood cells were examined. p � 0.01 (Rab5 versus control), p � 0.005 (Rab5Q67L versuscontrol), p � 0.1 (Rab5S22N versus control), according to Student’s t test are shown. D, endocytosis of a fluid-phase marker was not affected in thetransformants overexpressing EhRab5 wild-type and mutants. E. histolytica transformants were incubated in BI-S-33 medium containing 2 mg/mlFITC-dextran, and endocytosed FITC-dextran was measured using a fluorometer. Error bars in A–D represent S.D. of three independentexperiments. E, distribution of EhRab5S22N, EhRab5Q67L (green), and EhRab7A (red) after a 10-min incubation with red blood cells. Bars, 10 �m.Localization of EhRab5 and EhRab7A was examined with anti-EhRab5 and anti-Myc monoclonal antibody, respectively.


EhRab5 does not functionally complement the yeast �ypt51mutant. This is in good contrast to the yeast and mammaliancounterparts, which are virtually interchangeable; Ypt51p ex-pressed in a mammalian cell was properly localized to endo-somes and accelerated endocytosis (6). Fifth, compared withother organisms including mammals, plants, yeasts, and aparasitic protist Trypanosoma brucei, which have been shownto possess 2–5 Rab5 isotypes with distinct tissue and organelledistribution or developmental stage-specific expression (46,57–59), E. histolytica possesses only a single Rab5 gene basedon our thorough search of the genome data base (data notshown). Altogether, EhRab5 represents a unique Rab5 showingdiverse localization and functions.

We have identified and characterized an unprecedented vac-uole “PPV,” which is coassociated with EhRab5 and EhRab7Aat the early stage of its formation and becomes dissociated byEhRab5 during maturation. We have shown that PPV wereformed de novo in a very short time, and then acidified, in atime-dependent fashion during phagocytosis, by the fusion oflysosomes, which contain at least two independent degradativeproteins, i.e. CP2 and amoebapore A (Figs. 5–7). We proposethat PPV serves as a compartment for the temporal storage,processing, and/or activation of hydrolytic enzymes and lyticpeptides before fusion with phagosomes containing ingestedhost cells and microorganisms. In mammalian cells, a newlyformed phagosome is subjected to gradual maturation by con-tinuous exchange of their contents via sequential fusion withthe early and late endosomal compartments, leading to theformation of acidified phagolysosome (60). In contrast, in theamoeba, PPV apparently serves as a reservoir of digestiveenzymes (Fig. 7) and endosomal content (Fig. 4) prior to fusionwith phagosomes (Fig. 7). An immunoelectron micrographshowed that the vacuole containing amoebapore were enclosedby another membrane structure (Fig. 7B). Although multive-sicular vacuoles have been previously reported in Entamoeba(61), this is the first demonstration of a particular proteinwithin these multivesicular vacuoles. In mammalian and yeastcells, multivesicular bodies have been regarded as late endo-somes, in which proteins to be transported to lysosomal lumenare selectively packed into internal vesicles (62). These obser-vations imply that some proteins targeted to phagosomes areselectively included in PPV. We have recently identified thehomologue of retromer, which functions in retrieval of receptorproteins from late endosomes to the trans-Golgi network (63).3

The observation that one of retromer components, EhVps26, islocalized on PPV might imply that PPV had a similar functionto late endosomes/multivesicular bodies. As far as we areaware, such a “preparatory” organelle has not previously beendescribed and may represent a novel cellular compartment.

The formation of PPV was induced most efficiently by inter-action with red blood cells. A membrane ghost, but not a solublefraction, of red blood cells also induced the formation (data notshown). However, latex beads, yeasts, and Escherichia coli cellsinduced the formation of PPV to a much lesser extent (data notshown). Thus, Rab5 recruitment to PPV in this parasite mayoccur predominantly in a case of the engulfment of red bloodcells. PPV were also observed in cells that did not initiateengulfment of red blood cells (Fig. 3, E–L). These findingssuggest that interaction with red blood cells, but not engulf-ment per se, is sufficient for the induction of PPV formation.One intriguing hypothesis to explain why PPV formation isspecifically induced by red blood cells is that PPV is requiredfor the degradation and/or detoxification of the content of red

blood cells. It was previously reported that amoebae recognizessurface glycans with Gal�1–4GlcNAc terminal glycosphingo-lipid on red blood cells (64) by a Gal/GalNAc-inhibitable lectin(21, 22). The Gal�1–4GlcNAc terminal glycosphingolipid isabsent on the surface of latex beads, yeast, and E. coli (65, 66).It has also been demonstrated in macrophages that the phago-cytosis-induced response also depends on receptors (67). Forexample, phagocytosis via the Fc receptor lead to the produc-tion of proinflammatory molecules such as reactive oxygenintermediates, whereas phagocytosis involving mannose recep-tor produced proinflammatory cytokines including interleu-kin-1� and tumor necrosis factor-� (68). In contrast, phagocy-tosis via the complement receptor did not elicit release ofinflammatory mediators (69). Actin and microtubles wereshown to be important for the complement receptor system,whereas two regulatory proteins of actin cytoskeleton, vinculinand proxilin, were not necessary for the mannose receptorsystem, indicating diversity of the receptor-response relation-ship during phagocytosis (70).

In view of the signals necessary for PPV formation, we alsonoted that a phosphatidylinositol 3-kinase inhibitor, wortman-nin at 100 nM, abolished both ingestion, as previously reported(71), and PPV formation (data not shown). This finding alsosupports a tight correlation between ingestion of red blood cellsand formation of PPV. The fact that expression of wild-type orGTP mutant EhRab5 resulted in augmented or diminishedingestion of red blood cells, respectively, also supports thepremise that signal transduces from PPV to an initial site ofengulfment. However, whether a phagosome-associated phos-phatidylinositol 3-kinase is present in the amoeba, as shown inmammals (Vps34) (12) and what effector proteins (e.g. EEA1 inmammals) (72, 73) are recruited to the phagosomes of theamoeba in a phosphatidylinositol 3-kinase-dependent mannerremain unknown.

We have shown detailed quantitative data on how the mat-uration of PPV and phagosomes occur during erythrophagocy-tosis (Figs. 3Q, 6C, and 7, C and D). In contrast to the gradualand continuous acidification of PPV, which occurs in parallelwith recruitment of digestive enzymes to PPV, acidification ofphagosomes appears to be interrupted or, more likely, reversedby neutralization, which synchronizes with the dissociation ofEhRab7A from phagosomes (Fig. 7D). A few lines of evidencesuggest that neutralization of phagosomes takes place soonafter the content of PPV is transported to phagosomes. First,the percentage of acidified phagosomes remained unchangedbetween 20 and 50 min after ingestion (e.g. 43 and 38%, at 20and 50 min, respectively). Second, the percentage of acidifiedphagosomes was significantly lower than that of amoebapore-containing phagosomes at all time points (e.g. 58 and 71% at 20and 50 min).

We propose here a model by which EhRab5 and EhRab7Acoordinately regulate membrane fusion during phagocytosis.Upon the interaction of red blood cells with the amoeba plasmamembrane, independent EhRab5- or EhRab7A-associated ves-icles receive a signal, in a phosphatidylinositol 3-phosphate-de-pendent manner, presumably from the Gal/GalNAc-specificsurface lectin or a not yet identified plasma membrane recep-tor, which initiates subsequent sorting and reorganization ofthese compartments. EhRab5 vesicles start to heterotypicallyfuse with EhRab7A-associated vesicles, and then form PPV.PPV simultaneously fuse with lysosomes containing amoeba-pore and hydrolases. EhRab5 is then dissociated from PPVbefore the content of EhRab7A-associated PPV is targeted tophagosomes. Because the size of phagosomes did not increaseafter EhRab7A was transported from PPV to phagosomes, thedirect fusion between PPV and phagosome likely does not oc-

3 K. Nakada-Tsukui, Y. Saito-Nakano, V. Ali, and T. Nozaki, manu-script in preparation.


cur. We propose that transfer of the content of PPV involvesvesicular trafficking, i.e. budding from PPV followed by fusionof these vesicles to phagosomes. Once the content of PPV istransferred to phagosomes, EhRab7A is dissociated from pha-gosomes, whereas digestive proteins remain in phagosomes.Neutralization of phagosomes also takes place in close timingwith EhRab7A dissociation. After degradation of internalizedmaterials, membrane recycling from phagosomes also likelyoccurs via the budding of recycling vesicles (74). Finally, themolecular dissection of a unique function of EhRab5 and anovel EhRab5-associated compartment in this parasite mayshed light on the Entamoeba-specific phagocytic mechanismsclosely related to its virulence competence.

Acknowledgments—We are grateful to John Samuelson for providingthe genomic library, Iris Bruchhaus and Egbert Tannich for providingCP2 antibody, Barbara J. Mann and William A. Petri, Jr. for provid-ing GFP-expressing plasmid, Yuichi Takakuwa for providing anti-band3 antibody. We thank Marino Zerial and Akihiko Nakano for theirplasmids and yeast strains. We also thank Mami Okada, Mai Nude-jima, Yasuo Shigeta, Fumie Tokumaru, Osamu Fujita (NIID), andShin-ichiro Kawazu, (IMCJ) for technical assistance.

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