Differences in morphology of phagosomes and kinetics of acidification and degradation in phagosomes between the pathogenic Entamoeba histolytica and the non-pathogenic Entamoeba dispar.

Abstract

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 microscopy and live imaging with video microscopy between the virulent E. histolytica and the closely-related, but non-virulent 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 fluorescein isothiocynate-conjugated yeasts showed that phagosome acidification occurs within 2 min and persists for >12 h in both species. The acidity of phagosomes significantly differed between two species (4.58 +/- 0.36 or 5.83 +/- 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 specific inhibitor of vacuolar ATPase, concanamycin A, interrupted both the acidification 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 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.

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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).
DOI: 10.1002/cm.20087
*Correspondence to: Tomoyoshi Nozaki, Department of Parasitology,
Gunma University Graduate School of Medicine, 3-39-22 Showa-
machi, Maebashi, Gunma 371-8511, Japan.
E-mail: nozaki@med.gunma-u.ac.jp
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-
ences Foundation.
' 2005 Wiley-Liss, Inc.
Cell Motility and the Cytoskeleton 62:000–000 (2005)

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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
INTRODUCTION
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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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
Chemicals
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).
Phagocytosis Assays
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
counted manually.
Measurement of Phagosome pH
Phagosome pH was measured by ratiometry using
FITC-conjugatedyeasts
[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-
aspreviouslydescribed
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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
mentioned above.
Degradation of GFP-Yeasts and
GFP-L. amazonensis
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.
Electron Microscopy
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.
RESULTS
Morphological Differences of
P. aeruginosa-containing Phagosomes Between
Pathogenic and Nonpathogenic
Entamoeba Species
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
F1
F2
2A). We thus examined
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Fig. 1.
(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