Differences in Morphology of Phagosomes
and Kinetics of Acidification and Degradation
in Phagosomes Between the Pathogenic
Entamoeba histolytica and the
Nonpathogenic Entamoeba dispar
Biswa N. Mitra,1Tomoyoshi Yasuda,2Seiki Kobayashi,3Yumiko Saito-Nakano,2
and Tomoyoshi Nozaki1,4*
1Department of Parasitology, Gunma University Graduate School of Medicine,
Maebashi, Gunma 371-851, Japan
2Department of Parasitology, National Institute of Infectious Diseases,
Shinjuku-ku, Tokyo 162-8640, Japan
3Department of Parasitology, Keio University School of Medicine,
Tokyo 160-8582, Japan
4The Precursory Research for Embryonic Science and Technology,
Japan Science and Technology Agency, Tachikawa, Tokyo 190-0012, Japan
Phagocytosis plays an important role in the pathogenicity of the intestinal protozoan
parasite Entamoeba histolytica. We compared the morphology of phagosomes and
the kinetics of phagosome maturation using conventional light and electron micro-
scopy and live imaging with video microscopy between the virulent E. histolytica
and the closely-related, but nonvirulent E. dispar species. Electron micrographs
showed that axenically cultivated trophozoites of the two Entamoeba species
revealed morphological differences in the number of bacteria contained in a single
phagosome and the size of phagosomes. Video microscopy using pH-sensitive fluo-
rescein isothiocynate-conjugated yeasts showed that phagosome acidification occurs
within 2 min and persists for >12 h in both species. The acidity of phagosomes sig-
nificantly differed between two species (4.58 6 0.36 or 5.83 6 0.38 in E. histolytica
or E. dispar, respectively), which correlated well with the differences in the kinetics
of degradation of promastigotes of GFP-expressing Leishmania amazonensis. The
acidification of phagosomes was significantly inhibited by a myosin inhibitor,
whereas it was only marginally inhibited by microtubules or actin inhibitors. A spe-
cific inhibitor of vacuolar ATPase, concanamycin A, interrupted both the acidifica-
tion and degradation in phagosomes in both species, suggesting the ubiquitous role
of vacuolar ATPase in the acidification and degradation in Entamoeba. In contrast,
inhibitors against microtubules or cysteine proteases (CP) showed distinct effects on
degradation in phagosomes between these two species. Although depolymerization
of microtubules severely inhibited degradation in phagosomes of E. histolytica, it
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Received 28 March 2005; Accepted 21 June 2005
Published online in Wiley InterScience (www.interscience.wiley.com).
*Correspondence to: Tomoyoshi Nozaki, Department of Parasitology,
Gunma University Graduate School of Medicine, 3-39-22 Showa-
machi, Maebashi, Gunma 371-8511, Japan.
Contract grant sponsor: The Ministry of Education, Culture, Sports,
Science and Technology of Japan; Contract grant numbers: 15019120,
15590378, 16017307, and 16044250; Contract grant sponsors: The
Ministry of Health, Labour, and Welfare and The Japan Health Sci-
' 2005 Wiley-Liss, Inc.
Cell Motility and the Cytoskeleton 62:000–000 (2005)
did not affect degradation in E. dispar. Similarly, the inhibition of CP significantly
reduced degradation in phagosomes of E. histolytica, but not in E. dispar. These data
suggest the presence of biochemical or functional differences in the involvement of
microtubules and proteases in phagosome maturation and degradation between the
two species. Cell Motil. Cytoskeleton 62:000–000, 2005.
' 2005 Wiley-Liss, Inc.
Key words: phagocytosis; endocytosis; vacuolar ATPase; cysteine protease; amebiasis
Phagocytosis is an essential process by which pro-
fessional phagocytes (e.g., macrophages and neutrophils)
engulf invading pathogens, apoptotic cells, and other for-
eign particles [Leverrie et al., 2001; May and Machesky,
2001; Lee et al., 2003]. Phagocytosis triggers the activa-
tion of multiple transmembrane signaling pathways,
leading to the reorganization of the actin cytoskeleton
and the formation of a sealed intracellular compartment,
the phagosome [Tjelle et al., 2000]. The newly formed
phagosome undergoes a maturation process by a series
of fusions with endocytic compartments and eventually
lysosomes, and finally kills and degrades ingested sub-
stances within the phagolysosome [Desjardins et al.,
1994; Tjelle et al., 2000]. The coordinated maturation of
phagolysosomes by the acidification and recruitment of
hydrolases [Griffiths, 2004] is crucial for its microbicidal
action [Hackam et al., 1997] and the degradation of
phagosomal content [Grinstein et al., 1992]. A low pH
by itself is lethal for many microorganisms and also pro-
vides an optimal condition for the activation of lyso-
somal hydrolytic enzymes [Hackam et al., 1997]. It
was previously shown that vacuolar-type ATPases
(V-ATPases) accumulate in the phagosomal membrane
during maturation [Hackam et al., 1997; Tsukano et al.,
1999], and was, thus, thought to be the crucial determi-
nant in phagosome acidification [Hackam et al., 1997].
In macrophages, V-ATPases are involved in the killing
of bacteria and senescent erythrocytes [Grinstein et al.,
1992]. It was shown that proteases and other lysosomal
hydrolases become active to kill ingested pathogens
when the pH within phagolysosomes (late phagosomes)
reaches 5.0 [Lee et al., 2003]. The fusion of phagosomes
with lysosomes or other endocytic vesicles is microtu-
bule dependent and a prerequisite for phagosome matu-
ration because they facilitate fusion between phago-
somes and organelles of the endocytic pathway [Blocker
et al., 1996]. Besides professional phagocytes from
higher eukaryotes, some unicellular organisms, such as
the slime mold, Dictyostelium discoideum, and an enteric
parasite, Entamoeba histolytica, show an inherent phago-
cytosis ability. Entamoeba histolytica, a potentially inva-
sive enteric protozoan parasite, causes an estimated
50 million cases of amebiasis: amebic colitis, dysentery,
and extraintestinal abscesses [Petri, 2002], and 40,000–
100,000 deaths annually [WHO/Pan American Health,
1997]. The trophozoite of E. histolytica colonizes the
human gut and engulfs foreign cells, including microor-
ganisms and host cells. During tissue invasion, the troph-
ozoite first depletes the mucus of the intestinal paren-
chyma and then kills and phagocytoses the underlining
host cells [Ravdin et al., 1980]. A related Entamoeba
species, which is morphologically indistinguishable from
E. histolytica and shows 95% similarity to E. histolytica
within the protein coding sequences at the nucleotide
level [Willhoeft et al., 2000], also colonizes the human
gut and ingests microorganisms for its nutritional
requirements. However, when humans are infected with
E. dispar, the trophozoites remain as a harmless com-
mensal in the bowl lumen, without causing local inva-
sion leading to dysentery and liver abscesses. As these
two amoebas are similar in their genetic background, cell
biology, and host range, the systematic comparison
between E. histolytica and E. dispar constitutes an
important area of research to identify and analyze the
factors that might be important for its amebic pathoge-
nicity [Horstmann et al., 1992].
It has been demonstrated that the ability of ingest-
ing and killing microorganisms and their host cells
[Huston et al., 2003] is closely associated with an amoe-
ba’s pathogenesis. It was previously shown that mutants
deficient in phagocytosis were avirulent both in vitro and
in vivo [Orozco et al., 1983]. A number of amebic pro-
teins involved in phagocytosis and virulence were
previously identified, including galactose/N-acetylgalac-
tosamine-inhabitable lectin [Petri, 2002], cytoskeletal
proteins, and their associated regulatory molecules
[Voigt et al., 1999], cysteine proteases (CP) [Bruchhaus
et al., 1996; Que and Reed, 2000], pore-forming peptides
(i.e., amoebapores)[Leippe et al., 1994], and Rab
GTPases [Saito-Nakano et al., 2004]. Most of these pro-
teins and their encoding genes are present in both
E. histolytica and E. dispar with few exceptions, e.g.,
CP1 and CP5 [Bruchhaus et al., 1996]. The morphologi-
cal differences of phagosomes between the two species
have recently been demonstrated [Pimenta et al., 2002].
E. histolytica contained multiple bacteria in their phago-
somes, whereas phagosomes in E. dispar contained a sin-
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2 Mitra et al.
gle bacterium and some bacteria were freely present in
the cytoplasm (i.e., not enclosed by the membrane
structure) [Pimenta et al., 2002]. However, since the
E. histolytica and E. dispar xenic strains used in the
study were cultivated with uncharacterized and likely
different bacterial flora [Pimenta et al., 2002], it was not
clear that the observed differences in phagosome mor-
phology were attributable to inherited differences in the
phagosome structures and biogenesis between the two
species or to different culture conditions, e.g., co-cul-
tured bacteria species. In the present study, we examined
morphological differences of phagosomes containing a
homogeneous (not a mixture) line of Pseudomonas aeru-
ginosa, using electron and light microscopes. In addition,
we demonstrated the differences in the kinetics of phago-
some maturation, with video microscopy, between the
axenically cultivated E. histolytica and E. dispar strains.
MATERIALS AND METHODS
All chemicals of analytical grade were purchased
from Wako (Tokyo, Japan) unless otherwise stated. Con-
canamycin A, nocodazole, 2,3-butanedione monoxime
(BDM), and trans-epoxysuccinyl-L-leucylamido-(4-gua-
nidino) butane (E64) were purchased from Sigma-Aldrich
(Tokyo, Japan). 3,5-Dinitro-N4, N4-dipropylsulfanilamide
(Oryzalin) was purchased from AccuStandard (New
Haven, CT). Latrunculin A was purchased from Molecu-
lar Probes (Eugene, OR).
Cultivation of Parasites, Bacteria, and Yeast
Trophozoites of E. histolytica HM-1: IMSS cl6 were
cultured axenically in BI-S-33 as previously described
[Diamond et al., 1972]. E. dispar CYNO 09: TPC
[Kobayashi et al., 1998] were cultured in YIGADHA-S
medium, as described [Diamond et al., 1978; Kobayashi
et al., 2000]. Promastigotes of green fluorescence protein
(GFP)-expressing L. amazonensis [Chan et al., 2003], a
gift from K. P. Chang and S. Kawazu, were cultured in
199 medium (Nissui Pharmaceutical, Tokyo, Japan) sup-
plemented with 10% heat-inactivated fetal calf serum,
25 mM HEPES and 5 lg/ml tunicamycin. Pseudomonas
aeruginosa PA: KEIO strain [Nozaki et al., 1999] was
cultivated in BI-S-33 medium at 358C. GFP-expressing
Saccharomyces cerevisiae [Sato et al., 2001], a gift of Ko-
ichi Nihei and Akihiko Nakano, Riken, was grown in
MCD medium containing 0.67% yeast nitrogen base with-
out amino acids, 2% glucose, and 0.5% casamino acids
(Difco, Detroit, MI).
Approximately 5 3 104trophozoites were seeded
on a 0.79-cm2well of a collagen-coated glass-bottom
culture dish (MatTek Corporation, Ashland, MA), mixed
with 1 3 106fluorescein isothiocynate (FITC)-labeled
yeasts, GFP-expressing yeasts or GFP expressing-
L. amazonensis (1:20), enclosed with a cover slip, and
further cultured at 338C in a temperature control unit on
an AS Multi Dimension Workstation (AS-MDW, Leica
Microsystems, Wetzlar, Germany). At 20 min, 6–8
images (?40–50 trophozoites/image) were captured
under on a DM IRE2 inverted microscope with a HC
PLAN APO 203/0.70 objective (Leica) integrated in a
Leica AS-MDW system, and the internalized cells were
Measurement of Phagosome pH
Phagosome pH was measured by ratiometry using
[Ohkuma and Poole, 1978]. It has been shown that the
fluorescence spectrum of FITC changes as a function of
pH and is not affected by the FITC concentration (i.e.,
the FITC-labeling efficiency of yeasts), ionic strength, or
associated proteins (i.e., constituents of the media used
or the content of phagosomes) [Ohkuma and Poole,
1978]. FITC-yeasts (5 3 105; Molecular Probes, Eugene,
OR) were suspended in 75% PBS and 25% BI-S-33 or
YIGADHA-S medium supplemented with 137 mM
L-cysteine and 19 mM ascorbic acid and adjusted to pHs
ranging from 4.0 to 7.5, transferred to a collagen-coated
glass-bottom dish and covered with a cover slip. Fluores-
cence and phase contrast images of 10 continuous slices
(z-stack) with 1-lm intervals of FITC-yeasts were
acquired with a Roper Cool Snap HQ digital camera
(Roper Scientific, Duluth, GA) at two excitation wave
lengths (495 and 440 nm) on the system described above
with a BGR filter (#11513838). After the best focused
particles were manually chosen, the fluorescence inten-
sities with two excitation wavelengths of the 1.3 lm2-
circular region of interest (ROI) within a particle were
measured. The fluorescence signal of four ROI per par-
ticle of 20 randomly selected particles was determined at
each pH value. After the background fluorescence signal
intensities were subtracted, the ratio of the fluorescent
signal excited at 495 nm to that excited at 440 nm was
plotted against the pH value to obtain a standard curve.
For the measurement of phagosome pH, all experiments
using live trophozoites were performed using the same
mixture as mentioned above. For the short-term phago-
some acidification, ?5 3 104trophozoites were seeded
on the collagen-coated glass-bottom culture dish, mixed
with FITC-yeasts (5 3 105), enclosed with a cover slip,
and further cultured at 338C. Time-lapse video micro-
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Phagosome Biogenesis in E. histolytica and E. dispar3
scopy was performed at an interval of 30 s for 3 h and
ingested single FITC-yeast containing trophozoites were
selected, and the pH was measured using the standard
curve created above. For long-term phagosome acidifica-
tion, trophozoites (1 3 106) were settled in the wells of a
12-well cell culture plate (Corning Incorporated, Corn-
ing, NY) at 358C for 30 min, gently mixed with FITC-
yeast (5 3 107), sealed the plate, centrifuged for 5 min at
500g for artificial internalization of yeast, washed to
remove noninternalized particles, and cultured again in a
collagen-coated dish at 338C. Time-lapse video micro-
scopy was performed for 12 h at 30 min intervals, and
the pHs of 20–30 ingested particles were measured as
Degradation of GFP-Yeasts and
Approximately 5 3 104trophozoites of E. histolytica
or E. dispar, settled on the collagen-coated glass-bottom
culture dish, were mixed with 5 3 105GFP-yeasts or GFP-
L. amazonensis at a ratio of 1:10 and cultivated as described
above. Time-lapse video microscopy was performed with a
Leica AS MDW system as described above. The fluores-
cence intensity of the whole GFP-yeasts or GFP-Leishma-
nia with an excitation at 489 nm with the GFP filter
(#11513852) was measured by quantifying the intensity
within manually drawn ROI of the maximum projection
images captured at 15–30s intervals for 2.5 h.
Treatment of Parasites With Inhibitors
To test the effects of phagosome acidification and
degradation, ?4–6 3 105trophozoites of E. histolytica or
E. dispar were pretreated for 1 h with 10 lM concanamy-
cin A, a specific inhibitor for V-ATPase; 20–200 lM of
nocodazole, 100–200 lM of oryzalin, inhibitors for
microtubule polymerization [Makioka et al., 2000], 0.25–
0.50 lM of latrunculin A, an inhibitor of actin polymer-
ization [Makioka et al., 2001] or 20–40 mM of BDM, an
inhibitor of myosin ATPase [Borlak and Zwadlo, 2004] or
200 lM of E64, a CP inhibitor in 13 3 100 mm2screw-
capped Pyrex glass tubes at 358C in BI-S-33 or
YIGADHA-S medium, respectively.
E. histolytica and E. dispar trophozoites were har-
vested, washed, and resuspended in BI-S-33 and
YIGADHA-S medium, respectively. The concentrations
of trophozoites were maintained at ?1 3 105cells/ml in
13 3 100 mm2screw-capped Pyrex glass tubes. The
trophozoites were then incubated with 300 ll of a loga-
rithmic culture of P. aeruginosa at 358C for 18 h, washed
three times with PBS, pH 7.4, containing 2% glucose, fol-
lowed by centrifugation at 1500g for 2 min. The cell pellet
was resuspended and prefixed with 2% glutaraldehyde in
PBS for 1 h. After rinsing, the samples were postfixed
with 1% OsO4in PBS for 1 h, stained en bloc with 1%
uranyl acetate, dehydrated in a graded series of ethanol,
and embedded in Epon 812 (TAAB Laboratories Equip-
ment, England). Ultrathin sections were made on an
LKB-ultramicrotome (LKB-Produkter, Sweden). Sections
were stained with uranyl acetate and lead citrate and
examined with a Hitachi-H-700 electron microscope.
Morphological Differences of
P. aeruginosa-containing Phagosomes Between
Pathogenic and Nonpathogenic
To examine the morphological differences of
phagosomes between pathogenic and nonpathogenic
Entamoeba species, trophozoites of E. histolytica and
E. dispar were cultivated with P. aeruginosa for 18 h and
examined under electron and light microscopes. Both E.
histolytica (Fig.1 A and 1C) and E. dispar (Fig. 1B, 1D,
and 1E) trophozoites showed the general ultrastructural
architecture common to this group of parasites, as previ-
ously described [Lushbaugh and Miller, 1988; Espinosa-
Cantellano et al., 1998; Pimenta et al., 2002]. The cyto-
plasm lacks a typical rough endoplasmic reticulum, Golgi
apparatus, and mitochondria. The cytoplasm of both spe-
cies was filled with vesicles and vacuoles varying in size.
Some of these vacuoles contained ingested bacteria and
degraded bacterial debris. Ingested bacteria were always
found to be enclosed within the phagosome membrane in
both species, which contradicts the previous report
[Pimenta et al., 2002]. We failed to count the bacteria
ingested by the trophozoites using DAPI staining, as the
ingested bacteria in the E. histolytica phagosomes were
often degraded and, consequently, some phagosomes were
evenly stained with DAPI (Fig.
the number of bacteria ingested by the trophozoites on
electron micrographs where the membrane structures were
still recognizable after disintegration of the bacterial cell
membrane (Fig. 1). E. histolytica trophozoites ingested a
greater number of bacteria (56.0 6 15.5) than E. dispar
(45.5 6 2.1) [mean 6 standard deviation (S.D.)] per cell.
The average number of bacteria per phagosome in E. his-
tolytica and E. dispar was 2.45 6 0.07 and 1.74 6 0.38,
respectively (p<0.05 with Students t-test). The numbers
of phagosomes containing greater than four bacteria were
higher in E. histolytica than in E. dispar (5% and 14%,
respectively) (Fig. 2E). In E. histolytica, 5% of phago-
somes contained more than seven bacteria, while no phag-
osome contained more than seven bacteria in E. dispar
(data not shown). The average diameter of phagosomes
was slightly higher in E. histolytica than in E. dispar
2A). We thus examined
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4Mitra et al.
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(B, D, and E) cultivated with P. aeruginosa. The arrows indicate phagosomes containing multiple bacte-
ria in both species. Asterisks show phagosomes containing degraded P. aeruginosa in E. histolytica (C)
and E. dispar (E). Bars indicate 1 lm.
Electron micrographs of phagosomes in E. histolytica (A and C) and E. dispar trophozoites
Phagosome Biogenesis in E. histolytica and E. dispar5
(Fig. 2F). This was mainly due to the difference in the
average number of bacteria ingested by these two species,
because the average diameter of phagosomes similarly
increased in both types of amoebae as the number of bac-
Light microscopic observation of the DAPI-stained
trophozoites also revealed that bacteria in the E. his-
tolytica phagosomes appeared to be degraded extensively
(Fig. 2A–2B). A large proportion of phagosomes were
evenly stained and the morphology of the ingested bacte-
ria was no longer recognizable (arrows), while a majority
of ingested bacteria in E. dispar phagosomes retained
their cell morphology (Fig. 2C, arrowheads; also in E. his-
tolytica Fig. 2A).
Comparison of Phagocytosis of Fixed FITC-Yeast,
Live GFP-Yeast, and GFP-L. amazonensis
We compared the efficiency of internalization of a
number of natural and inert particles, i.e., chemically-
fixed FITC-conjugated yeasts, live GFP-yeasts, GFP-
L. amazonensis promastigotes, erythrocytes, and fluores-
cent beads, between E. histolytica and E. dispar. Live
GFP-Leishmania promastigotes were most efficiently
ingested among these particles (Fig.
3) (data of erythro-
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quantitative (E, F) differences in
the ingestion and degradation of
bacteria between the two Enta-
moeba species. A–D: Light micro-
scopic images of E. histolytica
(A, B) and E. dispar (C, D) tropho-
zoites that ingested bacteria and
were stained with DAPI (A, C).
Phase images are also shown (B,
D). Note that, in E. histolytica
trophozoites, the majority of bacte-
ria in phagosomes were lysed and
many phagosomes were evenly
stained with DAPI (arrows), where-
as, in E. dispar trophozoites, most
of the ingested bacteria remained
Nuclei are depicted with ‘‘N’’.
Bars represent 10 lm. E–F: The
number of bacteria ingested per
phagosomes (E) and the average
size of phagosomes (F) in E. histo-
lytica (cross-hatched bars) and E.
dispar (open bars) are shown. Error
bars represent S.D. of the measure-
ment of 120–170 phagosomes.
Qualitative (A–D) and
6 Mitra et al.
cytes and beads not shown) in both E. histolytica and
E. dispar. However, the efficiency was ?6-fold higher in
E. histolytica than in E. dispar. E. dispar failed to ingest
the live GFP-yeasts, even after 3 h of incubation (data
Distinct Kinetics of Phagosome Acidification in
E. histolytica and E. dispar
To examine the kinetics of phagosome acidifica-
tion, we used opsonized yeasts covalently labeled with
pH-sensitive FITC, and measured the phagosome pH
with ratiometry on high-resolution video microscopy.
Trophozoites attached on the glass surface were incu-
bated with and allowed to ingest FITC-yeasts. We cap-
tured images of trophozoites that ingested single yeast at
30 s intervals for 3 h to monitor the phagosome pH.
Upon internalization of yeast particles, phagosomes of
E. histolytica were acidified very rapidly (Fig.
Within 2 min, the pH of phagosomes decreased from that
of the extracellular medium (6.70 6 0.20) to 4.58 6
0.36 in E. histolytica. The phagosome remained acidified
(4.54 6 0.16) for at least 12 h (only data up to 30 min
are shown). In E. dispar, phagosomes were less acidified
than in E. histolytica. Upon ingestion, the pH dropped to
5.83 6 0.38 after 2 min and remained at 5.60 6 0.19 up
to 12 h in E. dispar (only data up to 30 min are shown).
The phagosome acidification occurred at a higher rate in
E. histolytica than in E. dispar; the initial acidification
occurred at the rate of a decrease of 1.06 and 0.44 pH/
min for the first 2 min in E. histolytica and E. dispar,
Phagosome Acidification of E. histolytica and
E. dispar is Mediated by V-ATPases
To see whether V-ATPase is involved in phago-
some acidification in Entamoeba, we examined the
effects of concanamycin A, a potent and specific proton
pump inhibitor [Rathman et al., 1996; Hackam et al.,
1997; Arora et al., 2000]. One hour pretreatment of
trophozoites with 10 lM concanamycin A almost com-
pletely abolished phagosomal acidification both in
E. histolytica and E. dispar (Fig. 4B). The pH of phago-
somes remained at 6.18 6 0.18 or 6.15 6 0.23 in
E. histolytica or E. dispar, respectively. These results are
consistent with the premise that V-ATPases play an
essential role for the initial acidification of phagosomes
and the maintenance of acidification in both species.
Inhibition of Microtubules, Actin, or Myosin Affects
the Kinetics of Acidification in E. histolytica
To investigate whether microtubules were required
for phagosome acidification, we treated trophozoites
with 20–200 lM nocodazole [Scheel et al., 1990; Bayer
et al., 1998], or 100–200 lM oryzalin [Makioka et al.,
2000], which are known to depolymerize microtubules,
for 1 h and then allowed trophozoites to ingest FITC-
yeasts. The acidification of phagosomes was slightly hin-
dered by nocodazole (Figs. 4B and
(Fig. 5B) in both E. histolytica and E. dispar. The pH
reached between 5.17 and 5.21 in the E. histotytica
trophozoites treated with the highest concentrations of
the drugs (Fig. 5A and 5B), while it reached 5.78 6 0.18
in E. dispar treated with 20 lM nocodazole (Fig. 4B)
(the results of treatment of E. dispar with oryzalin was
We also tested whether actin or myosin is also
involved in phagosome acidification in E. histolytica.
Treatment of trophozoites with 0.25–0.5 lM of latruncu-
lin A, an actin inhibitor [Makioka et al., 2001], resulted
in the immobilization of trophozoites and detachment
from the slide glass. The ingestion of the FITC-yeast par-
ticle was also significantly reduced (data not shown).
However, phagosome acidification was only slightly
inhibited (pH 4.97 6 0.08) (Fig. 5C). Treatment of
trophozoites with 20–40 mM BDM, a myosin inhibitor,
gave the most notable reduction of acidification (Fig. 5D,
pH 5.33 6 0.11). The initial phase of the rapid de-
crease in pH, in particular, was significantly perturbed.
The initial acidification occurred at the rate of 0.36 pH
5A) or oryzalin
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by E. histolytica (filled bars) and E. dispar (open bars), respectively. Par-
asites were incubated with opsonised FITC-conjugated yeasts, live GFP
expressing-yeasts, or GFP-expressing Leishmania promastigotes at a 1:20
ratio at 338C for 20 min, captured images, counting the ingested cells per
amoeba. The value is the average of three independent experiments. Error
bars represent the S.D. of the measurement of 250–300 particles.
Phagocytosis of FITC-yeast, GFP-yeast, or GFP-L. amazonensis
Phagosome Biogenesis in E. histolytica and E. dispar7
decrease per minute for the first 2.5 min in the tropho-
zoites treated with 40 mM BDM, while it occurred at
0.56, 0.60, or 0.56 in the cells treated with a highest con-
centration of nocodazole, oryzalin, or latrunculin A,
Distinct Kinetics of Degradation of GFP-Yeasts
or GFP-L. amazonensis Promastigotes in
Phagosomes Between E. histolytica and E. dispar
We next examined the kinetics of disintegration of
ingested particles followed by degradation of a marker
GFP by measuring the fluorescence signal of GFP
expressed by live yeasts or promastigotes of L. amazonen-
sis. A notable difference in the kinetics of GFP
degradation was observed in E. histolytica between GFP-
yeasts and GFP-Leishmania (Fig.
cence of GFP-Leishmania diminished rapidly (94.5% 6
0.71% of the initial GFP fluorescence was lost in the first
30 min), that of GFP-yeasts was more stable (only 26%
GFP fluorescence signal disappeared in 30 min). Since
GFP-expressing Leishmania is the best prey for the
amoeba trophozoites because of its high internalization
rate and stable high fluorescence compared with GFP-
expressing bacteria or yeasts, we used GFP-expressing
Leishmania promastigotes as a model to evaluate degrada-
tion in phagosomes. Degradation of GFP-Leishmania in E.
dispar was slower and less efficient than in E. histolytica.
E. dispar degraded only 30% of the initial fluorescent sig-
nal of ingested GFP-Leishmania in 30 min (Fig. 6A). As
E. dispar failed to ingest live GFP-yeasts, the kinetics of
GFP-yeast degradation was not determined in E. dispar.
6A). While the fluores-
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A: The changes of phagosome pH
in E. histolytica (closed circles) and
phozoites were mixed with FITC-
yeasts at a 1:10 ratio on a glass bot-
tom culture dish, enclosed with a
cover slip, and allowed to ingest
yeast particles at 338C. Tropho-
zoites containing a single particle
were selected and phagosome pH
was measured at 30 s intervals for 3
h with time-lapse video microscopy
on a Leica AS MDW, as described
in the MATERIALS AND METH-
ODS. Only data for the first 30 min
are shown. Error bars represent the
S.D. of 10 independent phago-
somes. B: Effects of concanamycin
A and nocodazole on phagosome
acidification in E. histolytica (filled
symbols and unbroken lines) and E.
dispar (open symbols and dotted
treated with 10 lM concanamycin
A (squares), 20 lM nocodazole
(triangles), or DMSO only (circles)
for 1 h, washed, and incubated, and
described above, except for the
time intervals (2.5 min). Error bars
represent the S.D. of eight inde-
Kinetics of acidification
8 Mitra et al.
Acidification of Phagosomes is Required for the
Efficient Degradation of GFP-L. amazonensis
We determined whether acidification is required for
GFP degradation within phagosomes. E. histolytica and E.
dispar trophozoites were pretreated with 10 lM concana-
mycin A for 1 h, and were then allowed to ingest live
GFP-L. amazonensis. In both E. histolytica and E. dispar,
the degradation of GFP-Leishmania was significantly
inhibited; after concanamycin A treatment, only 44.0 6
1.4 or 1.5% 6 2.1% of the initial GFP signal was lost in
30 min in E. histolytica and E. dispar, respectively (53%
or 95% relative reduction, respectively). Thus, the acidifi-
cation of phagosomes is required for efficient degradation
in phagosomes in both species, as shown for mammalian
cells [Arora et al., 2000]. Although acidification was
almost completely abolished by concanamycin A treat-
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some acidification in E. histolytica. Trophozoites were pretreated with 0, 50, 100, or 200 lM of nocoda-
zole (circles, triangles, squares, or diamonds, respectively) (A); 0, 100, or 200 lM of oryzalin (circles, tri-
angles, or squares, respectively) (B); 0, 0.25 or 0.50 lM latrunculin A (circles, triangles, or squares,
respectively) (C); and 0, 20, or 40 mM BDM (circles, triangles, or squares, respectively) (D). DW repre-
sents distilled water. Error bars represent S.D. of 6–10 phagosomes.
Effects of different concentrations of nocodazole, oryzalin, latrunculin A and BDM on phago-
Phagosome Biogenesis in E. histolytica and E. dispar9
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mastigotes in E. histolytica (filled symbols) and E. dispar (opened
symbols). A: Trophozoites of E. histolytica or E. dispar were incu-
bated with live GFP-yeasts or GFP-Leishmania promastigotes at a
1:10 ratio and images were captured at 30 s interval to measure the
GFP fluorescence intensity. Only data of 2.5 min intervals were
Degradation of GFP-expressing yeasts and Leishmania pro-
shown. Error bars represent the S.D. of 7–8 independent phagosomes.
B: Sequential images of representative experiments showing the deg-
radation of GFP-L. amazonensis promastigotes in an E. histolytica
(top panels) or E. dispar (bottom panels) trophozoite. Arrows indicate
the GFP-Leishmania promastigotes. Time (min, s) is shown at the
10 Mitra et al.
ment in both species, the inhibition of GFP-Leishmania
was more pronounced in E. dispar than in E. histolytica.
This may suggest the unique presence of an alternative
degradation machinery that does not require acidification
for its activation in E. histolytica.
Inhibition of CP or Microtubules Hampers GFP
Degradation in E. histolytica but Not in E. dispar
Inhibition of CPs by an irreversible, potent, and
highly selective CP inhibitor E64 [Katunuma and
Kominami, 1995; Sreedharan et al., 1996] blocked the
degradation of GFP-Leishmania only in E. histolytica
but not in E. dispar (Fig.7). In 30 min, only 39.0% 6
8.7% of GFP-Leishmania was degraded (59% reduction)
in E. histolytica, while E64 showed almost no effect on
the degradation in E. dispar. These data suggest the pres-
ence of not-yet-identified digestive enzymes other than
CP for degradation in phagosomes of E. dispar.
Disruption of microtubules by nocodazole significantly
inhibited the GFP degradation in E. histolytica (at 30 min
only 59% GFP was degraded; 38% reduction), which indi-
cates the importance of microtubules in degradation in phag-
osomes of E. histolytica (Fig. 7). In contrast, in E. dispar,
nocodazole showed no significant effect on degradation.
Morphological Differences in Phagosomes
Between Virulent and Avirulent
Electron micrographs of the trophozoites of axe-
nized E. histolytica and E. dispar strain cultivated with
P. aeruginosa revealed significant differences in both the
number of bacteria in a single phagosome and the aver-
age diameter of phagosomes between these two species
(Figs. 1 and 2). Previously, it was shown that E. dispar
trophozoites contain only a single bacterium in a single
phagosome and a fraction of bacteria were not enclosed
by membrane structures in the cytoplasm [Pimenta et al.,
2002]. Our results strongly argue against these obser-
vations: all ingested bacteria were enclosed in the phago-
somal membrane and phagosomes containing multiple
bacteria were found in E. dispar. Phagosomes of
E. histolytica are significantly larger in size and con-
tained a higher average number of bacteria than those in
E. dispar. These morphological differences are likely
associated with differences in the kinetics of phagosome
acidification and the efficiency of degradation of in-
gested substances in phagosomes demonstrated between
these two species (see below).
Differences in the Kinetics of Phagosome
Acidification Between Virulent and Avirulent
Entamoeba Species and Between Entamoeba
and Other Organisms
We have demonstrated the kinetics of acidification
of individual phagosomes (not as a whole cell) in
Entamoeba using highly sensitive and high-resolution
video microscopy and the pH-sensitive FITC-labeled
yeasts. Differences in the efficiency of phagosome acidifi-
cation between the two Entamoeba species indicate the
significant differences in nature of proton pumping across
the phagosome membrane and membrane trafficking,
leading to phagosome maturation. The abrupt pH
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Leishmania promastigotes in E. his-
tolytica and E. dispar. Trophozoites
were pretreated with concanamycin
A, E64, or nocodazole for 1 h,
washed and mixed with live GFP-
Leishmania promastigotes. Degrada-
tion was monitored as in Fig. 6A.
The fluorescent intensities of 10
independent phagosomes were meas-
Effects of inhibitors on the
Phagosome Biogenesis in E. histolytica and E. dispar11
decrease upon ingestion observed in both species suggests
that direct recruitment of V-ATPase to a newly formed
phagosome or, more likely, swift recruitment and fusion
of lysosomes in the initial phase of phagosome biogene-
sis, as previously shown in macrophages and neutrophils
[Styrt and Klempner, 1982; Lukacs et al., 1990]. Acidifi-
cation of endosomes was also previously studied in
E. histolytica [Meza and Clarke, 2004] and Dictyostelium
discoideum [Clarke et al., 2002a]. A very rapid acidifica-
tion of phagosomes we demonstrated in the present work
is in very good contrast to the rather slow acidification of
endosomes in E. histolytica [Meza and Clarke, 2004]. In
contrast, the persistence of the acidified environment is
shared by both phagosomes and endosomes, suggesting
the presence of a common mechanism for the sustained
acidification of these compartments.
The pH values of the phagosome and its homolo-
gous compartment (digestive vacuole) reported for mouse
macrophages, hemocytes of Mytilus edulis, amoeba
proteus, and D. discoideum vary in a range of 4.5–5.3
[Geisow et al., 1981; Kroschinski and Renwrantz, 1988;
Rupper et al., 2001]. In these organisms, the acidification
of phagosomes occurred within 7–15 min after ingestion
[Jensen and Bainton, 1973; Geisow et al., 1981; Bassoe
and Bjerknes, 1985; Rupper et al., 2001]. The acidifica-
tion of phagosomes in Entamoeba occurred more rapidly
(within 2 min) than these organisms (Fig. 4A).
The persistence of acidity in phagosomes for >12 h
in Entamoeba was surprising, as the maintenance of an
acidic pH is energetically expensive due to the continuous
expenditure of ATP. It has been shown in a few organisms,
including D. discoideum, that following the rapid acidifica-
tion of phagosomes upon the internalization of external par-
ticles, neutralization of phagosomes occurs after 30–60 min
[Rupper et al., 2001]. It was also shown that the digestive
vacuole of Paramecium caudatum began to neutralize
within 8 min after internalization [Fok et al., 1982]. Thus,
Entamoeba may represent a unique organism that sustains
phagosome acidity for an unusually long period, which may
be associated with its high capacity for phagocytosis and
degradation. The sustained acidity of the Entamoeba phago-
somes indicates the continuous presence of V-ATPase on
the phagosome membrane or, alternatively, the existence of
unidentified molecules for the maintenance of a pH gradient
across the phagosomal membrane in Entamoeba. We
should also mention that ?30%–35% of the ingested yeasts
were expelled by E. histolytica trophozoites without com-
plete degradation between 90 and 120 min after internaliza-
tion [Mitra and Nozaki, unpublished]. We reproducibly
observed that a small (0.5–0.7) pH increase occurred <5
min prior to expulsion (data not shown). Taken together, the
persistent acidification of phagosomes may occur only in
cases where the degradation in phagosomes is not com-
pleted, such as in inert latex particles or fixed bioparticles.
V-ATPase is Essential for the Phagosome
Acidification in both Entamoeba Species
Phagosome acidification was completely blocked
with the V-ATPase inhibitor in both Entamoeba species,
similar to mammalian cells, as previously shown [Arora
et al., 2000]. These data, together with our demonstration
of most of the homologs of V-ATPase subunits (i.e.,
A–H subunits of V1subcomplex and a–c subunits of V0
subcomplex, data not shown) in the genome databases
of the both species (http://www.tigr.org/tdb/e2k1/eha1/;
http://www.sanger.ac.uk/Projects/E_dispar/), indicate that
V-ATPase is primarily involved in the initial acidification
and the maintenance of acidic phagosomes in both Enta-
moeba species, as shown for D. discoideum [Rezabek
et al., 1997]. Some of these V-ATPase subunits were also
previously identified from E. histolytica [Descoteaux
et al., 1994]. In addition, our proteomic analysis of phago-
somes purified with sucrose step gradient centrifugation
using carboxylated latex beads also revealed three sub-
units of the V-ATPase V0subcomplex (a, b, and c) and
proteolipid [Okada et al., 2005]. The fact that the inhibi-
tion of V-ATPase severely retarded the degradation of
GFP expressed by the ingested Leishmania promastigotes
indicates that acidification is essential for degradation
It was shown that the inhibition of V-ATPase by
bafilomycin caused a reduction in phagocytosis [Ghosh
and Samuelson, 1997]. It was also shown that V-ATPase
(Vph1p) is involved in the virulence of the AIDS-related
opportunistic fungal pathogen, Cryptococcus neoformans
[Erickson et al., 2001]. Disruption of VPH1 resulted in
defects of four virulence factors, i.e., capsule, laccase, and
urease, and also growth [Erickson et al., 2001]. Thus,
V-ATPase seems to play a variety of roles in the virulent
mechanisms of pathogens.
Involvement of Microtubules, Actin, and Myosin
in the Phagosome Maturation
It is well established in general that the actin cytos-
keleton is essential for phagosome formation. Recent
data suggest that phagosome-bound actin is lost soon
after phagosome formation [Greenberg et al., 1991;
Rupper et al., 2001], indicating the role of the actin-rich
cytoskeleton in the early stages of phagosome matura-
tion. In E. histolytica, it has been demonstrated that
F-actin is present around phagosomes and colocalized
with myosin IB [Voigt et al., 1999]. Voigt et al. also
showed that myosin IB was also present in association
with phagosomes at later stages of phagocytosis, sug-
gesting its constitutive role in phagosome maturation
[Voigt et al., 1999]. Our study, using specific inhibitors
against microtubules, actin and myosin, indicates that
myosin plays a significant role in phagosome acidifica-
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12Mitra et al.
tion of this parasite, while microtubules and actin, which
are inhibitable by conventional inhibitors, are not
directly involved in the process. Our study also suggests
that additional unidentified mechanisms exist for phago-
some acidification in this organism.
Microtubules have been believed to play an essen-
tial role in phagosome maturation through cytoskeletal
reorganization and facilitating fusion between the phago-
somes and endosomes/lysosomes in various organisms,
e.g., [Desjardins et al., 1994; Blocker et al., 1996; Clarke
et al., 2002a]. However, in E. histolytica, the absence of
obvious microtubule-like structures in the cytoplasm, as
demonstrated by EM, suggests that the cytoplasmic
microtubules of this parasite may be particularly labile
to fixation techniques known to preserve microtubules in
other eukaryotic cells, or alternatively, microtubules are
present only in the nuclei [Vayssie et al., 2004]. It was
previously demonstrated that the transport of V-ATPase
to early endosomes in Dictyostelium occurs by the rapid
fusion of endosomes mediated by microtubules [Clarke
et al., 2002a, b]. In macrophages, the depolymerization
of microtubules severely affected degradation of opson-
ized Staphylococcus aureus [Damiani and Colombo,
2003]. As the two Entamoeba species revealed similar
morphological features on the cytoskeleton (this study),
the differential effects of the microtubule inhibitor on
degradation in phagosomes between the two species
were unexpected. Although the microtubule inhibitors
only slightly reduced the phagosome acidification, noco-
dazole notably reduced degradation in phagosomes of
E. histolytica, while almost no inhibition of phagosome
acidification or degradation was found in E. dispar. In
the mouse, one of the V-ATPases, which contains the a3
isoform among the four isoforms (a1–a4) of V0a subunit
[Toyomura et al., 2000], has been shown to localize to
late endosome/lysosomes, and be associated with micro-
tubules and lysosomal marker protein, Lamp2 (Toyo-
mura et al., 2003), as shown by the depolymerization of
microtubules resulting in the dispersion of a3-associated
V-ATPase and Lamp2 [Toyomura et al., 2003]. So, a
disparity in the effects on microtubules and V-ATPase in
E. dispar may suggest a lack of interaction between
microtubules and V-ATPase in E. dispar. Alternatively,
E. dispar was relatively resistant to nocodazole, com-
pared to E. histolytica. It was previously shown that the
sensitivity to nocodazole varied among cell types
[Gruenberg and Howell, 1989; van Deuer et al., 1995]. It
has also been shown that the disassembly of microtu-
bules by nocodazole depends on the temperature and
duration of treatment [Cirillo et al., 1999].
Involvement of CP in Degradation in Phagosomes
It is conceivable to speculate that acidification for
efficient degradation within phagosomes is essential for
the activation of hydrolytic enzymes. Entamoeba kills or
digests bacteria and host cells (in case of E. histolytica)
within their phagolysosomes via oxygen-independent
mechanisms, using a variety of digestive proteins, includ-
ing CP, lysozymes, and amoebapores [Bruchhaus et al.,
1996; Leippe, 1997]. It was shown that the digestion of
internalized collagen in fibroblasts largely depends on the
activity of CP, e.g., cathepsin B [Everts et al., 1996].
Acidification triggers amoebapore activation in E. histoly-
tica [Bruhn et al. 2003]. Our proteomic analysis of phago-
some proteins in E. histolytica revealed a variety of hydro-
lytic enzymes and degradative proteins including, CP1, 2,
4 and 5, phospholipases, dipeptidylaminopeptidase, b-
hexosaminidase, and lysozymes [Okada et al., 2005]. It is
conceivable that continuous acidification is required for
the activation of these various hydrolytic enzymes, which
are recruited at different times (Okada et al, unpublished)
in the course of phagosome maturation.
Degradation in the E. histolytica phagosomes
occurs somewhat faster (?3–4 fold) compared with the
E. dispar phagosomes, which is consistent with the pre-
vious finding that the CP activity in E. histolytica is
10–1000-fold higher than in E. dispar (Bruchhaus et al.,
2003). This also indicates, together with our finding that
the CP inhibitor did not inhibit degradation in phago-
somes of E. dispar that CP may not be the major pro-
tease for digestion in phagosomes in this species. It is
also possible that the active site assembly of CPs has dif-
ferent requirements depending on the type of CPs and
that E64 selectively binds to substrates with a particular
conformation of the active site. Therefore, it is conceiv-
able that some of CPs from E. dispar might be relatively
resistant to E64. Altogether, differences in the efficiency
of internalization, degradation in phagosomes, and
enzymes involved in the degradation between the two spe-
cies are likely associated with the efficiency of removal
and degradation of human cells on intestinal mucosa,
which partially determines the outcome of infection.
Efficient Leishmania Degradation in
We have shown that Entamoeba trophozoites are
able to internalize and degrade Leishmania promasti-
gotes more efficiently than yeasts (Fig. 5A) or erythro-
cytes (data not shown). It is known that Leishmania is
able to protect itself from phagolysosome degradation in
macrophages by inhibiting phagosome-endosome fusion
[Desjardins and Descoteaux, 1997], hydrolytic enzymes
[Sacks et al., 2000], cell signaling pathways [Cunning-
ham, 2002], nitric oxide production [Holm et al., 2001]
and cytokine production [Piedrafita et al., 1999] with the
lipophosphoglycan molecule. Thus, the inability of
Leishmania promastigote’s survival in Entamoeba sug-
gests that the target proteins of Leishmania lipophospho-
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Phagosome Biogenesis in E. histolytica and E. dispar 13
glycan or downstream effectors that Leishmania utilizes
for the impediment of phagosome maturation are absent in
Entamoeba. Conversely, Entamoeba possesses a receptor
to interact with the Leishmania cell surface, leading to the
activation of a downstream signaling pathway necessary
for further phagosome maturation. Since the Entamoeba
parasite does not presently encounter Leishmania in its life
cycle, the intestinal microorganisms that Entamoeba nor-
mally ingests in mammalian intestines likely possess
ligands with structures shared by Leishmania.
The authors thank Shin-ichiro Kawazu, Interna-
tional Medical Center of Japan, Tokyo, and K.P. Chang,
Department of Microbiology and Immunology, Finch
University of Health Sciences, Chicago, for the GFP-
expressing Leishmania amazonensis strain, and Ko-ichi
Nihei and Akihiko Nakano, Riken, for a GFP-expressing
Saccharomyces cerevisiae strain. We also thank Kumiko
Nakada-Tsukui, Mami Okada, Mai Nudejima, Yasuo
Shigeta, and Fumie Tokumaru for their technical assis-
tance and helpful discussion. This work was supported in
part by a grant for Precursory Research for Embryonic
Science and Technology (PRESTO), the Japan Science
and Technology Agency, a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture,
Sports, Science and Technology of Japan to T.N.
(15019120, 15590378, 16017307, 16044250), a grant for
Research on Emerging and Re-emerging Infectious Dis-
eases from the Ministry of Health, Labour and Welfare,
and a grant for the Project to Promote the Development
of Anti-AIDS Pharmaceuticals from the Japan Health
Sciences Foundation to T.N.
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