R E V I E W S
Biology and Center for
Tropical and Global
Pathobiology and Center for
University ofIllinois at
§Instituto de Biofisica Carlos
Federal do Rio de Janeiro,
Rio de Janeiro,Brazil.
||Laboratório de Biologia
Celular e Tecidual da
Universidade Estadual do
Correspondence to R.D.
NATURE REVIEWS | MICROBIOLOGY
VOLUME 3 | MARCH 2005 | 251
Infection with trypanosomatid parasites, including
African and American trypanosome species and para-
sites from the Leishmaniaand Phytomonasgenera,are
among the most widespread human,animal and plant
parasitic diseases worldwide and are responsible for
large socio-economic losses,especially in developing
countries.Trypanosoma cruziis the aetiological agent of
Chagas disease or American trypanosomiasis1.At least
20 species of Leishmaniaare known to infect humans,
causing cutaneous,mucocutaneous and visceral leish-
maniasis2. African sleeping sickness is caused by
Trypanosoma brucei gambienseand Trypanosoma brucei
rhodesiense3.Most of these trypanosomatid species
also infect animals,and in some areas,such as in sub-
Saharan Africa,such infection has precluded using
some domestic animals to provide food3.Plant try-
panosomatids — known as Phytomonas spp.— can
also result in devastating diseases,including phloem
necrosis in many coffee plant species, heart rot in
coconut plants and sudden wilt in oil palm4.
Trypanosomatids belong to the family Trypano-
somatidae and are of the order Kinetoplastida, and
are characterized by their different cell morphologies
during stages of the life cycle,the most important of
which are shown in FIG.1a.Trypanosomatids contain
organelles that are typical of most eukaryotic organ-
and the endoplasmic reticulum,and have well-developed
endocytic and secretory pathways. Some of the try-
panosomatid organelles have features that are unique to
this group (FIG.1b; BOX 1).Trypanosomatids were also
the first cells in which acidocalcisomes were identified5,6.
Acidocalcisomes are dense acidic organelles — both in
terms of weight and as shown by electron microscopy
— with a high concentration of phosphorus present as
pyrophosphate and polyphosphate complexed with
calcium and other elements7. Acidocalcisomes are
related to organelles that were previously known as
volutin or metachromatic granules8and polyphosphate
vacuoles9,and which were thought to contain nucleic
acids and/or to function as storage granules10
(TIMELINE).The discovery that trypanosomatid acido-
calcisome membranes contain several pumps and
exchangers suggested a metabolic function.After their
identification in trypanosomatids,acidocalcisomes
were found in several microorganisms such as
Toxoplasma gondii11,which is the aetiological agent
of toxoplasmosis, Plasmodium spp.12–14, which are
the causative agents of malaria, the green alga
Chlamydomonas reinhardtii15and the slime mould
Dictyostelium discoideum16.The recent identification of
acidocalcisomes in bacteria17,18and human platelets19
indicates that these organelles have been conserved
from bacteria to humans.
ACIDOCALCISOMES — CONSERVED
FROM BACTERIA TO MAN
Roberto Docampo*,‡,Wanderley de Souza§,Kildare Miranda§,||,Peter Rohloff‡and
Abstract | Recent work has shown that acidocalcisomes, which are electron-dense acidic
organelles rich in calcium and polyphosphate, are the only organelles that have been conserved
during evolution from prokaryotes to eukaryotes. Acidocalcisomes were first described in
trypanosomatids and have been characterized in most detail in these species. Acidocalcisomes
have been linked with several functions, including storage of cations and phosphorus,
polyphosphate metabolism, calcium homeostasis, maintenance of intracellular pH homeostasis
and osmoregulation. Here, we review acidocalcisome ultrastructure, composition and function
in different trypanosomatids and other organisms.
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Acidocalcisomes are rounded organelles with an
8-nm-thick membrane.They can be stained with dyes
such as acridine orange5,6or cycloprodigiosin20that
accumulate in acidic compartments and they can be
observed as cytoplasmic granules in Giemsa-stained
The organelle contains an amorphous and electron-
dense material (FIG.2),but the amount seen is dependent
on the method by which the sample is prepared for elec-
tron microscopy.Using standard staining methods for
transmission electron microscopy,part of the dense
material can be lost,leaving either an empty vacuole
(FIG.2e)or a thin layer ofdense material that sticks to the
inner face ofthe membrane.In some trypanosomatids7,21
and bacteria17,the dense material adheres to one side of
the membrane as an inclusion (FIG. 2a). In some
Phytomonasspecies,the electron-dense material seems to
be arranged in a concentric pattern22(FIG.2d).Electron-
dense material is also seen to precipitate in cells that are
fixed using potassium pyroantimoniate23or potassium
fluoride (K.M.et al.,unpublished observations),both of
which are known to precipitate calcium.Acidocalcisomes
are best preserved in cells that are fixed using cryotech-
niques, such as physical fixation by high-pressure
freezing followed by freeze substitution,where they
seem completely filled with an electron-dense
material24(FIG.2b),or when frozen sections are directly
observed at low temperature in the electron
microscope25(FIG.2c).Another useful method to observe
acidocalcisomes is to allow whole cells to dry onto
carbon- or formvar-coated grids in the transmission
electron microscope25,especially if it is equipped with
an energy filter,so that electron spectroscopic images
(contrast-tuned images) can be obtained22,24,26,27(FIG.3a–d).
The general morphology of acidocalcisomes varies
according to the species and the cultivation medium.
Generally,the organelles are spherical structures with
an average diameter of ~0.2 µm in T.cruzi20,24(FIG.3c),
and human platelets19,butthey can be 0.6 µm in some
Leishmania spp.26or 0.05 µm in merozoites of
Plasmodium falciparum29.In some organisms,such as
in some Leishmania26and Phytomonas isolates22,27,
acidocalcisomes are elongated and polymorphic.In
trypanosomatids,the organelles are usually distributed
throughout the cells,but seem to preferentially localize
to the central portion of the cell body or in close
proximity to the contractile vacuole30.They can also
localize to the flagellum,only occasionally in epimas-
tigotes of T. cruzi24but frequently in promastigotes
of Blastocrithidia culicis27(FIG.3a).In trypomastigotes of
T. cruzi, they are preferentially localized to the ante-
rior portion (the region of the parasite from which
the flagellum emerges)24.In other cells15–19,they are
usually randomly distributed,although in some bacte-
ria they can be close to one pole17.In addition,acidocal-
cisomes are sometimes aligned (FIG.3c,d),which might
indicate an interaction with cytoskeletal components.
In some electron microscopy images apparent budding
of new acidocalcisomes can be observed (FIG. 3c,d).
Figure 1 | Schematic representation of trypanosomatids. a | The main cellular forms of
trypanosomatids as defined by cell shape, flagellum presence and attachment, and position of
the basal body, kinetoplast and nucleus. In general, the epimastigote and promastigote forms of
digenetic trypanosomatids are found in the vector, the trypomastigote form is found in the
mammalian host and the amastigote form is intracellular. b | Schematic representation of
longitudinal section of an epimastigote form of T. cruzi. Part b is modified from a drawing by Flavia
Moreira-Leite, University of Oxford.
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R E V I E W S
Several pumps and exchangers and at least one channel
have been identified in the acidocalcisome membrane
Calcium pumps. A Ca2+-ATPase that is sensitive to
vanadate and present in an acidic compartment was
first identified in experiments using permeabilized
T.brucei5and T.cruzi6cells and later detected in iso-
lated acidocalcisomes from both parasite species20,28.
Genes encoding acidocalcisomal Ca2+-ATPases were
identified in T. cruzi (tca1)23, T. brucei (TbPMC1)31,
T.gondii32and D.discoideum16,33.The T.cruzi,T.brucei,
and T. gondii genes were able to complement yeast
mutants that were deficient in the vacuolar Ca2+-ATPase
gene PMC1,providing evidence of their functionality.
Close contact between the acidocalcisome and the
nucleus,lipid inclusions,mitochondria and subpellicular
microtubuleshas also been observed24.
The number of acidocalcisomes varies from species
to species and even among the developmental stages of
the same species.For instance,in T.cruzi,amastigote
forms (FIG.3c)contain more acidocalcisomes (about 40
distributed throughout the cell) than epimastigotes and
trypomastigotes24.A MORPHOMETRIC STUDY in different
trypanosomatids showed that,although the numbers
and sizes of acidocalcisomes vary,the volume of the cell
that is occupied by acidocalcisomes remains ~2%.The
size of the organelle seems to be inversely proportional
to the number of organelles present — acidocalcisomes
are often large when present in low numbers and small
when there are many present27.
A cytoskeletal structure of
microtubules that forms flagella
An invagination of the plasma
membrane that is used to
incorporate external material.
The diameter of acidocalcisomes
in electron microscopy sections
is measured and their volume is
calculated assuming that they
are perfect spheres.
Box 1 | Trypanosomatid model systems
Although typanosomatids contain organelles and metabolic pathways that seem to be absent from prokaryotes and other
eukaryotes,some ofthe organelles and metabolic routes first discovered in trypanosomatids were later found in other
organisms.For this reason,and owing to the global burden oftrypanosomatid diseases,these organisms are now used as
model systems in cell biology.
GPI-linked surface molecules
The main cell surface molecules oftrypanosomatids are rich in glycosylphosphatidylinositol (GPI)-anchored
glycoproteins and GPI-related glycolipids.Early composition and structural studies on Trypanosoma bruceivariant
surface glycoprotein93,which is responsible for antigenic variation in these parasites,were important for understanding
this new type ofmembrane attachment molecule94.
Cytoskeleton and cellular organization
The cytoskeleton ofthe uniflagellated trypanosomatids,which determines their cell shape,has a subpellicular array of
microtubules that are crosslinked to each other and to the plasma membrane95.The organization ofthe helical pattern
ofthe microtubules after cell division under the influence ofa pre-existing cytoskeletal structure (by the flagellar
connector) is one ofthe few examples ofcytotaxis96.In addition to a conventional AXONEME,the flagellum of
trypanosomatids has an associated structure known as the paraflagellar rod.It is formed by a complex array offilaments
and is involved in flagellar motility21.
Trypanosomatids are highly polarized cells.All their endocytic and exocytic functions occur through the flagellar pocket
and,in some cases,the CYTOSTOME21.
DNA and RNA biology
The single trypanosomatid mitochondrion contains a kinetoplast,which is a specific structure that is found adjacent to
the basal body ofthe flagellum and contains approximately 5–20% ofthe total cellular DNA.The DNA-rich kinetoplast
can be stained and viewed using light microscopy,and was the first extranuclear DNA to be discovered,long before
mitochondria were shown to contain DNA97.Kinetoplast DNA is a large network ofseveral thousand similar copies of
minicircles and a few dozen copies ofmaxicircles98.The maxicircle DNA encodes ribosomal RNAs and a few
mitochondrial proteins,in common with the mitochondrial DNA ofother eukaryotes.Many maxicircle transcripts
undergo RNA editing,a process first discovered in trypanosomatids99,whereas the minicircles encode for small guide
RNAs that control the specificity ofediting100.
Many trypanosome mRNAs are trans-spliced — the transfer ofsplice leader sequences or mini-exons to the
polycystronic mRNAs101.This process,together with polyadenylation,functions to cleave polycistronic transcripts and
attach a cap to mRNAs,and has subsequently been found in nematodes,euglenoids,trematodes and chordates102.
In all trypanosomatids most glycolytic enzymes are found in specialized peroxisomes known as glycosomes103,which
contain typical peroxisomal enzymes and develop by a biogenesis pathway similar to peroxisomes.The
compartmentalization ofthe glycolytic pathway in these organelles is important for glycolysis regulation and is unique to
One unique metabolic feature oftrypanosomatids is their substitution oftrypanothione (a glutathione–spermidine
conjugate)105for glutathione in many reactions involved in protection against oxidative stress,such as trypanothione
reductase and trypanothione-dependent peroxidase activities106.
Several trypanosomatids contain a contractile vacuole that functions in water extrusion and osmoregulation74,75.
Although the vacuole is important for free-living kinetoplastids like Bodospp.76,it is also relevant for parasites such as
Trypanosoma cruzi30,66,which are exposed to wide variations in osmolarity during their life cycle.
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are K+-stimulated (type I) and can be used as markers
for acidocalcisome purification15,16,28,41–43,46.Although
they are not restricted to the acidocalcisome,they are
concentrated in this organelle.The T.cruziV-H+-PPase
is also found in the Golgi complex and in the plasma
Na+/H+and Ca2+/H+exchangers.A Na+/H+exchanger
has been found in T.bruceiprocyclic forms48,49and in
L.donovanipromastigotes50.This exchanger is sensitive
to 3,5-dibutyl-4-hydroxy toluene (BHT) but tolerant to
5-(N-ethyl-N-isopropyl) amiloride (EIPA),in contrast
to other Na+/H+exchangers. A Ca2+/H+exchanger is
thought to be involved in Ca2+release when Na+is added
to the organelles in situ48–50or in vitro28,and it might
function in the release of Ca2+from the organelles
because second messengers,such as inositol trisphos-
phate (InsP3),did not release Ca2+from intracellular
Ca2+stores ofthese organisms51–53.Although the isolated
organelles from T.bruceiprocyclic trypomastigotes28
and L.donovanipromastigotes43have a Na+/H+exchanger,
this exchanger is absent from acidocalcisomes of
T. cruzi20. They have different properties because
ADP stimulates the T. brucei49exchanger but not the
Aquaporins.A water channel,or aquaporin,has been
found in T.cruzi acidocalcisomes30.This protein can
function as a water channel in Xenopusoocytes but is
unable to transport glycerol.The T.cruziaquaporin is
localized to both the contractile vacuole complex and
the acidocalcisome, which might indicate a role in
The matrix of the acidocalcisome is electron-dense and,
in common with volutin granules,this was thought to
be due to the high concentrations of phosphorus com-
pounds inside this compact structure.Incubating fixed
These Ca2+-ATPases are closely related to the family of
plasma membrane calcium ATPases (PMCA) and localize
to acidocalcisomes.Interestingly,T.bruceihas a second
PMCA-type Ca2+-ATPase (TbPMC2) that localizes to
the plasma membrane31,whereas the T.cruziPMCA-
type Ca2+-ATPase is present in both acidocalcisomes
and plasma membranes23.The acidocalcisomal Ca2+-
ATPases of T. cruzi23, T. brucei31, T. gondii32and
D. discoideum16,33, and the vacuolar Ca2+-ATPases of
yeast34and Entamoeba histolytica35form a subcluster
among the conserved core sequences of all PMCA-
type Ca2+-ATPases.A common feature of these pumps
is the lack of a calmodulin-binding domain,which is
found in other PMCA-type Ca2+-ATPases.
Proton pumps. Two proton pumps — a vacuolar-
type H+-ATPase (V-H+-ATPase) and a vacuolar-type
H+-pyrophosphatase (V-H+-PPase) — have been found
in acidocalcisomes from different microorganisms.
The V-H+-ATPase was identified in permeabilized
T.brucei5and T.cruzi6cells owing to its sensitivity to
bafilomycin A1, which is a specific inhibitor of this
proton pump36,and this finding was later confirmed in
experiments using intact cells of T.cruzi6,T.brucei37,
Leishmania amazonensis38and T. evansi39. Acido-
calcisomeV-H+-ATPases were also found in T.gondii40,
C. reinhardtii15,D.discoideum16and human platelets19.
The V-H+-ATPase co-localizes in acidocalcisomes with
the vacuolar-type Ca2+-ATPase in T. cruzi23but is
absent from the endocytic pathway of these parasites40,
which is in contrast to its presence in this pathway in
A V-H+-PPase has been detected in T. cruzi41,
Phytomonas françai22, T. gondii44, P. falciparum13,
Plasmodium berghei14,C.reinhardtii15and D.discoideum16.
This enzyme also localizes in acidocalcisomes in all
these species (FIG. 2e). The V-H+-PPase from T. cruzi
functions in yeast45.The acidocalcisomal V-H+-PPases
granules that stain pink
with basic blue dyes
are first described in
Meyer8changes the term to
volutin because he realizes
that it is a chemical, and
devises detection tests
based on granule stability and
staining with methylene blue.
The term ‘volutans granules’ is
used123to describe granules
that occur in plant, fungal and
bacterial cells and that have
staining properties similar to
those of the metachromatic
granules of Spirillum volutans.
Volutin granules are
identified with Meyer’s tests
in unicellular eukaryotes,
Wiame127shows that the
presence or absence of a
yeast chemical determines
the metachromasia of their
Vacuoles morphologically identified
as polyphosphate vacuoles in
Trypanosoma cyclops are studied
by X-ray microanalysis and shown
to contain high concentrations of P,
Ca and Zn129.
chemical as ‘Graham salt’,
an inorganic long-chain
granules are renamed
An intracellular organelle
capable of transporting protons
and calcium is identified in
T. brucei5and T. cruzi6and is
named the acidocalcisome.
organelles (volutin granules)
of T. cruzi are shown to be
the same organelles as the
• Acidocalcisomes are
found in Toxoplasma
• Na+/H+and Ca2+/H+
exchangers are found
in acidocalcisomes of
In T. cruzi, Mg, K, Ca and P
are found to be localized in
organelles that are identified
as polyphosphate vacuoles55.
Timeline | Major developments in the history of acidocalcisome research
• A vacuolar H+-pyrophosphatase is
found in acidocalcisomes of
T. cruzi41and P. falciparum13, and an
acidocalcisomal Ca2+-ATPase is
identified in T. cruzi23.
• Acidocalcisomes are purified from
L. donovani43and T. brucei28.
189519021904 19071947 19521977 19881994 1996 19971998
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R E V I E W S
as polyP3,polyP4and polyP5.31P NMR spectra ofpurified
acidocalcisomes of T.cruzi,T.brucei and Leishmania
majorindicate that the polyphosphate has an average
chain length of 3.2 phosphates59. Based on the total
concentration of polyphosphates in different stages of
T. cruzi60and the relative volumes of the acidocalci-
somes at each stage of the life cycle (0.86%,2.3% and
0.26% of the total cell volume of epimastigotes,
amastigotes and trypomastigotes,respectively24),and
assuming that these compounds are mainly concen-
trated in acidocalcisomes, the concentration in the
organelles can be calculated as 3–8 M.This is consistent
with the detection of solid-state condensed phosphates
by magic-angle spinning NMR techniques and with the
very high electron density of acidocalcisomes in situ61.
Other components of these organelles,such as carbohy-
drates25or lipids,could be involved in maintaining this
Acidocalcisomes also contain high concentrations of
free amino acids — 1,250 ±297 nmol per mg protein
were found in epimastigotes ofT.cruzi.The basic amino
acids arginine and lysine account for almost 90% of the
amino acid pool ofthe acidocalcisomes,whereas whole-
cell extracts contain high concentrations of neutral and
acidic amino acids62.
The low sulphur content detected by elemental
analysis (TABLE 1)indicates that few proteins are present in
acidocalcisomes,but a few acidocalcisome enzymes have
been detected:polyphosphate kinase has been detected in
T.cruzi60,an acidocalcisome exopolyphosphatase has
been detected in L. major63and a soluble inorganic
pyrophosphatase has been identified in T.brucei64.
Storage function.Acidocalcisomes in several microor-
ganisms are the main storage compartment for calcium,
compounds (inorganic pyrophosphate and polyphos-
phate) and basic amino acids.Most ofthese compounds
are present at millimolar or molar concentrations.Ca2+
ions,and possibly other cations,are imported by the
Ca2+/H+countertransporting ATPase and can be released
from acidocalcisomes when alkalinizing agents,such as
the ionophores monensin and nigericin or NH4Cl,are
applied to intact cells6,11,12,14,37,38,43,60,65or isolated acido-
calcisomes20,28.In T.brucei,monensin-induced Ca2+that
is released from acidocalcisomes is rapidly taken up by
the mitochondria65.Hypoosmotic stress or alkalinizing
agents are also able to produce hydrolysis of short- and
long-chain polyphosphate60,but the resulting inorganic
phosphate (Pi) is not released to the cytosol or to the
extracellular medium, which results in swelling of
acidocalcisomes66.By removing water from the cytosol,
this process helps the cells to recover their volume66.
Although inorganic pyrophosphate is a byproduct
of biosynthetic reactions (for example, synthesis of
nucleic acids,coenzymes and proteins,activation of
fatty acids and isoprenoid synthesis) in which hydrolysis
by inorganic pyrophosphatases makes these reactions
thermodynamically favourable,none of these pathways
have been found inacidocalcisomes.Why is inorganic
T. cruzi54or T. evansi39cells with a pyrophosphatase
removed the electron-dense matrix,which indicates that
inorganic pyrophosphate (PPi) is an important compo-
nent of the structure of this organelle.The matrix has
been studied using electron microscopy,31P NMR and
The following elements are concentrated in the
calcium15–18,24,25,29,32,55–57.Zinc has been found in acido-
calcisomes from the trypanosomatids24,25,55–57,T.gondii32
and C.reinhardtii15,whereas sodium and potassium are
also frequently detected in trypanosomatids (TABLE 1).
Iron has been found in acidocalcisomes of T.cruzithat
were isolated from the bloodstream57,and in P.françai22,
in L.amazonensis26and in several trypanosomatids that
have been cultivated in complex medium27.
The structure and composition of acidocalcisomes
varies with cultivation conditions.For example,pro-
mastigotes of L.amazonensishave spherical iron-free
acidocalcisomes ifgrown in a semi-defined medium but
contain polymorphic and iron-rich organelles when
grown using iron-rich complex medium26. However,
when different trypanosomatids are cultivated under
similar conditions,they can contain different elemental
compositions in their acidocalcisomes27,indicating that
the elemental composition does not depend exclusively
on the cultivation conditions but also on species-specific
characteristics.Therefore,it seems that in some species
the acidocalcisomes operate mostly as a storage compart-
ment for other elements (for example,zinc) rather than
calcium27.A remarkable property ofthe acidocalcisomes
is the low variation in the concentration ofthe elements
within organelles ofthe same cells,independent oftheir
All acidocalcisomes that have been described so far
contain high concentrations of phosphorus in the
form of inorganic pyrophosphate and polyphosphate
(polyP) (TABLE 2).Trypanosomatids54,59and T.gondii46
are especially rich in short-chain polyphosphates such
• 31P NMR studies show that polyphosphate,
inorganic phosphate and pyrophosphate are the
only phosphorus compounds in acidocalcisomes59.
• The first gene for a non-plant, non-bacterial V-H+-
PPase, localized to acidocalcisomes, is identified in
T. cruzi and functionally expressed in yeast45.
• A V-H+-PPase in T. gondii is localized to
• Acidocalcisomes are identified in malaria parasites14.
• An acidocalcisomal
identified in L. major63.
• Acidocalcisomes are
purified from T. gondii46.
are identified and
• An acidocalcisomal
identified in T. gondii32.
are identified and
• R. rubrum acidocalcisomes are identified18. Human
platelet-dense granules are found to contain
polyphosphate and to be similar to acidocalcisomes19.
• An aquaporin is identified in T. cruzi acidocalcisomes30.
• An inorganic pyrophosphatase is found in
acidocalcisomes of T. brucei and the role of
acidocalcisomes and the contractile vacuole in
osmoregulation is identified66.
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the form of polyphosphate reduces the osmotic effect
of large pools of this crucial nutrient element.A rapid
increase in the concentrations of short- and long-chain
polyphosphate was detected during T. cruzi trypo-
mastigote to amastigote differentiation (within 2–4
hours) and during the lag phase of epimastigote growth
(within 12–24 hours)60. Concentrations rapidly
decreased after epimastigotes resumed growth60.The
changes observed in the content of polyphosphate in
T.cruziepimastigotes when inorganic phosphate is pre-
sent in the growth medium at high concentrations
could indicate a requirement for these compounds as
energy sources for resuming growth, whereas the
changes observed during differentiation might indicate
an adaptation to the intracellular life of amastigotes60.
The concentrations of both short- and long-chain
polyphosphate also rapidly decreased on exposure of
epimastigotes to hypoosmotic stress,whereas concen-
trations increased after hyperosmotic stress60. This
might indicate a role for storage of inorganic phosphate
in the acidocalcisomes in the adaptation of the parasites
to environmental stress.
pyrophosphate stored in these organelles? Is it a byprod-
uct of the polyphosphate hydrolysis or an intermediate
for polyphosphate synthesis? Only three reactions are
known to use inorganic pyrophosphate in trypanoso-
matids — one is catalysed by the pyruvate phosphate
dikinase located in the glycosomes67,another is catalysed
by the V-H+-PPase that is responsible for acidification of
acidocalcisomes41,42and a third is catalysed by an inor-
ganic pyrophosphatase located in the matrix ofacidocal-
cisomes64.As inorganic pyrophosphate is charged and
polar,it must presumably be transported through the
acidocalcisomal membrane by a transporter,in common
with the transmembrane transporters that shuttle inor-
ganic pyrophosphate between intracellular and extra-
cellular compartments in mammalian tissues68.A similar
channel in the acidocalcisomal membrane could trans-
port inorganic pyrophosphate into the acidocalcisome
after synthesis in the cytosol or other compartments,or
could transport inorganic pyrophosphate out into the
cytosol,where it could be a substrate for the V-H+-PPase.
Polyphosphate (BOX 2)accumulates to high concen-
trations in acidocalcisomes60.Storage of phosphate in
Figure 2 | Thin sections of acidocalcisomes of trypanosomatid parasites prepared by different transmission electron
microscopy methods and of hydrogenosomes of Tritrichomonas foetus. Acidocalcisomes of epimastigote forms of
Trypanosoma cruzi submitted to chemical fixation followed by conventional embedding in epoxide resin (a), cryofixation by high-
pressure freezing followed by freeze substitution and epoxide embedding (b) and cryofixation by immersion in ethane, cryosection
and observation at low temperature (c) (parts a and b are reproduced with permission from REF.24 © (2000) Springer; part c is
reproduced with permission from REF.25 © (1997) American Society for Biochemistry and Molecular Biology). Note that the electron-
dense material is better preserved with the use of cryomethods. Part a shows an acidocalcisome with an electron-dense inclusion
and a vacuole of the endocytic pathway filled with endocytic tracers. Part b shows that a membrane surrounds the acidocalcisome.
Phytomonas françai submitted to routine fixation and embedding in epoxide resin (d) (reproduced with permission from REF.22 ©
(2004) Cambridge Univ. Press). Note the arrangement of the electron-dense material in concentric patterns in the acidocalcisome.
e | Cryo-immunoelectron microscopy of Leishmania amazonensis using antibodies raised against the V-H+-PPase (reproduced with
permission from REF.26 © (2004) Springer). f | Tritrichomonas foetus hydrogenosome. Cells were fixed according to a
glutaraldehyde-osmium tetroxide-potassium ferrocyanide procedure with 5 mM CaCl2added to all solutions (reproduced with
permission from REF.130 © (1983) Society of Protozoologists). The electron-dense reaction product is visible in a vesicle-like
structure (hydrogenosome vesicle) separated from the rest of the hydrogenosome, which has a double membrane. The scale bars
represent 150 nm, 100 nm, 100 nm, 100 nm, 200 nm and 300 nm in parts a–f, respectively.
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to osmotic stress was also shown by changes in the
sodium and chlorine content ofthe acidocalcisomes after
hypoosmotic stress57.A link between acidocalcisomes and
the contractile vacuole complexes ofC.reinhardtii15and
D.discoideum16,which are involved in water extrusion in
hypoosmotic medium,was also demonstrated.The con-
tractile vacuole complex is composed of two compart-
ments — a collection of tubules and vesicles called the
SPONGIOME,and a larger vacuole known as the bladder73.
Early observations74of epimastigotes of T.cruziby
phase-contrast microscopy had detected the presence of
a contractile vacuole complex as a group of small vac-
uoles that fuse after they enlarge.The PULSATION PERIOD
was between 60 and 75 seconds74.Electron micrographs
of the contractile vacuole and surrounding spongiome
of other kinetoplastids have also been published75,76.
Re-investigation of the presence of a contractile vacuole
in trypanosomatids resulted in the identification of an
aquaporin that is located in both the acidocalcisomes
and the contractile vacuole complex of T. cruzi30.
Microtubule- and cyclic AMP-mediated fusion of aci-
docalcisomes to the contractile vacuole complex results
in translocation of aquaporin and the resulting water
movement,which,in addition to swelling of acidocalci-
somes,is responsible for the decrease in volume that is
not accounted for by efflux of osmolytes66.Additional
evidence for a role of acidocalcisomes in osmoregula-
tion resulted from studies on T. brucei64. The use of
RNAi to reduce the expression of the acidocalcisomal
soluble pyrophosphatase (TbVSP1) resulted in try-
panosomes that were deficient in polyphosphate and in
their response to hypoosmotic stress64.
pH homeostasis.It has been proposed that acidic intra-
cellular compartments regulate intracellular pH69and
are important under pathological conditions70,71.
Polyphosphate could be involved in intracellular pH
regulation because it has been shown that H+generation
from polyphosphate hydrolysis can neutralize a pH
change ofup to 2.5 pH units in S.cerevisiae72.
A role for acidocalcisomes in regulation of intra-
cellular pH in T. brucei was shown by the phenotype
changes that occurred in cells in which the acidocalci-
some V-H+-PPase activity was reduced by RNA interfer-
ence42.pHhomeostasis failed in these cells when they
were exposed to an external basic pH >7.4,and the same
cells recovered from intracellular acidification at a slower
rate and to a more acidic final intracellular pH42.
Osmoregulation.Osmoregulation is essential for DIGENETIC
TRYPANOSOMATIDSas osmotic stress occurs in both the insect
vector and the vertebrate host.The regulatory volume
decrease mechanism,which involves the cellular release
of ions and osmolytes,including amino acids,enables
adaptation of trypanosomatids to hypoosmotic stress.
However,a considerable amount of volume recovery
could not be accounted for by release ofamino acids and
ions,and it was proposed that acidocalcisomes might be
involved in this process62,66.
Rapid hydrolysis or synthesis of acidocalcisomal
polyphosphate occurs when epimastigotes of T.cruzi
are exposed to hypoosmotic or hyperosmotic stress
conditions, respectively60, indicating a link between
acidocalcisomes and osmotic homeostasis.A role for aci-
docalcisomes in the response of L.majorpromastigotes
Trypanosomes that have two
hosts,in contrast to
which only have one host.
Tubules and vacuoles that are
connected to the contractile
The period of time between
contractions of the contractile
Figure 3 | Morphology of acidocalcisomes in whole trypanosomatids. Electron spectroscopic imaging (contrast tuning) of
whole cells adhered to formvar-coated grids showing the shape, size and distribution of the acidocalcisomes (black spots) in
different developmental forms of trypanosomatid species. a | Promastigote of Blastocrithidia culicis (scale bar 2 µm). Reproduced
with permission from REF.27 © (2000) Elsevier. b | Choanomastigote of Crithidia deanei (scale bar 1 µm). c | Amastigote of
Trypanosoma cruzi (scale bar 1.5 µm). Reproduced with permission from REF.24 © (2000) Springer. d | Epimastigote of T. cruzi
(scale bar 2 µm). Reproduced with permission from REF.131 © (2000) Academia Brasileira de Ciências.
258 | MARCH 2005 | VOLUME 3
R E V I E W S
coatamer and adaptor molecules, have been found
in trypanosomatids78. However, little is known
about trafficking of proteins to the acidocalcisome.
Acidocalcisomes are not labelled with endocytic mark-
ers79. The presence of putative N-terminal leader
sequences in the T.cruziacidocalcisomal V-H+-PPase
(TcPPase) indicates that this protein is processed in the
endoplasmic reticulum and trafficked to a location or
locations within the secretory pathway,from where it is
transported to the acidocalcisome45.N-terminal leader
sequences have also been found in the acidocalcisomal
V-H+-PPase80and Ca2+-ATPase32from T.gondii.Two
recent studies have shown that defects in the biogenesis
ofacidocalcisomes result in distinct cellular phenotypes.
RNAi-mediated downregulation of a kinesin-like pro-
tein from T.brucei(TbKIFC1) resulted in acidocalci-
somes that were deficient in Ca2+release81,and it was
suggested that this motor protein could be associated
either with shuttle vesicles or macromolecular com-
plexes moving to acidocalcisomes81.In a mutant form of
L.major lacking the first enzyme in the sphingolipid
biosynthesis pathway (serine palmitoyl transferase),the
acidocalcisomes were shown to be ‘empty’by electron
microscopy and were devoid of long-chain polyphos-
phate,indicating that biogenesis of acidocalcisomes is
linked to sphingolipid metabolism82.
Acidocalcisomes and related organelles
Acidocalcisomes are now known to be similar to volutin
granules,which were the first subcellular structures to
be recognized in bacteria8.Volutin granules were later
identified in algae and protists,and named polyphos-
phate vacuoles because this polymer is present at
high concentrations in this organelle (TIMELINE).After
their identification in trypanosomatids,the presence
of acidocalcisomes in organisms previously known to
contain volutin granules, such as the apicomplexan
parasite T.gondii11,32,46,80,the green alga C.reinhardtii15
and the slime mould D.discoideum16,was confirmed.
Bacterial volutin granules were thought to lack an
internal structure or limiting membranes10.Recently
however,volutin granules that are surrounded by a
membrane have been observed in Agrobacterium
tumefaciens17and Rhodospirillum rubrum18using trans-
mission electron microscopy.The membrane-bound
volutin granules were stained with dyes that indicate the
presence of acidic compartments and have also been
shown to contain membrane-bound enzymes such as
The digestive vacuole of P.falciparumtrophozoites
contains H+and Ca2+pumps (V-H+-ATPase,V-H+-PPase
and Ca2+-ATPase)83,84that are similar to those of acido-
calcisomes of other protozoa.Similarly,D.discoideum16
and T.cruzi30contain acidocalcisomes and a contractile
vacuole,both ofwhich contain H+and Ca2+pumps.The
plant vacuole and the vacuole from yeast and other
fungi have several similarities to the acidocalcisomes,
such as an acidic nature,an abundance of polyphos-
phate and free basic amino acids,and the presence of
proton and calcium pumps, Na+/H+and Ca2+/H+
Biogenesis of acidocalcisomes
Genetics and genomic sequencing have revealed that
the regulation ofvesicular transport in trypanosomatids
is partially conserved in other eukaryotes,particularly
the early steps in the secretory pathway (reviewed in
REFS 77,78). In trypanosomatids, the endoplasmic
reticulum is contiguous with the nuclear envelope,a
Golgi apparatus consisting of a stack of 3–10 cisternae
and a polymorphic trans-Golgi network78.Components
of the vesicle budding,transport and fusion machinery,
including N-ethylmaleimide-sensitive fusion protein
(NSF), multiple Rab proteins and subunits of the
ADP + Pi
Mg, Zn, Fe,
Figure 4 | Schematic representation of a typical acidocalcisome. Ca2+uptake occurs in
exchange for H+by a reaction catalysed by a vacuolar Ca2+-ATPase that is inhibited by vanadate.
A H+gradient is established by a bafilomycin A1(Baf A1)-sensitive vacuolar H+-ATPase and an
amino-methylene-diphosphonate (AMDP)-sensitive vacuolar H+-PPase (V-H+-PPase). Ca2+
release occurs in exchange for H+and is favoured by sodium–proton exchange. An aquaporin
allows water transport. Other transporters (for example, for Mg, Zn, Fe, inorganic phosphate (Pi)
and pyrophosphate (PPi), arginine and lysine) are probably present. The acidocalcisome is rich in
pyrophosphate, short- and long-chain polyphosphate (polyP), magnesium, calcium, sodium, and
zinc. An exopolyphophatase (PPX), a pyrophosphatase (PPase) and a polyphosphate kinase
(PPK) are also present. Not all these enzymes are necessarily present in all acidocalcisomes
described, and the internal concentration of elements may also vary.
Table 1 | Elemental analysis of acidocalcisomes
161 ± 18
646 ± 19
1,390 ± 13
10 ± 1
2 ± 1
37 ± 2
171 ± 6
148 ± 6
148 ± 58
515 ± 179
1,216 ± 316
–3.1 ± 25.2
68 ± 34
237 ± 80
39 ± 18
74 ± 64
Concentrations of elements are expressed as nmol per mg dry weight. Elemental analysis is of
acidocalcisomes from T. cruzi epimastigotes25and L. major promastigotes56.
NATURE REVIEWS | MICROBIOLOGY
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compound phytic acid92.The evolutionary relationship
ofthese ‘organelles within organelles’to acidocalcisomes
is intriguing and deserves further study.
Acidocalcisomes were found in bacteria more than one
hundred years ago but their study,as well as the study of
their main constituent, polyphosphate, has been
neglected for many years. The conservation of this
organelle from bacteria to man indicates that it has
important functions that await discovery.Further stud-
ies are necessary to understand the biogenesis and
function of acidocalcisomes in different organisms,
why they have been conserved and how widely the
organelle is distributed.Phylogenetic relationships of
various acidocalcisomal enzymes need to be established
as sequence comparisons are important indicators of
the evolution of these organelles.We do not know how
acidocalcisomes are distributed in daughter cells after
cell division or why morphological changes occur in
acidocalcisomes ofsome trypanosomatids.Intracellular
pyrophosphate,polyphosphate,cations and basic amino
acids are accumulated in large amounts in acidocalci-
somes,but the mechanisms by which these compounds
are transported into the organelle and the reasons for
their accumulation are largely unknown.This is an excit-
ing area of research,not least because these organelles
have different characteristics in different organisms,
which indicates that they could be targets for new drugs.
Animal cells contain organelles that are similar to
bacterial and unicellular eukaryotic acidocalcisomes,
notably the dense granules in human platelets,which
contain high concentrations of intracellular pryophos-
phate and polyphosphate and belong to the same class
of organelles as acidocalcisomes19.This indicates that
acidocalcisomes evolved before the prokaryotic and
eukaryotic lineages diverged,and have been conserved
during evolution in both bacteria and humans.
Two known organelles — the hydrogenosome and
the protein storage vacuole — have internal compart-
ments that are similar to acidocalcisomes. Hydro-
genosomes are membrane-bound organelles that
were first identified in the parabasalid flagellate
Tritrichomonas foetus86and which are evolutionarily
related to the mitochondria87,88.Hydrogenosomes of
T.foetus contain a peripheral vesicle that,like the aci-
docalcisome,is electron-dense,contains large amounts
of phosphorus,calcium,magnesium,iron and other
elements89,and is able to accumulate zinc when cells are
cultivated in its presence90(FIG.2f).The hydrogenosomal
vesicles of the rumen anaerobic fungus Neocallimastix
frontalis91are physiologically similar.Protein storage
vacuoles store large concentrations of proteins during
plant-seed development and maturation.They contain
a membrane-bound acidic and electron-dense vacuole
known as the globoid,which,like the acidocalcisome,is
characterized by the presence of a V-H+-PPase and an
aquaporin (γ-TIP) and which is rich in the phosphorus
Box 2 | Polyphosphate
Polyphosphate is a linear chain ofinorganic phosphate moieties (from a few to several hundred moieties) linked by
high-energy phosphoanhydride bonds,and it is ubiquitous from bacteria to mammals107,108.
Polyphosphate has several functions in bacteria — for example,it can be a phosphate store or an energy source to
replace ATP,and can have roles in cation sequestration and storage,cell membrane formation and function,
transcriptional control,regulation of enzyme activities,response to stress and stationary phase,and the structure of
channels and pumps107,108.As the metabolic turnover ofATP is considerably higher than that of polyphosphate109,it
has been suggested110that polyphosphate is not an efficient supply of energy and that it has a regulatory role.Similar
functions in adaptation to stress have been assigned to polyphosphate in eukaryotic cells such as yeast72,111,fungi112
In many organisms,the mobilization ofpolyphosphate is mainly due to the action ofenzymes that catalyse the
synthesis and degradation ofthis polymer — the polyphosphate kinase and the endo- and exopolyphosphatases,
respectively107,108.Until recently,the only genes to encode exopolyphosphatases116and endopolyphosphatases117in
eukaryotes were from Saccharomyces cerevisiae,with the exception ofa putative polyphosphate kinase gene (DdPPK1)
in Dictyostelium discoideum107,118and a second polyphosphate kinase in D.discoideum(DdPPK2),which might be
localized to the acidocalcisome118,119.DdPPK2 has a similar amino acid sequence to,and characteristics of,actin-related
proteins,which in turn are similar to muscle actins.Actin inhibitors such as phalloidin and DNase I also inhibit
DdPPK2-mediated synthesis ofpolyP.So,this particular actin-related protein complex is an enzyme that can polymerize
into an actin-like filament concurrent with its synthesis ofa polyphosphate chain in a fully reversible reaction119.It is
interesting to note that actin-like proteins have been found in the electron-dense granules of Entamoeba histolytica,
which are similar to acidocalcisomes in both morphology and composition120,and that immunofluorescence with
antibodies against actin revealed a granular pattern in different trypanosomatids121.
Table 2 | Polyphosphate (polyP) compounds in different life cycle stages of T. cruzi and L. major
54.3 ± 0.3
3.1 ± 1.4
0.82 ± 0.005
25.5 ± 5.1
0.13 ± 0.01
21.4 ± 3.0
55.9 ± 5.6
T. cruzi data are from REF.60 and L. major data are fromREF.63.
260 | MARCH 2005 | VOLUME 3
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1. Urbina, J. A. & Docampo, R. Specific chemotherapy of
Chagas disease: controversies and advances. Trends
Parasitol. 19, 495–501 (2003).
Croft, S. L. & Coombs, G. H. Leishmaniasis — current
chemotherapy and recent advances in the search for new
drugs. Trends Parasitol. 19, 502–508 (2003).
Fairlamb, A. Chemotherapy of human African
trypanosomiasis: current and future prospects. Trends
Parasitol. 19, 488–494 (2003).
Camargo, E. P. Phytomonas and other trypanosomatid
parasites of plants and fruits. Adv. Parasitol. 42, 29–112
Vercesi, A. E., Moreno, S. N. J. & Docampo, R. Ca2+/H+
exchange in acidic vacuoles of Trypanosoma brucei.
Biochem. J. 304, 227–233 (1994).
Docampo, R., Scott, D. A., Vercesi, A. E. & Moreno, S. N. J.
Intracellular Ca2+storage in acidocalcisomes of
Trypanosoma cruzi. Biochem. J. 310, 1005–1012 (1995).
Docampo, R. & Moreno, S. N. J. Acidocalcisome: a novel Ca2+
storage compartment in trypanosomatids and apicomplexan
parasites. Parasitol. Today15, 443–448 (1999).
Meyer, A. Orientierende untersuchungen über verbreitung.
Morphologie, und chemie des volutins. Bot. Zeit. 62,
Kornberg, A. Inorganic polyphosphate: toward making a
forgotten polymer unforgettable. J. Bacteriol. 177, 491–496
10. Jensen, T. E. in Ultrastructure of Microalgae (ed. Berner, T.)
7–50 (CRC Press, Boca Raton, Florida, 1993).
11. Moreno, S. N. J. & Zhong, L. Acidocalcisomes in Toxoplasma
gondiitachyzoites. Biochem. J. 313, 655–659 (1996).
12. Garcia, C. R. et al. Acidic calcium pools in
intraerythrocytic malaria parasites. Eur. J. Cell Biol. 76,
13. Luo, S., Marchesini, N., Moreno, S. N. J. & Docampo, R.
A plant-like vacuolar H+-pyrophosphatase in Plasmodium
falciparum. FEBS Lett. 460, 217–220 (1999).
14. Marchesini, N., Luo, S., Rodrigues, C. O., Moreno, S. N. J. &
Docampo, R. Acidocalcisomes and a vacuolar H+-
pyrophosphatase in malaria parasites. Biochem. J. 347,
15. Ruiz, F. A., Marchesini, N., Seufferheld, M., Govindjee, &
Docampo, R. The polyphosphate bodies of
Chlamydomonas reinhardtii possess a proton pumping
pyrophosphatase and are similar to acidocalcisomes.
J. Biol. Chem. 276, 46196–46203 (2001).
16. Marchesini, N., Ruiz, F. A., Vieira, M. & Docampo, R.
Acidocalcisomes are functionally linked to the contractile
vacuole of Dictyostelium discoideum. J. Biol. Chem. 277,
17. Seufferheld, M. et al. Identification in bacteria of organelles
similar to acidocalcisomes of unicellular eukaryotes. J. Biol.
Chem. 278, 29971–29978 (2003).
First report of membrane-bound acidocalcisomes in
bacteria, which were identified by X-ray microanalysis,
subcellular fractionation and fluorescence and electron
18. Seufferheld, M., Lea, C. R., Vieira, M., Oldfield, E. &
Docampo, R. The H+-pyrophosphatase of Rhodospirillum
rubrum is predominantly located in polyphosphate-rich
acidocalcisomes. J. Biol. Chem. 279, 51193–51202
19. Ruiz, F. A., Lea, C. R., Oldfield, E. & Docampo, R.
Human platelet dense granules contain polyphosphate
and are similar to acidocalcisomes of bacteria and
unicellular eukaryotes. J. Biol. Chem. 279, 44250–44267
Shows that human platelet dense granules have
morphological and structural similarities to
acidocalcisomes, and contain polyphosphate that
was released on thrombin stimulation.
20. Scott, D. A. & Docampo, R. Characterization of isolated
acidocalcisomes of Trypanosoma cruzi. J. Biol. Chem. 275,
The purification method of acidocalcisomes using
iodixanol gradient centrifugation was developed.
21. De Souza, W. Basic cell biology of Trypanosoma cruzi.
Curr. Pharm. Design 8, 269–285 (2002).
22. Miranda, K. et al. Acidocalcisomes of Phytomonas françai
possess distinct morphological characteristics and contain
iron. Microsc. Microanal. 10, 647–655 (2004).
23. Lu, H.-G. et al. Ca2+content and expression of an
acidocalcisomal calcium pump are elevated in intracellular
forms of Trypanosoma cruzi. Mol. Cell. Biol. 18, 2309–2323
First report of a PMCA-type Ca2+-ATPase in
acidocalcisomes. The gene was cloned, sequenced,
expressed and shown to complement yeast deficient
in PMC1, and the protein was shown to localize in
24. Miranda, K., Benchimol, M., Docampo, R. & de Souza, W.
The fine structure of acidocalcisomes of Trypanosoma cruzi.
Parasitol. Res. 86, 373–384 (2000).
25. Scott, D. A., Docampo, R., Dvorak, J. A., Shi, S. &
Leapman, R. D. In situ compositional analysis of
acidocalcisomes of Trypanosoma cruzi. J. Biol Chem. 272,
Quantitative analysis of the elemental composition of
acidocalcisomes. On the basis of the response to
ionophores it was established that acidocalcisomes
correspond to the electron-dense organelles
previously identified in trypanosomes.
26. Miranda, K. et al. Dynamics of polymorphism of
acidocalcisomes in Leishmania parasites. Histochem. Cell
Biol. 121, 407–418 (2004).
27. Miranda, K., Docampo, R., Grillo, O. & de Souza, W.
Acidocalcisomes of trypanosomatids have species-specific
elemental composition. Protist 155, 395–405 (2004).
28. Rodrigues, C. O., Scott, D. A. & Docampo, R.
Characterization of a vacuolar pyrophosphatase in
Trypanosoma brucei and its localization to acidocalcisomes.
Mol. Cell. Biol. 19, 7712–7723 (1999).
29. Ruiz, F. A., Luo, S., Moreno, S. N. J. & Docampo, R.
Polyphosphate content and fine structure of
acidocalcisomes of Plasmodium falciparum.
Microsc. Microanal. 10, 563– 567 (2004).
30. Montalvetti, A., Rohloff, P. & Docampo, R. A functional
aquaporin co-localizes with the vacuolar proton
pyrophosphatase to acidocalcisomes and the contractile
vacuole complex of Trypanosoma cruzi. J. Biol. Chem. 279,
31. Luo, S., Rohloff, P., Cox, J., Uyemura, S. A. & Docampo, R.
Trypanosoma brucei plasma membrane-type Ca2+-ATPase 1
(TbPMC1) and 2 (TbPMC2) genes encode functional Ca2+-
ATPases localized to the acidocalcisomes and plasma
membrane, and essential for Ca2+homeostasis and growth.
J. Biol. Chem. 279, 14427–14439 (2004).
32. Luo, S., Vieira, M., Graves, J., Zhong, L. & Moreno, S. N. J.
A plasma membrane-type Ca2+-ATPase co-localizes with a
vacuolar H+-pyrophosphatase to acidocalcisomes of
Toxoplasma gondii. EMBO J. 20, 55–64 (2001).
Identification of a Ca2+-ATPase and its
co-localization with the V-H+-PPase in
acidocalcisomes of T. gondii.
33. Moniakis, J., Coukell, M. B. & Forer, A. Molecular cloning of
an intracellular P-type ATPase from Dictyostelium that is
up-regulated in calcium-adapted cells. J. Biol. Chem. 270,
34. Cunningham, K. W. & Fink, G. R. Calcineurin-dependent
growth control in Saccharomyces cerevisiae mutants
lacking PMC1, a homolog of plasma membrane Ca2+
ATPases. J. Cell Biol. 124, 351–363 (1994).
35. Ghosh, S. K., Rosenthal, B., Rogers, R. & Samuelson, J.
Vacuolar localization of an Entamoeba histolytica
homologue of the plasma membrane ATPase (PMCA). Mol.
Biochem. Parasitol. 108, 125–130 (2000).
36. Bowman, E. J., Siebers, A. & Altendorf, K. Bafilomycins: a
class of inhibitors of membrane ATPases from
microorganisms, animal cells, and plant cells. Proc. Natl
Acad. Sci. USA 85, 7972–7976 (1988).
37. Scott, D. A., Moreno, S. N. J. & Docampo, R. Ca2+storage
in Trypanosoma brucei: the influence of cytoplasmic pH and
importance of vacuolar acidity. Biochem. J. 310, 789–794
38. Lu, H.-G. et al. Intracellular Ca2+pool content and signaling
and expression of a calcium pump are linked to virulence in
Leishmania mexicana amazonensis J. Biol. Chem. 272,
39. Mendoza, M. et al. Physiological and morphological
evidences for the presence of acidocalcisomes in
Trypanosoma evansi: single cell fluorescence and 31P NMR
studies. Mol. Biochem. Parasitol. 125, 23–33 (2002).
40. Benchimol, M. et al. Functional expression of a vacuolar-
type H+-ATPase in the plasma membrane and intracellular
vacuoles of Trypanosoma cruzi. Biochem. J. 332, 695–702
41. Scott, D. A. et al. Presence of a plant-like proton-pumping
pyrophosphatase in acidocalcisomes of Trypanosoma cruzi.
J. Biol. Chem. 273, 22151–22158 (1998).
First report and biochemical characterization of a
V-H+-PPase in a unicellular eukaryotic parasite.
42. Lemercier, G. et al. A vacuolar-type H+pyrophosphatase
governs maintenance of functional acidocalcisomes and
growth of the insect and bloodstream forms of
Trypanosoma brucei. J. Biol. Chem. 277, 37369–37376
43. Rodrigues, C. O., Scott, D. A. & Docampo, R. Presence of a
vacuolar H+-pyrophosphatase in promastigotes of Leishmania
donovaniand its localization to a different compartment from
the vacuolar H+-ATPase. Biochem J. 340, 759–766 (1999).
44. Rodrigues, C. O. et al. Vacuolar proton pyrophosphatase
activity and pyrophosphate (PPi) in Toxoplasma gondii as
possible chemotherapeutic targets. Biochem. J. 349,
45. Hill, J., Scott, D. A., Luo, S. & Docampo, R. Cloning and
functional expression of a gene encoding a vacuolar-type
proton-translocating pyrophosphatase from Trypanosoma
cruzi. Biochem. J. 351, 281–288 (2000).
First cloning and functional expression of a
V-H+-PPase from an organism that is neither a
bacteria or plant.
46. Rodrigues, C. O., Ruiz, F. A., Rohloff, P., Scott, D. A. &
Moreno, S. N. J. Characterization of isolated
acidocalcisomes from Toxoplasma gondii tachyzoites
reveals a novel pool of hydrolysable polyphosphate. J. Biol.
Chem. 277, 48650–48656 (2002).
47. Martinez, R. et al. A proton pumping pyrophosphatase in the
Golgi apparatus and plasma membrane vesicles of
Trypanosoma cruzi. Mol. Biochem. Parasitol. 120, 205–213
48. Vercesi, A. E. & Docampo, R. Sodium-proton exchange
stimulates Ca2+release from acidocalcisomes of
Trypanosoma brucei. Biochem. J. 315, 265–270 (1996).
49. Vercesi, A. E., Grijalba, M. T. & Docampo, R. Inhibition of
Ca2+release from Trypanosoma brucei acidocalcisomes by
3,5-dibutyl-4-hydroxytoluene (BHT): role of the Na+/H+
exchanger. Biochem. J. 328, 479–482 (1997).
50. Vercesi, A. E., Rodrigues, C. O., Catisti, R. & Docampo, R.
Presence of a Na+/H+exchanger in acidocalcisomes of
Leishmania donovani and their alkalization by anti-
leishmanial agents. FEBS Lett. 473, 203–206 (2000).
51. Moreno, S. N. J., Docampo, R. & Vercesi, A. E. Calcium
homeostasis in procyclic and bloodstream forms of
Trypanosoma brucei. Lack of inositol 1,4,5-trisphosphate-
sensitive Ca2+release. J. Biol. Chem. 267, 6020–6026
52. Moreno, S. N. J., Vercesi, A. E., Pignataro, O. P. &
Docampo, R. Calcium homeostasis in Trypanosoma cruzi
amastigotes. Presence of inositol phosphates and lack of an
inositol 1,4,5-trisphosphate-sensitive calcium pool. Mol.
Biochem. Parasitol. 52, 251–262 (1992).
53. Docampo, R., Moreno, S. N. J. & Vercesi, A. E. Effect of
thapsigargin on calcium homeostasis in Trypanosoma cruzi
trypomastigotes and epimastigotes. Mol. Biochem.
Parasitol. 59, 305–314 (1993).
54. Urbina, J. A. et al. Trypanosoma cruzi contains major
pyrophosphate stores and its growth in vitro and in vivo is
blocked by pyrophosphate analogs. J. Biol. Chem. 274,
Pyrophosphate is found in trypanosomes at greater
concentrations than ATP and localized in
acidocalcisomes, which establishes pyrophosphate
metabolism as a therapeutic target.
55. Dvorak, J. A., Engel, J. C., Leapman, R. D., Swyt, C. R. &
Pella, P. A. Trypanosoma cruzi: elemental composition
hetereogeneity of cloned stocks. Mol. Biochem. Parasitol.
31, 19–26 (1988).
56. LeFurgey, A., Ingram, P. & Blum, J. J. Elemental composition
of polyphosphate-containing vacuoles and cytoplasm of
Leishmania major. Mol. Biochem. Parasitol. 40, 77–86
57. LeFurgey, A., Ingram, P. & Blum J. J. Compartmental
responses to acute osmotic stress in Leishmania major
result in rapid loss of Na+and Cl–. Comp. Biochem. Physiol.
Mol. Integr. Physiol. 128, 385–394 (2001).
58. Correa A. F., Andrade, L. R. & Soares. M. J. Elemental
composition of acidocalcisomes of Trypanosoma cruzi
bloodstream trypomastigote forms. Parasitol Res. 88,
59. Moreno, B. et al. 31P NMR spectroscopy of Trypanosoma
brucei, Trypanosoma cruzi and Leishmania major: Evidence
for high levels of condensed inorganic phosphates. J. Biol.
Chem. 275, 28356–28362 (2000).
60. Ruiz, F. A., Rodrigues, C. O. & Docampo, R. Rapid changes
in polyphosphate content within acidocalcisomes in
response to cell growth, differentiation, and environmental
stress in Trypanosoma cruzi. J. Biol. Chem. 276,
61. Moreno, B. et al. Magic-angle spinning 31P NMR
spectroscopy of condensed phosphates in parasitic
protozoa: visualizing the invisible. FEBS Lett. 523, 207–212
62. Rohloff, P., Rodrigues, C. O. & Docampo R. Regulatory
volume decrease in Trypanosoma cruzi involves amino acid
efflux and changes in intracellular calcium. Mol. Biochem.
Parasitol. 126, 219–230 (2003).
63. Rodrigues, C. O., Ruiz, F. A., Vieira, M., Hill, J. E. &
Docampo, R. An acidocalcisomal exopolyphosphatase
from Leishmania major with higher affinity for short-term
polyphosphate. J. Biol. Chem. 277, 50899–50906 (2002).
NATURE REVIEWS | MICROBIOLOGY
VOLUME 3 | MARCH 2005 | 261
R E V I E W S
64. Lemercier, G. et al. A pyrophosphatase regulating
polyphosphate metabolism in acidocalcisomes is essential
for Trypanosoma brucei virulence in mice. J. Biol. Chem.
279, 3420–3425 (2004).
65. Xiong, Z.-H., Ridgley, E. L., Enis, D., Olness, F. & Ruben, L.
Selective transfer of calcium from an acidic compartment to
the mitochondrion of Trypanosoma brucei: measurements
with targeted aequorin. J. Biol. Chem. 272, 31022–31028
66. Rohloff, P., Montalvetti, A. & Docampo, R. Acidocalcisomes
and the contractile vacuole complex are involved in
osmoregulation in Trypanosoma cruzi. J. Biol. Chem. 279,
The role of acidocalcisomes and the contractile
vacuole of trypanosomes in osmoregulation is
67. Bringaud, F., Baltz, D. & Baltz, T. Functional and molecular
characterization of a glycosomal PPi-dependent enzyme in
trypanosomatids: pyruvate, phosphate dikinase. Proc. Natl
Acad. Sci. USA 95, 7963–7968 (1998).
68. Ho, A. M., Johnson, M. D. & Kingsley, D. M. Role of the
mouse ank gene in control of tissue calcification and arthritis.
Science 289, 265–270 (2000).
69. Wadsworth, S. J. & Van Rossum, G. D. V. Role of vacuolar
adenosine triphosphate in the regulation of cytosolic pH in
hepatocytes. J. Membrane Biol. 142, 21–34 (1994).
70. Bronk, S. F. & Gores, G. J. Efflux of protons from acidic
vesicles contributes to cytosolic acidification of hepatocytes
during ATP depletion. Hepatology 14, 626–633 (1991).
71. Madshus, I. H., Tonnessen, T. I., Olsnes, S. & Sandvig, K.
Effect of potassium depletion of Hep 2 cells on intracellular
pH and on chloride uptake by anion antiport. J. Cell Physiol.
131, 6–13 (1987).
72. Castro, C. D., Koretsky, A. P. & Domach, M. M. NMR-
observed phosphate trafficking and polyphosphate
dynamics in wild-type and vph1-1 mutant Saccharomyces
cerevisiae in response to stresses. Biotechnol. Prog. 15,
73. Allen, R. D. & Naitoh, Y. Osmoregulation and contractile
vacuoles of protozoa. Int. Rev. Cytol. 215, 351–394
74. Clark, T. B. Comparative morphology of four genera of
trypanosomatidae. J. Protozool. 6, 227–232 (1959).
75. Linder, J. C. & Staehelin, L. A. A novel model for fluid
secretion by the trypanosomatid contractile vacuole
apparatus. J. Cell Biol. 83, 371–382 (1979).
76. Attias, M., Vommaro, R. C. & de Souza, W. Computer aided
three-dimensional reconstruction of the free-living protozoan
Bodo sp. (Kinetoplastida:Bodonidae). Cell Struct. Funct. 21,
77. McConville, M. J., Mullin, K. A., Ilgoutz, S. C. & Teasdale. R. D.
Secretory pathway of trypanosomatid parasites. Microbiol.
Mol. Biol. Rev. 66, 122–154 (2002).
78. Morgan, G. W., Hall, B. S., Denny, P. W., Field, M. C. &
Carrington, M. The endocytic apparatus of the
kinetoplastida. Part II: machinery and components of the
system. Trends Parasitol. 118, 540–546 (2002).
79. Docampo, R. & Moreno, S. N. J. (2001)
The acidocalcisome. Mol. Biochem. Parasitol. 114,
80. Drozdowicz, Y. M. et al. Isolation and characterization of
TgVP1, a type I vacuolar H+-translocating pyrophosphatase
from Toxoplasma gondii. The dynamics of its subcellular
localization and the cellular effects of a diphosphonate
inhibitor. J. Biol. Chem. 278, 1075–1085 (2003).
81. Dutoya, S. et al. A novel C-terminal kinesin is essential for
maintaining functional acidocalcisomes in Trypanosoma
brucei. J. Biol. Chem. 276, 49117–49124 (2001).
82. Zhang, K. et al. Leishmania salvage of host sphingolipids
accompanied by remodeling to form parasite-specific inositol
phosphoceramide is required for acidocalcisome biogenesis
and parasite survival. Mol. Microbiol. (in the press).
A role for sphingolipid biosynthesis in acidocalcisome
biogenesis is established.
83. Saliba, K. J. et al. Acidification of the malaria parasite’s
digestive vacuole by a H+-ATPase and a H+-
pyrophosphatase. J. Biol. Chem. 278, 5605–5612 (2003).
84. Biagini, G., Bray, P. G., Spiller, D. G., White, M. R. H. &
Ward, S. A. The digestive food vacuole of the malaria
parasite is a dynamic intracellular Ca2+store. J. Biol. Chem.
278, 27910–27915 (2003).
85. Maeshima, M. Tonoplast transporters: organization and
function. Annu. Rev. Plant Physiol. 52, 469–497 (2001).
86. Lindmark, D. G. & Müller, M. Hydrogenosome, a
cytoplasmic organelle of the anaerobic flagellate
Tritrichomonas foetus, and its role in pyruvate metabolism.
J. Biol. Chem. 248, 7724–7728 (1973).
87. Bui, E. T., Bradley, P. J. & Johnson, P. J. A common
evolutionary origin for mitochondria and hydrogenosomes.
Proc. Natl Acad. Sci. USA 93, 9651–9656 (1996).
88. Dyall, S. D. & Johnson, P. J. Origins of hydrogenosomes
and mitochondria. Evolution and organelle biogenesis.
Curr. Opin. Microbiol. 3, 404–411 (2000).
89. Ribeiro, K., C., Benchimol, M. & Farina, M. Contribution of
cryofixation and freeze-substitution to analytical
microscopy: a study of Tritrichomonas foetus
hydrogenosomes. Microsci. Res. Tech. 53, 87–92 (2001).
90. Benchimol, M., Aquino Almeida, J. C., Lins, U., Rodrigues
Gonçalves, N. & de Souza, W. Electron microscopy study
of the effect of Zn on Tritrichomonas foetus. Antimicrob.
Agents Chemother. 37, 2722–2726 (1993).
91. Biagini, G. A., van der Giezen, M., Hill, B., Winters, C. &
Lloyd, D. Ca2+accumulation in the hydrogenosomes of
Neocallimastix frontalis L2: a mitochondrial-like physiological
role. FEMS Microbiol. Lett. 149, 227–232 (1997).
92. Jiang, L. et al. The protein storage vacuole: a unique
compound organelle. J. Cell Biol. 155, 991–1002 (2001).
93. Ferguson, M. A. J., Haldar, K. & Cross, G. A. M.
Trypanosoma brucei variant surface glycoprotein has a
sn-1,2-dimyristoylglycerol membrane anchor at its COOH
terminus. J. Biol. Chem. 260, 4963–4968 (1985).
94. Ferguson, M. A. J. The structure, biosynthesis and
functions of glycosylphosphatidylinositol, and the
contributions of trypanosome research. J. Cell Sci. 112,
95. Gull, K. The cytoskeleton of trypanosomatid parasites.
Annu. Rev. Microbiol. 53, 629–655 (1999).
96. Moreira-Leite, F. F., Sherwin, T., Kohl, L. & Gull, K.
A trypanosome structure involved in transmitting
cytoplasmic information during cell division. Science 294,
97. Ziemann, H. Eine methode der doppelfärbung bei
flagellaten, pilzen, spirillen und bakterien, sowie bei
einigen amöben. Zentralbl. Bakteriol. Parasitenkd.
Infektionskr. Hyg. 24, 945–955 (1898).
98. Shapiro, T. A. & Englund, P. The structure and replication
of kinetoplast DNA. Annu. Rev. Microbiol. 49, 117–143
99. Benne, R. et al. Major transcript of the frameshifted coxII
gene from trypanosome mitochondria contains four
nucleotides that are not encoded in the DNA. Cell 46,
100. Blum, B., Bakalara, N. & Simpson, L. A model for RNA
editing in kinetoplastid mitochondria: ‘guide’ RNA
molecules transcribed from maxicircle DNA provide the
edited information. Cell 60, 189–198 (1990).
101. Boothroyd, J. C. & Cross, G. A. M. Transcripts encoding
for variant surface glycoproteins of Trypanosoma brucei
have a short, identical exon at their 5′ end. Gene 20,
102. Liang, X.-H., Haritan, A., Uliel, S. & Michaeli, S. Trans and
cis splicing in trypanosomatids: mechanism, factors, and
regulation. Eukaryot. Cell 2, 830–840 (2003).
103. Opperdoes, F. R. & Borst, P. Localization of nine glycolytic
enzymes in a microbody-like organelle in Trypanosoma
brucei: the glycosome. FEBS Lett. 80, 360–364 (1977).
104. Parsons, M. Glycosomes: parasites and the divergence of
peroxisomal function. Mol. Microbiol. 53, 717–724 (2004).
105. Fairlamb, A. H., Blackburn, P., Ulrich, P., Chait, B. T. &
Cerami, A. Trypanothione: a novel
bis(glutathionyl)spermidine cofactor for glutathione
reductase in trypanosomatids. Science 227, 1485–1487
106. Muller, S., Liebau, E., Walter, R. D. & Krauth-Siegel, R. L.
Thiol-based redox metabolism of protozoan parasites.
Trends Parasitol. 19, 320–328 (2003).
107. Kornberg, A., Rao, N. N., & Ault-Riché, D. Inorganic
polyphosphate: a molecule of many functions. Annu. Rev.
Biochem. 68, 89–125 (1999).
108. Kulaev, I. & Kulakovskaya, T. Polyphosphate and
phosphate pump. Annu. Rev. Microbiol. 54, 709–734
109. Chapman, A. G. & Atkinson, D. E. Adenine nucleotide
concentrations and turnover rates. Their correlation with
biological activity in bacteria and yeast. Adv. Microbiol.
Physiol. 15, 253–306 (1977).
110. Rao, N. N. & Kornberg, A. Inorganic polyphosphate
supports resistance and survival of stationary-phase
Escherichia coli. J. Bacteriol. 178, 1394–1400 (1996).
111. Castro, C. D., Meehan, A. J., Koretsky, A. P. & Domach,
M. M. In situ31P nuclear magnetic resonance for
observation of polyphosphate and catabolite responses of
chemostat-cultivated Saccharomyces cerevisiae after
alkalinization. Appl. Environ. Microbiol. 61, 4448–4453
112. Yang, Y. C., Bastos, M., & Chen, K. Y. Effects of osmotic
stress and growth stage on cellular pH and
polyphosphate metabolism in Neurospora crassa as
studied by 31P nuclear magnetic resonance spectroscopy.
Biochim. Biophys. Acta 1179, 141–147 (1993).
113. Pick, U. & Weiss, M. Polyphosphate hydrolysis within
acidic vacuoles in response to amine-induced alkaline
stress in the halotolerant alga Dunaliella salina.
Plant Physiol. 97, 1234–1240 (1991).
114. Weiss, M., Bental, M. & Pick, U. Hydrolysis of
polyphosphates and permeability changes in response to
osmotic shocks in cells of the halotelerant alga Dunaliella.
Plant Physiol. 97, 1241–1248 (1991).
115. Pick, U., Zeelon, O. & Weiss, M. Amine accumulation in
acidic vacuoles protects the halotolerant alga Dunaliella
salina against alkaline stress. Plant Physiol. 97, 1226–1233
116. Wurts, H., Shiba, T. & Kornberg, A. The gene for a major
exopolyphosphatase of Saccharomyces cerevisiae.
J. Bacteriol. 177, 898–906 (1995).
117. Sethuraman, A., Rao, N. N. & Kornberg, A. The
endopolyphosphatase gene: essential in Saccharomyces
cerevisiae. Proc. Natl Acad. Sci. USA 98, 8542–8547
118. Kornberg, A. Biochemistry matters. Nature Struct. Mol. Biol.
6, 493 (2004).
119. Gomez-Garcia, M. R. & Kornberg, A. Formation of an actin-
like filament concurrent with the enzymatic synthesis of
inorganic polyphosphate. Proc. Natl Acad. Sci. USA 101,
A polyphosphate kinase of possible acidocalcisome
localization is identified as a complex of actin-related
120. Leon, G. et al. Electron probe analysis and biochemical
characterization of electron-dense granules secreted by
Entamoeba histolytica. Mol. Biochem. Parasitol. 85,
121. Mortara, R. Studies on trypanosomatid actin. I.
Immunochemical and biochemical identification.
J. Protozool. 36, 8–13 (1989).
122. Babes, V. Beobachtungen über die metachromatischen
körperchen, sporenbildung, verzwiegung, kolben- und
kapsel-bildung pathogener bakterien. Zentralbl. Bakteriol.
Parasitenkd. Infektionskr. Hyg. 20, 412–420 (1895).
123. Grimme, A. Die wichtigsten methoden der bakterenfärbung
in ihrer wirkung auf die membran, den protoplasten und die
einschlüsse der bakterienzelle. Zentralbl. Bakteriol.
Parasitenkd. Infektionskr. Hyg. 32, 161–165 (1902).
124. Kunze, W. Uber Orcheobius herpobdellae schuberg et
kunze. Arch. Protistenk. 9, 382–390 (1907).
125. Swellengrebel, N. H. La volutine chez les trypanosomes.
C. R. Soc. Biol. Paris 64, 38–43 (1908).
126. Erdnmann, R. Kern und metachromatische körper bei
sarkosporidien. Arch. Protistenk. 20, 239–243 (1910).
127. Wiame, J. H. Etude d’une substance polyphosphorée,
basophile et métachromatique chez les levures. Biochim.
Biophys. Acta 1, 234–255 (1947)
128. Ebel, J. P. Recherches sur les polyphosphates contenus
dans diverses cellules vivantes. II. Etude chromatographique
et potentiométrique des polyphosphates de levure. Bull.
Soc. Chim. Biol. 34, 330 (1952).
129. Vickerman, K. & Tetley, L. Recent ultrastructural studies on
trypanosomes. Ann. Soc. Belge Méd. Trop. 57, 441–455
130. Benchimol, M. & de Souza, W. Fine structure and
cytochemistry of the hydrogenosome of Tritrichomonas
foetus. J. Protozool. 30, 422–425 (1983).
131. de Souza, W. et al. Two special organelles found in
Trypanosoma cruzi. An. Acad. Bras. Ciênc. 72, 421–432
Work in our laboratories was funded by the US National Institutes of
Health (to R.D. and S.N.J.M.), the Burroughs Wellcome Fund (to R.D.
and S.N.J.M.) and Programa de Núcleos de Excelência (to W.S.).
Competing interests statement
The authors declare no competing financial interests.
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