Acidocalcisomes of
Phytomonas françai
Possess Distinct
Morphological Characteristics and Contain Iron
Kildare Miranda,
1,2
Claudia O. Rodrigues,
2
Joachim Hentchel,
3
Anibal Vercesi,
2,4
Helmut Plattner,
3
Wanderley de Souza,
1
and Roberto Docampo
2,
*
1
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio
de Janeiro, Av. Brigadeiro Trompovski, s/n., bloco G, Cidade Universitária, 21949-900, Rio de Janeiro, RJ, Brazil
2
Laboratory of Molecular Parasitology, Department of Pathobiology and Center for Zoonoses Research, University of Illinois
at Urbana–Champaign, Urbana, IL 61802
3
Department of Biology, University of Konstanz, 78457 Konstanz, Germany
4
Laboratório de Bioenergética, Núcleo de Medicina e Cirugia Experimental, Universidade Estadual Campinas,
Campinas-SP, Brazil
Abstract: Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and
apicomplexan parasites, and recently found in other unicellular eukaryotes. The aim of this study was to
identify the presence of acidocalcisomes in the plant trypanosomatid Phytomonas françai. Electron-dense
organelles of P. françai were shown to contain large amounts of oxygen, sodium, magnesium, phosphorus,
potassium, calcium, iron, and zinc as determined by X-ray microanalysis, either in situ or when purified using
iodixanol gradient centrifugation or by elemental mapping. The presence of iron is not common in other
acidocalcisomes. In situ, but not when purified, these organelles showed an elongated shape differing from
previously described acidocalcisomes. However, these organelles also possessed a vacuolar H
1
-pyrophosphatase
~V-H
1
-PPase! as determined by biochemical methods and by immunofluorescence microscopy using anti-
bodies against the enzyme. Together, these results suggest that the electron-dense organelles of P. françai are
homologous to the acidocalcisomes described in other trypanosomatids, although with distinct morphology
and elemental content.
Key words: Phytomonas, acidocalcisomes, elemental mapping, pyrophosphatase, calcium, transmission electron
microscopy
INTRODUCTION
Infection of plants with trypanosomatids was shown as
early as 1909, when Lafont ~1909! described the presence of
flagellate protozoa in lactiferous plants. Plant trypanosoma-
tids include those that live in the latex of various families of
lacticiferous plants, in the phloem vessels of palms, coffee
trees, and other plants, in the fruit sap and seeds of several
plant families, and in flowers ~Catarino et al., 2001!. Most of
them occur as promastigotes and a few as choanomastigotes
and are transmitted to their plant hosts by phytophagous
insects. It has been generally considered that parasites found
in lactiferous tubes are not pathogenic. One exception is
Phytomonas françai, which infects cassava ~Manihot escu-
lenta!, a plant of economic interest, inducing the poor
development of its root system and chlorosis of aerial parts
~Kitajima et al., 1986!. In addition, some species of intra-
phloematic trypanosomatids are important plant patho-
gens, causing diseases such as phloem necrosis in coffee
~Stahel, 1931; Vermeulen, 1963!, heart rot in coconut
~Parthasarathy et al., 1976! and sudden wilt in oil palm ~Dol-
let et al., 1977!, and are responsible for the destruction of
many plantations in Central and South America ~Camargo,
1999!.
Protozoan parasites can cause diseases that affect mil-
lions of people, animals ~Anonymous, 1997!, and plants
~Camargo, 1999! worldwide, leading to great social and
economic losses. The current chemotherapy against these
parasites faces many problems that include low specificity
and/or high toxicity of the drugs used and drug resistance.
Therefore, it is important to search for biochemical targets
that could be used for rational development of improved
therapies. The understanding of the mechanisms involved
in the control of Ca
21
homeostasis in parasitic protozoa has
gained attention in recent years ~for reviews, see Benaim,
1996; Docampo & Moreno, 1996!. Cell viability requires
perfect functioning of these mechanisms, and disruption of
Ca
21
homeostasis by toxins can lead to cell death ~Berridge,
Received March 15, 2003; accepted May 6, 2003.
*Corresponding author. E-mail: rodoc@staff.uiuc.edu
Microsc. Microanal. 10, 647–655, 2004
DOI: 10.1017/S1431927604040887
MicroscopyAND
Microanalysis
© MICROSCOPY SOCIETY OF AMERICA 2004
First publ. in: Microscopy and Microanalysis 10 (2004), pp. 647-655
Konstanzer Online-Publikations-System (KOPS)
URL: http://www.ub.uni-konstanz.de/kops/volltexte/2007/4234/
URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-42341
1997!. In addition, Ca
21
is known to be involved in the
invasion of host cells by different parasites, a process that is
crucial for maintaining their life cycles ~Moreno et al., 1994;
Lu et al., 1997; Vieira & Moreno, 2000!.
Previous studies concerning Ca
21
homeostasis in try-
panosomatids have demonstrated that most of these cells
have three intracellular Ca
21
pools that comprise the endo-
plasmic reticulum, mitochondria, and acidocalcisomes
~Docampo & Vercesi, 1989a, 1989b; Vercesi et al., 1991a,b;
Docampo & Moreno, 1999, 2001!. Mitochondrial Ca
21
up-
take occurs by an electrophoretic mechanism, is inhibited
by antimycin A, FCCP and ruthenium red, and is stimulated
by respiratory substrates, phosphate and acetate. This pool
has a high capacity and a low affinity for Ca
21
and is able
to buffer external Ca
21
at concentrations in the range
of 0.6–0.7 mM, as occurs with vertebrate mitochondria
~Docampo & Vercesi, 1989a, 1989b!.Ca
21
uptake by the
endoplasmic reticulum occurs via a Ca
21
-ATPase and is
inhibited by high concentrations of vanadate and antical-
modulin agents. This pool has a low capacity and a high
affinity for Ca
21
and is able to buffer external Ca
21
at
concentrations in the range of 0.05–0.1 mM ~Vercesi et al.,
1991b!. Acidocalcisomes are acidic vacuoles that contain a
considerable fraction of intracellular Ca
21
, which can be
released by nigericin, a K
1
/H
1
ionophore, via alkalinization
of the interior of the vacuole ~Vercesi et al., 1994; Docampo
et al., 1995!. They possess a vacuolar-type H
1
-ATPase
and/or a vacuolar H
1
-pyrophosphatase for proton uptake, a
Ca
21
/H
1
countertransporting ATPase for Ca
21
uptake, and
aCa
21
/nH
1
antiporter for Ca
21
release ~Docampo &
Moreno, 1999, 2001!.ANa
1
/H
1
antiporter that may partici-
pateinCa
21
release from these organelles has also been
described in Trypanosoma brucei ~Vercesi & Docampo, 1996,
Vercesi et al., 1997; Rodrigues et al., 1999a! and L. donovani
~Vercesi et al. 2000!.
In this work, we provide evidence for the presence of
acidocalcisomes in the plant trypanosomatid Phytomonas
françai. These acidocalcisomes are morphologically differ-
ent, although functionally similar, to acidocalcisomes of
other trypanosomatids, being characterized by their high
iron content and elongated shape in situ.
MATERIALS AND METHODS
Cell Cultures
Promastigotes of P. françai isolated from cassava ~Vainstein
& Roitman, 1985! were grown in Warren’s medium ~Wa r-
ren, 1960! at 288C supplemented with 10% ~v/v! heat inac-
tivated fetal bovine serum. At 2–3 days after inoculation,
cells were collected by centrifugation and washed twice in
250 mM sucrose ~for TEM preparations! or in Dulbecco’s
phosphate buffered saline ~PBS!. Cells were suspended in
Dulbecco’s PBS for cell fractionation and physiological as-
says. Protein was determined by using the Bio-Rad Coomas-
sie blue method.
Chemicals
Dulbecco’s PBS, ethylene glycol-bis~b-aminoethyl ether!-
N,N,N
'
,N
'
-tetraacetic acid ~EGTA!, 2-hydroxyethyl-1-
piperazineethanesulfonic acid ~HEPES!, protease inhibitors,
and nigericin were purchased from Sigma Chemical Com-
pany ~St. Louis, MO!. Acridine orange ~AO! was obtained
from Molecular Probes ~Eugene, OR!. Polyclonal antibodies
raised against a keyhole limpet hemocyanin-conjugated syn-
thetic peptide corresponding to the hydrophilic loop XII
~antibody PAB
HK
or 326! of plant V-H
1
-PPase ~Zhen et al.,
1997! and aminomethylenediphosphonate ~AMDP!~Zhen
et al., 1994! were kindly provided by Prof. Philip Rea,
University of Pennsylvania ~Philadelphia, PA!. All other
reagents were of analytical grade.
Conventional Transmission Electron Microscopy
Cells were washed in Dulbecco’s PBS, pH 7.2, fixed in 2.5%
glutaraldehyde, 4% paraformadehyde in 0.1 M cacodylic
acid buffer, postfixed in 1% OsO
4
plus 0.8% ferrocyanide
and5mMCaCl
2
in 0.1 M cacodylic acid buffer for 30 min,
dehydrated in acetone series, and embedded in Polibed 812
epoxide resin. Sections of 70 nm were obtained and stained
for 40 min in 5% aqueous uranyl acetate and for 5 min in
lead citrate. Observation was made in a Zeiss 900 transmis-
sion electron microscope operating at 80 kV.
Imaging of Unfixed Whole Cells
Cells were washed and resuspended in 250 mM sucrose.
Drops were applied to 200 mesh formvar/carbon-coated
copper grids, allowed to adhere for 10 min, carefully blotted
dry, and observed in an energy-filtering LEO EM 912 trans-
mission electron microscope operating at 120 kV. Electron
spectroscopic images were recorded at an energy loss of
60 eV using a spectrometer slit width of 20 eV.
Electron-Probe X-Ray Microanalysis
Energy-dispersive X-ray spectra were recorded from the
acidocalcisomes present in whole cells adhered to formvar/
carbon-coated copper grids. As control, spectra were col-
lected from regions of the cytoplasm adjacent to acido-
calcisomes and from formvar/carbon film. Specimens were
analyzed in a LEO-912 transmission electron microscope
operating at 80 kV. X rays were collected for 300 s using a
Si~Li! detector with an ultrathin window in a 0–10-keV
energy range with a resolution of 10 eV/channel. Analyses
were performed using an Oxford Link ISIS system attached
to the microscope.
648
Kildare Miranda et al.
Elemental Mapping
X-ray mappings were acquired in a LEO 912 Omega
equipped with a scanning device. The microscope was oper-
ated at 80 kV using a tungsten filament, in the scanning
transmission ~STEM! imaging mode, spot size was 40 nm,
and emission current ;10 mA. X rays were collected by a
Li-drifted Si-detector ~frontarea30mm
2
!equipped with
an ATW atmospheric window. Analyses were performed
using a Link multichannel energy analyzer and Link ISIS
3.00 software ~Oxford Instruments, Wiesbaden, Germany!.
Immunofluorescence Microscopy
Cells fixed in freshly prepared 4% formaldehyde were al-
lowed to adhere to poly~L-lysine!-coated coverslips, perme-
abilized with 0.3% Triton X-100 for 3 min, and blocked
with 50 mM ammonium chloride and 3% bovine serum
albumin in PBS. Immunofluorescence was carried out using
a 1:100 dilution of anti-V-H
1
-PPase antibody and a fluores-
cein isothiocyanate-coupled goat anti-rabbit IgG secondary
antibody ~1:100!. Fluorescence images were obtained with
an Olympus BX-60 fluorescence microscope fitted with a
493-nm excitation filter, a CCD camera, and an image
analysis system.
Isolation of Electron-Dense Organelles
Electron-dense organelles were isolated by the iodixanol
method similar to that described before for the isolation of
acidocalcisomes of Trypanosoma cruzi ~Scott & Docampo,
2000!. Cells were washed twice with PBS, pH 7.2, and
suspended in lysis buffer ~20 mM HEPES, pH 7.2, 50 mM
KCl, 125 mM sucrose, 0.5 mM ethylene diamine tetraacetic
acid ~EDTA!, 5 mM dithiothreitol, 0.1 mM 4-~2-amino-
ethyl!benzenesulphonyl fluoride, 10 mM pepstatin A, 10 mM
leupeptin, and 10 mM E-64!. Cell pellets of Phytomonas
were mixed with approximately 23 wet weight silicon car-
bide ~Aldrich! and ground with a pestle and mortar until
cell lysis reached 90%. The lysate was subsequently sus-
pended in lysis buffer and centrifuged 10 min at 580 3 g to
remove the silicon carbide, unbroken cells, and debris. The
supernatant was centrifuged for 10 min at 15,000 3 g, and
the pellet obtained was suspended in 4 ml of lysis buffer
with the aid of a 22-gauge needle and applied to a discon-
tinuous gradient of iodixanol, with 4-ml steps of 24, 28, 34,
37, and 40% iodixanol diluted in lysis buffer. The gradient
was centrifuged at 50,000 3 g in a Beckman SW 28 rotor for
60 min. The resulting fractions were suspended in lysis
buffer and used in physiological assays. The fraction pel-
leted on the bottom of the tube was also used for electron
microscopy preparations.
Proton Pump Activity
Pyrophosphate-driven proton transport was assayed by
measuring changes in the absorbance of acridine orange
~A
493
-A
530
! in an SLM-Aminco DW 2000 dual wavelength
spectrophotometer. Cells were incubated at 308C in 2.5 ml
130 mM KCl standard reaction medium containing in addi-
tion 2 mM MgSO
4
, 10 mM HEPES, 50 mM EGTA, 1 mM
oligomycin, 100 mM sodium pyrophosphate, 3 mM acridine
orange, and different inhibitors where indicated. Each exper-
iment was repeated at least three times with different cell
preparations, and Figure 4, below, show representative
experiments.
RESULTS
In contrast to other eukaryotic cells, most of the calcium in
different trypanosomatids such as T. cruzi ~Docampo et al.,
1995; Scott et al., 1997; Miranda et al., 2000!, T. brucei
~Vercesi et al., 1994; Scott et al., 1998; Rodrigues et al.,
1999a!, Leishmania mexicana amazonensis ~Lu et al., 1997;
Vannier-Santos et al., 1999! and Leishmania donovani ~Rod-
rigues et al., 1999b! is concentrated in the acidocalcisomes.
Acidocalcisomes have been morphologically defined in rou-
tine TEM preparations as empty vacuoles with a single
membrane, circular shape, and electron-dense cores associ-
ated with the inner face of their membranes ~Scott et al.,
1997, 1998; Rodrigues et al., 1999a, 1999b; Miranda et al.,
2000!. This aspect as empty vacuoles is mainly attributed to
the extraction of their mineral content during the conven-
tional processing for transmission electron microscopy,
which involves steps of fixation, dehydration, and embed-
ding of the material ~Miranda et al., 2000!. When cryo-
techniques such as high-pressure freezing followed by freeze-
substitution, cryosectioning followed by freeze-drying, or
even when whole intact cell mounting techniques for TEM
are applied, the acidocalcisomes appear as spherical electron-
dense organelles with the whole matrix filled by an electron-
dense material, which is mainly composed of oxygen, sodium,
magnesium, phosphorus, potassium, calcium, and zinc as
probed by energy-dispersive X-ray microanalysis ~Scott et al.,
1997; Miranda et al., 2000!.
To investigate the presence of acidocalcisomes in P.
françai, unfixed whole cells were air dried on formvar/
carbon-coated TEM grids and observed in an energy-
filtering TEM. This method was chosen to prevent and/or
minimize the extraction of the mineral content of the
acidocalcisomes during the routine TEM specimen prepara-
tion. Electron spectroscopic images showed a large number
~208 6 25! of dense organelles spread throughout the para-
site cell without any preferential location ~Fig. 1A!.In
contrast to the parasites thus far studied, the electron-dense
organelles of P. françai were not all spherical. They appeared
as pleomorphic structures with an elongated shape varying
from 45 nm to 130 nm in width and 80 nm to 270 nm in
length ~Fig. 1B!. Energy dispersive X-ray microanalysis
showed considerable amounts of oxygen, sodium, magne-
sium, phosphorus, potassium, calcium, iron, and zinc in all
Acidocalcisomes of P. françai 649
dense organelles analyzed ~Fig. 1C!. X-ray microanalysis
performed on different regions of the cytoplasm ~Fig. 1D!
and on fomvar/carbon film ~Fig. 1E! did not show the
calcium, iron, and zinc peaks characteristic of the electron
dense organelles. Except for the presence of iron, the elemen-
tal composition of these organelles was similar to that
found in acidocalcisomes of T. cruzi ~Scott et al., 1997;
Miranda et al., 2000!, T. brucei ~Rodrigues et al., 1999a!, and
L. donovani ~Rodrigues et al., 1999b!, thus providing evi-
dence that, from the morphological point of view, the dense
organelles of P. françai correspond to iron-rich acidocalci-
somes. Elemental mapping showed that most of the magne-
sium, phosphorus, calcium, iron, and zinc is located within
the acidocalcisomes ~Fig. 2!. In thin sections, these organ-
elles appeared membrane limited, with the membrane sur-
rounding electron-dense inclusions ~Fig. 3A–G!. As observed
in whole cells, many vacuoles displayed an elongated shape
~Fig. 3B,E,F!. Some presented two or more inclusions
~Fig. 3F! whereas others appeared completely filled, with the
electron-dense material arranged in concentric patterns,
suggesting the sequential deposition of material in the or-
ganellar matrix ~Fig. 3E,G!. Elongated structures resembling
endoplasmic reticulum with an enlarged matrix also contain-
ing an electron-dense material were frequently seen ~Fig. 3C!.
X-ray microanalysis confirmed that these organelles have
the same composition as the dense organelles seen in whole-
cell preparations ~Fig. 3D!.
Immunolocalization using polyclonal antibodies raised
against a plant vacuolar proton pyrophosphatase showed a
strong reaction with small intracellular structures in P.
françai ~Fig. 4A,B!, indicating the presence of a H
1
PPase
in these parasites. Cell fractionation performed using the
iodixanol method, described before for the isolation of the
acidocalcisomes of T. cruzi ~Scott & Docampo, 2000!,re-
sulted in fractions in which it was possible to detect PP
i
-
driven H
1
uptake ~Fig. 4C,D!. Addition of PP
i
was followed
by a decrease in acridine orange absorbance, which was
reversed by the addition of AMDP, a pyrophosphate analog
which is known to specifically inhibit the H
1
pyrophos-
phatase, and by the K
1
/H
1
ionophore nigericin ~Fig. 4C!.As
Figure 1. Presence of calcium-rich organelles in whole promastigotes. A: Electron spectroscopic image ~ESI! of whole
unstained cells showing numerous electron-dense organelles spread throughout the cell. B: High magnification showing
the heterogeneity of shapes of dense organelles in P. françai. Note that some are almost spherical ~arrows! whereas others
appear as elongated structures ~arrowheads!. C: Typical X-ray spectrum of a dense organelle. Peaks of calcium, iron,
phosphorus, sodium, magnesium, oxygen, carbon, and zinc, similar to that already described for the acidocalcisomes of
different trypanosomatids ~except for the presence of iron!, can be observed. Occasionally potassium could also be
found in some organelles. D: X-ray spectrum collected from a cytoplasmic region adjacent to the dense organelles.
E: Control X-ray spectrum collected from the formvar/carbon film. Ti comes from the specimen holder and copper
from the electron microscope grid. Bars: A: 1.3 mm; B: 1 mm.
650 Kildare Miranda et al.
shown in Figure 4D, the maximum activity was found in
fraction 14 ~bottom of the gradient!. When this fraction
was applied to formvar/carbon-coated electron microscope
grids, it was shown to consist mainly of electron-dense
organelles with the same morphology ~Fig. 4E! and com-
position ~Fig. 4F! as the dense organelles found in whole
cells.
DISCUSSION
Previous studies applying X-ray microanalysis and/or ele-
mental mapping have reported the presence of iron-rich
organelles in different trypanosomatids. Trypanosoma cy-
clops contains a prominent iron-containing vacuole only
visible by light microscopy when cells are grown in the
presence of hemoglobin, and for this reason they were
thought to represent a phagosome or storage body con-
taining haem derived from the incomplete digestion of
hemoglobin ~Vickerman & Tetley, 1977!. Herpetomonas
samuelpessoai also exhibit iron-rich electron-dense organ-
elles similar to those found in T. cyclops, where iron may
result from the degradation of hemin, an essential compo-
nent of the culture media ~Carvalho & de Souza, 1977!.
Elemental mapping of epimastigotes revealed substantial
differences in the iron content in different strains of T. cruzi
and in the color of the cell pellets ~cells containing a high
iron content displayed a brown color whereas cells with a
low iron content displayed a white color; Dvorak et al.,
1988!. A subsequent quantitative work done in cryosections
of T. cruzi identified two electron dense organelles in the
epimastigote form ~Scott et al., 1997!. One was rich in
calcium, phosphorus, and zinc but with no iron, thus corre-
sponding to the acidocalcisome, and the other was calcium
free, less electron dense if compared to acidocalcisomes, and
contained high concentrations of iron and chloride. On the
basis that epimastigotes are cultured in the presence of
hemin and possess organelles of the endo-/lysosomal sys-
tem ~reservosomes! that are acidic and of similar size, the
authors suggested that the iron-rich vesicles could corre-
spond to the reservosomes ~Scott et al., 1997!. Accordingly,
a later work where X-ray spectra were collected from reser-
vosomes of epimastigotes that had incorporated gold-
labeled albumin showed the presence of iron in these
organelles ~Miranda et al., 2000!. Therefore, it is generally
believed that the presence of iron in trypanosomatids is
directly related to the degradation of incorporated iron-
containing nutrients and growth factors ~i.e., hemin, hemo-
globin, and hematin! that are prelocalized in the newly
formed endocytic compartments.
In P. françai, one remarkable characteristic of the aci-
docalcisomes is the presence of a high concentration of
iron, a feature that, up to now, was unique to this parasite,
except for its presence in acidocalcisomes of bloodstream
forms of T. cruzi ~Correa et al., 2002!. Recent results from
our laboratories have indicated that iron is also present in
the acidocalcisomes of other members of the Trypanosoma-
tidae family when cells are grown under similar conditions
to those used to grow P. françai in this work ~unpubl.
Figure 2. Elemental mapping of whole cells. Elemental mapping performed in whole cells shows that most of the
calcium is localized within the acidocalcisomes comineralized with phosphorus, magnesium, sodium, iron, and zinc.
Sulfur, although present, is not located within the acidocalcisomes. Bar: 1.1 mm.
Acidocalcisomes of P. françai
651