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Rab5-associated Vacuoles Play a Unique Role in Phagocytosis of the
Enteric Protozoan Parasite Entamoeba histolytica*
Received for publication, April 4, 2004, and in revised form, August 27, 2004
Published, 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‡¶储
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-Universita¨ t Kiel, Olshausenstrasse 40,
24118 Kiel, Germany, and the ¶Precursory Research for Embryonic Science and Technology, Japan Science and
Technology 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 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 ma-
tured by fusion with lysosomes containing a digestive
hydrolase, cysteine proteinase, and a membrane-perme-
abilizing 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, suggest-
ing 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, ex-
pression 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 pack-
aging 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 molec-
ular mechanism in professional phagocytes, e.g. macrophage,
has been extensively studied at the molecular level (1, 2). A
number of steps including cell surface binding to ligands and
the activation of a signaling pathway leading to F-actin polym-
erization have been identified as essential for phagocytosis. In
addition, membrane trafficking plays an important role in the
controlled maturation of phagosomes. The maturation is ac-
companied by sequential fusion with the endocytic compart-
ment to form a phagolysosome, and is orchestrated by small
GTPase, Rab proteins, which act as molecular switches regu-
lating the fusion of vesicles with target membranes through the
conformational change between active (GTP-bound) and inac-
tive (GDP-bound) forms (3). It has been reported that Rab5 and
Rab7 play an important role in the maturation of phagosomes
in macrophages (4).
Rab5 was initially shown to be localized to early endosomes
and the plasma membrane, and involved in endocytosis and the
endosome fusion (5, 6). Rab5 was also observed on nascent
phagosomes, and has been implicated to play an important role
in 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 in
the former case, and reduced activity for killing of ingested
bacteria because of the inhibition of phagosome maturation in
the latter case (8, 9). In addition to Rab5 per se, some of the
Rab5 effectors that were implicated in endosome fusion, e.g.
EEA1 and phosphatidylinositol 3-kinase (Vps34) (10, 11), also
have been identified on the phagosome membrane, suggesting
that phosphoinositide metabolism is important for phagosome
maturation as seen in the endocytic pathway (12, 13). Rab7 has
been implicated in late endosomal membrane trafficking in the
endocytic pathway (14), and also in the late stage of phagosome
maturation (4, 13). Although a specific role for Rab7 during
phagocytosis has not yet been well demonstrated, some intra-
cellular microorganisms have been reported to be capable of
blocking the maturation and acidification of phagosomes by
interfering with Rab7 (15, 16). It has also been recently dem-
onstrated that a novel effector protein, RILP, is recruited to the
phagosomal membrane by Rab7, which promotes fusion be-
tween phagosomes and lysosomes (17).
* This work was supported in part by a grant for Precursory Research
for Embryonic Science and Technology (PRESTO), Japan Science and
Technology Agency, Grant-in-aid for Scientific Research 15790219 (to
Y. S.-N.) and 15019120 and 15590378 (to T. N.) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan, a grant
for Research on Emerging and Re-emerging Infectious Diseases from
the Ministry of Health, Labor, and Welfare, a grant for the Project to
Promote Development of Anti-AIDS Pharmaceuticals from the Japan
Health Sciences Foundation (to T. N.), and a grant from the Deutsche
Forschungsgemeinschaft (to M. L.). The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank
TM
/EBI Data Bank with accession number(s) AB054582
(EhRab5) and AB054583 (EhRab7A).
储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: nozaki@nih.go.jp and nozaki@med.
gunma-u.ac.jp.
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
by guest, on February 19, 2013www.jbc.orgDownloaded from
Besides professional phagocytes from higher eukaryotes,
some unicellular organisms such as Dictyostelium discoideum
and Entamoeba histolytica show an inherent ability of phago-
cytosis. E. histolytica, an enteric protozoan parasite that causes
an 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 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 (20). Although a
number of amoebic molecules involved in attachment, phago-
cytosis, and degradation of microorganisms and host cells have
been identified including galactose/N-acetylgalactosamine
(Gal/GalNAc)-inhibitable lectin (21, 22), cytoskeletal proteins
and their associated regulatory molecules (23–25), cysteine
proteinases (CP),
1
and pore-forming peptides (i.e. amoeba-
pores) (26, 27), the molecular mechanism of phagocytosis in
this parasite remains largely unknown.
We presumed that Rab proteins also play an essential and
central role in the regulation of phagocytosis and endocytosis in
E. histolytica. We and other groups (28 –31) 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 (29). Putative EhRab7 and
EhRab11 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 molecular
mechanism of Rab proteins involved in the phagosome biogen-
esis in Entamoeba, we characterized, in the present study, two
amebic Rab proteins, EhRab5 and EhRab7A, that show signif-
icant 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 endo-
cytosis. Second, in contrast to mammalian Rab5, which is im-
mediately recruited to phagosomes after the engulfment of
bacteria or beads, EhRab5 is not recruited directly to phago-
somes, 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 impor-
tant role in the transport of the major membrane-permeabiliz-
ing peptide amoebapore. Therefore, in conjunction with
EhRab7A, EhRab5 plays a key role in the biogenesis of phago-
somes by regulating the formation of PPV and transport of
membranolytic and hydrolytic factors during phagocytosis in
this parasite.
EXPERIMENTAL PROCEDURES
Organism and Culture—E. histolytica trophozoites of HM-I:IMSS cl
6 (32) were cultured axenically in BI-S-33 medium at 35 °C as described
previously (33).
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 (28). A
full-length EhRab7A gene was obtained by reverse transcriptase-PCR
using oligonucleotide primers designed based on sequences previously
reported (30, 34). 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 characteriza-
tion 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
(35), BglII and XhoI sites between cysteine synthase 5⬘- and 3⬘-flanking
regions to insert a gene of interest, cysteine synthase 3⬘-flanking re-
gions and neomycin resistance gene flanked by the 5⬘and 3⬘regions of
actin gene, obtained from pA5⬘A3⬘NEO (36), 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
(37). 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 (38), 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 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 to
6 –20
g/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 Molec-
ular Probes (Eugene, OR). Anti-amoebic CP2 and human band 3 rabbit
antibodies were gifts from Iris Bruchhaus and Egbert Tannich (40), and
Yuichi Takakuwa (41), respectively. The production of anti-amoebapore
A antibody was previously described (42).
Indirect Immunofluorescence—Amoeba transformants in a logarith-
mic 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 10
7
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 mM3,3⬘-diaminobenzidine, 0.048% H
2
O
2
,
and 50 mMTris-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 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 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 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
TM
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 histolytica
Rab7A.
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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
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 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-dimen-
sionally reconstituted and only a selected plane containing a PPV or a
GFP-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 as
described above. Samples were prefixed with 3.7% paraformaldehyde,
PBS for 20 min, and then incubated with 0.1 Mglycine, 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% OsO
4
, PBS for 30 min followed by
dehydration, 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 stained
with uranyl acetate and examined with a Hitachi-H-700 electron
microscope.
Measurement of FITC-dextran Uptake—Transformants were cul-
tured 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
FIG.1.Plasmids used to express epitope-tagged EhRab5 and EhRab7A in E. histolytica.A, construction and schematic representation
of the plasmids. All plasmids shown are derivatives of pBluescript KS II⫹.CS5⬘,CS3⬘,Act5⬘,Act3⬘,orNeo,5⬘upstream or 3⬘downstream from
the cysteine synthase gene, 5⬘upstream or 3⬘downstream from the actin gene, or the neomycin resistance gene, respectively. Only representative
constructs to express wild-type EhRab5 and EhRab7 and GFP are shown. B, nucleotide and amino acid sequences of selected regions of the
expression cassette for EhRab5, EhRab7A, and GFP are shown. Nucleotide (nt) number of genes deposited under accession numbers (in
parentheses) is shown. Amino acid sequences are shown above nucleotide sequences. (AS), (ASSKKKPL), or (PG) depict inserted amino acids
because of engineered restriction sites shown below the nucleotide sequences. An asterisk (*) depicts the stop codon. Restriction sites are
underlined.EhRab5, EhRab7A, and GFP open reading frame are italicized.
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with 6 ml of ice-cold PBS containing 2% glucose, and solubilized with 50
mMTris-HCl, pH 7.0, containing 0.1% Nonidet P-40, and 10
g/ml of
trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64). Fluo-
rescence emission at 520 nm was measured with excitation at 490 nm
on 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 them
EhRab5 and EhRab7A, respectively. EhRab5 and EhRab7A
showed 45 and 48% identity to mammalian Rab5 and Rab7,
respectively. The effector region and
␣
2 helix loop, which are
important to the specificity of Rab proteins (45), 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 ⌬ypt51/⌬vps21 mutant (46, 47) and ⌬ypt7
mutant (14) 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 ⌬ypt51/⌬vps21 cells
(data not shown). Neither did overexpression of EhRab7A in
the ⌬ypt7 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 ex-
pressed 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 epitope-
tagged 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-over-
lapping 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 vacu-
oles (4.0 ⫾0.9
m 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 sur-
rounded 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 (4).
To unequivocally demonstrate the dynamics of the matura-
tion 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 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 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)
FIG.2.Comparison of amino acid sequences of Rab5 and Rab7
from E. histolytica, human, and yeast. A, sequence alignment of
EhRab5, Homo sapiens Rab5a, and Saccharomyces cerevisiae Ypt51p.
B, sequence alignment of EhRab7A, H. sapiens Rab7, and S. cerevisiae
Ypt7p. Sequences were aligned by DNASIS (Hitachi Software Engi-
neering Co.). Amino acid residues conserved among at least two species
are shown in reverse type. The GTP-binding consensus sequences, the
effector 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.
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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 red
blood cells, but is not stained by diaminobenzidine because of
loss of its content. To exclude this possibility, we used an
antibody raised against a major component of the membrane
cytoskeleton of red blood cells, band 3 (48). The anti-band 3
antibody clearly reacted with red blood cells in phagosomes
(Fig. 3V,red,arrow), whereas none of the EhRab5-positive PPV
was reacted with the antibody (green,arrowheads). In addition,
localization of red blood cells by diaminobenzidine staining or
anti-band 3 antibody agreed very well (Fig. 3, Vand W). These
results 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 it
was also observed in wild-type amoebae at a comparable fre-
quency, as detected by the antibodies raised against recombi-
FIG.3. Subcellular localization of
EhRab5 and EhRab7A changed dur-
ing red blood cell phagocytosis. A–P,
subcellular localization of EhRab5 and
EhRab7A was examined by immuno-
fluorescence assay using the amoeba
transformant co-expressing 3HA-tagged
EhRab5 and 3Myc-tagged EhRab7A in
the absence of red blood cells (A–D), or
after 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 monoclonal
antibody (red;B,F,J, and N), respec-
tively. Merged images of EhRab5 and
EhRab7A (C,G,K, and O) and phase-
contrast images under transmission light
(D,H,L, and P) are also shown. Large
arrowheads 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 associated
with EhRab5. A thin arrow (L) indicates
an engulfed red blood cell associated with
neither EhRab5 nor EhRab7A. Q, quanti-
tative analysis of EhRab5 and EhRab7A
localization to PPV and phagosomes dur-
ing erythrophagocytosis. The number of
EhRab5/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), and
EhRab7A-negative phagosomes (filled
bars;phagosome) per cell is shown at 5,
10, and 30 min after the addition of red
blood cells. R–U, subcellular localization
of EhRab7A was examined by immunoflu-
orescence assay using wild-type amoebae
and anti-EhRab7A antibody in the ab-
sence of red blood cells (Rand S) or after
a 10-min (Tand U) incubation with red
blood cells. Panels S and Ushow phase
images of panels R and T, respectively.
Arrowheads in Tand Udepict PPV. V and
W, three-dimensional sections of the
amoeba containing red blood cell showing
the presence of red blood cells in phago-
somes, but not in PPV. Localization of
PPV and red blood cells was examined
with anti-EhRab5 antibody (green,arrow-
heads) and anti-band 3 antibody (arrows,
red), respectively. Among 17 z-sections
(1-
m intervals) obtained with confocal
laser scanning microscopy, only one rep-
resentative xy section, together with se-
lected xz (green line), and yz (red line)
sections, are shown. Wshows a phase im-
age of V.
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nant EhRab5 and EhRab7A (Fig. 3, R-U; data of EhRab5 not
shown).
EhRab5 Is Not Associated with Endosomes or Lysosomes but
Exhibits 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-phase
marker, FITC-dextran, and EhRab5. Amoebae were either incu-
bated with FITC-dextran for 10 min to label the early endosomes
or incubated with FITC-dextran for 10 min and further chased
without FITC-dextran for 45 min to label the late endosomes, and
then 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 (data
not shown). These findings imply that the EhRab5-positive com-
partment is neither early nor late endosomes.
When amoebae were simultaneously incubated with red
blood cells and FITC-dextran for 10 min, 30% of PPV contained
endocytosed FITC-dextran (Fig. 4B). When the amoebae were
pulsed with FITC-dextran for 10 min and chased for 45 min to
label the late endosomes, and further incubated with red blood
cells for 10 min, the extent of colocalization of FITC-dextran
and PPV was comparable (26%) (data not shown). These results
suggest that PPV fuse with both early and late endosomes
during maturation.
To assess where and how PPV are formed during phagocy-
tosis, we examined the dynamics of EhRab5 using the amoeba
transformant expressing GFP-EhRab5 under time-lapse mi-
croscopy. Images of 18 planes of the z-section with 1.5-
m
intervals to cover from the top to the bottom of the cell were
recorded at 2.85-s intervals. This allowed us to evaluate the
detailed dynamism of PPV formation. After a few minutes of
coincubation with red blood cells, an EhRab5-positive vacuole
suddenly emerged in less than 20 s. Neither plasma membrane
invagination nor ruffling were observed during this period,
suggesting that PPV forms de novo (Fig. 5).
We also excluded a possibility that PPVs are micropinosomes
or phagosomes. First, the fact that only a minor proportion of
PPV contained FITC-dextran at 10 min (Fig. 4B) suggests that
PPVs are not formed by invagination of the plasma membrane-
like macropinosomes, which form by the closure of membrane
ruffles and contain a fluid-phase marker (49, 50). Second, PPV
is 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 1
and 5 min, respectively. Third, the major Gal/GalNAc lectin on
the plasma membrane was abundantly demonstrated in pha-
gosomes by proteomic analysis of phagosome proteins during
the course of phagosome maturation (from 0 min to 2 h after
ingestion)
2
but was not demonstrated on PPV by immunofluo-
rescence study using a specific monoclonal antibody against
heavy or intermediate lectin subunits (data not shown). These
results strongly argue against two possibilities: 1) PPV origi-
nates from the plasma membrane, and 2) PPV is a remnant of
phagosomes.
Acidification of phagosomes has been shown to occur by
fusion with late endosomes and lysosomes in mammalian cells
(52). We examined by using LysoTracker Red, a membrane-
diffusible probe accumulated in acidic organelles (53), whether
the PPV and phagosomes of the amoeba are acidified during
maturation. Amoebae were pulsed with LysoTracker and then
subjected 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 with
red blood cells when EhRab7A-positive PPV were formed, only
20 –30% of PPV contained LysoTracker, suggesting that PPV
were only partially acidified in the early stage (Fig. 6, B,upper
panels, and C, data at 5 min not shown). After 30 – 40 min, a
large 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 of
internalized host cells and microorganisms, e.g. CP (26), amoe-
bapores (27), lysozyme (54), and phospholipases (55), we tested
whether amoebapore A and CP2 were transported to phago-
somes via PPV. Immunostaining of amoebapore and CP2 using
specific antisera showed similar patterns to those obtained
with 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, and
T. Nozaki, submitted for publication.
FIG.4. Immunofluorescent micrographs showing cross-talk
between early endosomes and PPV. A, the amoebae were pulsed
with 2 mg/ml FITC-dextran (green) for 10 min, washed with PBS, and
then subjected to immunofluorescence assay using anti-HA antibody to
probe 3HA-tagged EhRab5 (red). Yellow arrowheads indicate endocy-
tosed FITC-dextran. B, the amoebae were pulsed with FITC-dextran in
the 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 endocytosed
FITC-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 mixed
with red blood cells, and then images of a stack of 18 sections along the
z-axis (every 1.5
m) were immediately recorded every 2.85 s. From
each time point, a representative section showing EhRab5-associated
vesicle or vacuole during the course of PPV formation was chosen to
show the de novo generation of PPV at a site indicated by the arrow-
heads. Times in seconds are also shown. Bars,10
m.
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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 during
erythrophagocytosis. At 10 min, 80% of acidified EhRab7A-
positive PPV were associated with amoebapore (Fig. 7, A,10
min, and C). At 30 min, all acidified EhRab7A-positive PPV
remained 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,30
min). As the number of total phagosomes increased during
incubation with red blood cells, the number of amoebapore- or
LysoTracker-positive phagosomes increased in parallel (Fig.
7D). However, the number of EhRab7A-associated phagosomes
did not increase after 30 min; the percentage of EhRab7A-
positive phagosomes transiently increased at 20 min and then
decreased (i.e. 20, 31, 25, and 19% at 10, 20, 30, and 50 min),
consistent with the notion that EhRab7A was dissociated from
phagosomes at this stage. The kinetics of CP2 was indistin-
guishable from that of amoebapore (data not shown). These
results indicate that amoebapore and CP2 were transported
from 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 an
anti-amoebapore A antibody further documented detailed lo-
calization of amoebapore in the PPV (Fig. 7B). At the 10-min
addition of red blood cells, gold particles were detected on an
amorphous 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 for
endocytosed FITC-dextran (Fig. 4B).
Expression of EhRab5 Wild Type or Mutants Influences Cell
Growth, Ingestion of Red Blood Cells, and Amoebapore Trans-
port to Phagosomes but Not Endocytosis—To further examine
the specific role of EhRab5 and PPV, we introduced a consti-
tutively active GTP form (EhRab5Q67L) or an inactive GDP
form (EhRab5S22N) mutant of EhRab5 into wild-type amoeba.
Introduction of neither wild-type EhRab5 nor EhRab5S22N
affected the amoeba growth compared with the vector control
independent of coexpression of EhRab7A (Fig. 8A). In contrast,
expression of EhRab5Q67L unexpectedly caused a severe
growth defect. This is the first case of a growth defect caused by
the expression of a mutant Rab5.
We also studied the effects of expression of wild-type and
mutant EhRab5 on phagocytosis of red blood cells. The number
of red blood cells engulfed by the amoebae at 10, 20, or 30 min
was counted (Fig. 8B). Expression of wild-type EhRab5 accel-
erated engulfment of red blood cells by 1.4 –2.2-fold, whereas
expression of either the EhRab5Q67L or EhRab5S22N mutant
inhibited the efficiency of phagocytosis by 50 –70% compared
with the control transformant.
Next, we assessed whether expression of EhRab5 wild-type
or mutants influences the transport of cargo proteins, e.g.
amoebapore, to phagosomes. Efficiency of the amoebapore
transport 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 engulfed
red blood cells colocalized with amoebapore at 30 min of incu-
bation, whereas 87 ⫾2.3% of the ingested red blood cells
colocalized with amoebapore in wild-type EhRab5-expressing
cells (p⬍0.01). In contrast, the expression of EhRab5Q67L
reduced efficiency of the amoebapore transport to 45 ⫾3.0%
(p⬍0.05), whereas no significant change was observed in the
EhRab5S22N-expressing transformant (58 ⫾2.8%, p⬎0.1).
These data indicate that overexpression of wild-type EhRab5 or
the EhRab5Q67L mutant increased or interfered with the
amoebapore transport to phagosomes, respectively. Neither
fluid-phase nor receptor-medicated endocytosis (56), as indi-
FIG.6.Acidification of EhRab5/EhRab7A-associated PPV and
phagosomes. A, localization of EhRab5 (green) and LysoTracker (red)
(left panel), and EhRab7A (green) and LysoTracker (red)(right panel)in
the absence of red blood cells. Amoebae were pulsed with LysoTracker
overnight and subjected to immunofluorescence assay with anti-HA (for
EhRab5) or anti-Myc (for EhRab7A) monoclonal antibody. B, localiza-
tion of EhRab7A (green) and LysoTracker (red) at 5 and 30 min of red
blood cell incubation. White arrowheads (5 and 30 min) indicate
EhRab7A-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 LysoTracker
and 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 of
LysoTracker-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 independent
experiments.
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cated by FITC-dextran (Fig. 8D) or lactoferrin (data not shown)
internalization, respectively, was influenced by expression of
wild-type or mutant EhRab5 up to 3 h. Together with the lack
of colocalization of EhRab5 and FITC-dextran shown above
(Fig. 4A), these results also support the premise that EhRab5 is
unlikely involved in endocytosis. In addition, the efflux of the
internalized fluid-phase marker was not affected in EhRab5
mutants (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 and
EhRab5S22N, 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 blood
cells, in contrast to wild-type EhRab5, EhRab5Q67L- or
EhRab5S22N-associated vacuoles were not observed; their lo-
calization appeared to be identical to that at steady state (Fig.
FIG.7. Amoebapore A (AP-A) and
cysteine proteinase 2 (CP2) were
transported 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 presence
of red blood cells (l0 and 30 min). White
arrowheads indicate EhRab7A-positive
PPV that contain neither LysoTracker
nor AP-A. Yellow arrowheads indicate
PPV containing both LysoTracker and
AP-A. White arrows indicate phagosomes
associated with AP-A, but not with Lyso-
Tracker. Yellow arrows indicate phago-
somes that contain both LysoTracker and
AP-A. Bars,10
m. B, an immunoelectron
micrograph using the amoebapore anti-
body after a 10-min incubation of red
blood cells. Localization of amoebapore in-
dicated by 5-nm of gold particles (arrow
heads) was partially localized in the vac-
uole of 2.5
m in diameter. Luminal mem-
brane structure found in the vacuole was
indicated with an arrow.Bar,1
m. C,
colocalization of LysoTracker and AP-A
on PPV. The percentages of PPV that con-
tained both LysoTracker and AP-A (filled
bars), or LysoTracker only (gray bars)at
10 and 30 min of red blood cell incubation
are shown. D, quantitative analysis of
phagosome maturation. The number of
total phagosomes, and the AP-A-, Lyso-
Tracker-, or EhRab7A-associated phago-
somes is shown at 10 –50 min of red blood
cell incubation.
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8E). These results, together with the data shown above, sug-
gest that EhRab5 is essential for the formation of PPV, which
is required for the efficient phagocytosis and transport of the
degradative 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 divergent
from that of the mammalian and yeast homologues. First,
whereas the mammalian and yeast Rab5/Ypt51p play a role in
endocytosis, EhRab5 is involved exclusively in phagocytosis,
but not in endocytosis. This has been shown by the absence of
colocalization 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 dominant
active EhRab5 mutant (Fig. 8D). Second, the localization of
EhRab5 and its association to 3–5-
m translucent PPV, which
has not been described in other organisms, are unique to this
organism. In the mammalian cells, Rab5 is localized to the
early endosomes, and early endosomes directly fuse with pri-
mary phagosomes during phagocytosis (4, 7), whereas EhRab5
is not localized to phagosomes at any stages of phagocytosis in
the amoeba (Fig. 3). Instead, EhRab5 is localized, in conjunc-
tion with EhRab7A, to PPV before these vacuoles fuse with
phagosomes containing red blood cells. Third, in contrast to
mammals, 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 or
GDP mutant showed a similar defect in erythrophagocytosis
and PPV formation (Fig. 8). This may indicate that require-
ment of GTP hydrolysis by Rab5 for membrane fusion may
differ 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. histolytica
transformants 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 were
calculated from three independent experiments performed in triplicate. B, overexpression of EhRab5 wild-type and mutants influenced by
phagocytosis of red blood cells. E. histolytica transformants overexpressing EhRab5/EhRab7A, EhRab5S22N/EhRab7A, or EhRab5Q67L/EhRab7A
and the control transformant were incubated with red blood cells for the indicated times. Phagocytosed red blood cells in these transformants were
counted under a microscope. C, the transport of AP-A to red blood cell-containing phagosomes was affected by the overexpression of EhRab5
wild-type and EhRab5Q67L. The efficiency of AP-A transport was evaluated by calculating percentages of phagocytosed red blood cells colocalizing
with AP-A. Two hundred phagosomes containing red blood cells were examined. p⬍0.01 (Rab5 versus control), p⬍0.005 (Rab5Q67L versus
control), p⬎0.1 (Rab5S22N versus control), according to Student’s ttest are shown. D, endocytosis of a fluid-phase marker was not affected in the
transformants overexpressing EhRab5 wild-type and mutants. E. histolytica transformants were incubated in BI-S-33 medium containing 2 mg/ml
FITC-dextran, and endocytosed FITC-dextran was measured using a fluorometer. Error bars in A–Drepresent S.D. of three independent
experiments. 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.
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EhRab5 does not functionally complement the yeast ⌬ypt51
mutant. This is in good contrast to the yeast and mammalian
counterparts, which are virtually interchangeable; Ypt51p ex-
pressed in a mammalian cell was properly localized to endo-
somes and accelerated endocytosis (6). Fifth, compared with
other organisms including mammals, plants, yeasts, and a
parasitic protist Trypanosoma brucei, which have been shown
to possess 2–5 Rab5 isotypes with distinct tissue and organelle
distribution or developmental stage-specific expression (46,
57–59), E. histolytica possesses only a single Rab5 gene based
on our thorough search of the genome data base (data not
shown). Altogether, EhRab5 represents a unique Rab5 showing
diverse localization and functions.
We have identified and characterized an unprecedented vac-
uole “PPV,” which is coassociated with EhRab5 and EhRab7A
at the early stage of its formation and becomes dissociated by
EhRab5 during maturation. We have shown that PPV were
formed de novo in a very short time, and then acidified, in a
time-dependent fashion during phagocytosis, by the fusion of
lysosomes, which contain at least two independent degradative
proteins, i.e. CP2 and amoebapore A (Figs. 5–7). We propose
that PPV serves as a compartment for the temporal storage,
processing, and/or activation of hydrolytic enzymes and lytic
peptides before fusion with phagosomes containing ingested
host cells and microorganisms. In mammalian cells, a newly
formed phagosome is subjected to gradual maturation by con-
tinuous exchange of their contents via sequential fusion with
the early and late endosomal compartments, leading to the
formation of acidified phagolysosome (60). In contrast, in the
amoeba, PPV apparently serves as a reservoir of digestive
enzymes (Fig. 7) and endosomal content (Fig. 4) prior to fusion
with phagosomes (Fig. 7). An immunoelectron micrograph
showed that the vacuole containing amoebapore were enclosed
by another membrane structure (Fig. 7B). Although multive-
sicular vacuoles have been previously reported in Entamoeba
(61), this is the first demonstration of a particular protein
within these multivesicular vacuoles. In mammalian and yeast
cells, multivesicular bodies have been regarded as late endo-
somes, in which proteins to be transported to lysosomal lumen
are selectively packed into internal vesicles (62). These obser-
vations imply that some proteins targeted to phagosomes are
selectively included in PPV. We have recently identified the
homologue of retromer, which functions in retrieval of receptor
proteins from late endosomes to the trans-Golgi network (63).
3
The observation that one of retromer components, EhVps26, is
localized on PPV might imply that PPV had a similar function
to late endosomes/multivesicular bodies. As far as we are
aware, such a “preparatory” organelle has not previously been
described 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 soluble
fraction, of red blood cells also induced the formation (data not
shown). However, latex beads, yeasts, and Escherichia coli cells
induced the formation of PPV to a much lesser extent (data not
shown). Thus, Rab5 recruitment to PPV in this parasite may
occur predominantly in a case of the engulfment of red blood
cells. PPV were also observed in cells that did not initiate
engulfment of red blood cells (Fig. 3, E–L). These findings
suggest 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 is
specifically induced by red blood cells is that PPV is required
for the degradation and/or detoxification of the content of red
blood cells. It was previously reported that amoebae recognizes
surface 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 is
absent 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). For
example, phagocytosis via the Fc receptor lead to the produc-
tion of proinflammatory molecules such as reactive oxygen
intermediates, 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 of
inflammatory mediators (69). Actin and microtubles were
shown to be important for the complement receptor system,
whereas two regulatory proteins of actin cytoskeleton, vinculin
and proxilin, were not necessary for the mannose receptor
system, indicating diversity of the receptor-response relation-
ship during phagocytosis (70).
In view of the signals necessary for PPV formation, we also
noted 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 also
supports a tight correlation between ingestion of red blood cells
and formation of PPV. The fact that expression of wild-type or
GTP mutant EhRab5 resulted in augmented or diminished
ingestion of red blood cells, respectively, also supports the
premise that signal transduces from PPV to an initial site of
engulfment. However, whether a phagosome-associated phos-
phatidylinositol 3-kinase is present in the amoeba, as shown in
mammals (Vps34) (12) and what effector proteins (e.g. EEA1 in
mammals) (72, 73) are recruited to the phagosomes of the
amoeba in a phosphatidylinositol 3-kinase-dependent manner
remain 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, Cand D). In contrast to the gradual
and continuous acidification of PPV, which occurs in parallel
with recruitment of digestive enzymes to PPV, acidification of
phagosomes appears to be interrupted or, more likely, reversed
by neutralization, which synchronizes with the dissociation of
EhRab7A from phagosomes (Fig. 7D). A few lines of evidence
suggest that neutralization of phagosomes takes place soon
after the content of PPV is transported to phagosomes. First,
the percentage of acidified phagosomes remained unchanged
between 20 and 50 min after ingestion (e.g. 43 and 38%, at 20
and 50 min, respectively). Second, the percentage of acidified
phagosomes was significantly lower than that of amoebapore-
containing phagosomes at all time points (e.g. 58 and 71% at 20
and 50 min).
We propose here a model by which EhRab5 and EhRab7A
coordinately regulate membrane fusion during phagocytosis.
Upon the interaction of red blood cells with the amoeba plasma
membrane, independent EhRab5- or EhRab7A-associated ves-
icles receive a signal, in a phosphatidylinositol 3-phosphate-de-
pendent manner, presumably from the Gal/GalNAc-specific
surface lectin or a not yet identified plasma membrane recep-
tor, which initiates subsequent sorting and reorganization of
these compartments. EhRab5 vesicles start to heterotypically
fuse with EhRab7A-associated vesicles, and then form PPV.
PPV simultaneously fuse with lysosomes containing amoeba-
pore and hydrolases. EhRab5 is then dissociated from PPV
before the content of EhRab7A-associated PPV is targeted to
phagosomes. Because the size of phagosomes did not increase
after EhRab7A was transported from PPV to phagosomes, the
direct 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.
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cur. We propose that transfer of the content of PPV involves
vesicular trafficking, i.e. budding from PPV followed by fusion
of these vesicles to phagosomes. Once the content of PPV is
transferred to phagosomes, EhRab7A is dissociated from pha-
gosomes, whereas digestive proteins remain in phagosomes.
Neutralization of phagosomes also takes place in close timing
with EhRab7A dissociation. After degradation of internalized
materials, membrane recycling from phagosomes also likely
occurs via the budding of recycling vesicles (74). Finally, the
molecular dissection of a unique function of EhRab5 and a
novel EhRab5-associated compartment in this parasite may
shed light on the Entamoeba-specific phagocytic mechanisms
closely related to its virulence competence.
Acknowledgments—We are grateful to John Samuelson for providing
the genomic library, Iris Bruchhaus and Egbert Tannich for providing
CP2 antibody, Barbara J. Mann and William A. Petri, Jr. for provid-
ing GFP-expressing plasmid, Yuichi Takakuwa for providing anti-band
3 antibody. We thank Marino Zerial and Akihiko Nakano for their
plasmids and yeast strains. We also thank Mami Okada, Mai Nude-
jima, Yasuo Shigeta, Fumie Tokumaru, Osamu Fujita (NIID), and
Shin-ichiro Kawazu, (IMCJ) for technical assistance.
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