FEMS Microbiology Letters 234 (2004) 117–123
www.fems-microbiology.org
The pentose phosphate pathway in Trypanosoma cruzi
Dante A. Maugeri, Juan J. Cazzulo
*
Instituto de Investigaciones Biotecnologicas/INTECH, Universidad Nacional de General San Martin/CONICET, Av. Gral Paz s/n, INTI, edificio 24,
Av. General Paz y Albarellos, Casilla de Correo 30, 1650 San Martin, Buenos Aires, Argentina
Received 29 December 2003; received in revised form 3 March 2004; accepted 8 March 2004
First published online 19 March 2004
Abstract
The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was
demonstrated in epimastigotes by measuring the evolution of 14 CO2 from [1-14 C] or [6-14 C]D -glucose. Glucose consumption through
the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP
are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP
enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).
Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Trypanosoma cruzi; Chagas disease; Pentose phosphate pathway; Oxidative stress; Subcellular localisations
1. Introduction
Trypanosoma cruzi, the parasitic flagellate which causes
the American Trypanosomiasis, Chagas disease, actively
catabolizes glucose through the classical Embden–Meyerhof pathway, coupled to the production of reduced catabolites, succinate and L -alanine, which are excreted into
the medium [1]. At variance with all other eukaryotic cells,
where glycolysis takes place in the cytosol, Trypanosomatids have the first six enzymes of the glycolytic pathway,
from hexokinase (HK) to glyceraldehyde 3-phosphate
dehydrogenase, located inside a peroxisome-like organelle,
Abbreviations: PPP, pentose phosphate pathway; G6PDH,
glucose-6-phosphate dehydrogenase; 6PGDH, 6-phosphogluconate
dehydrogenase; lactonase, 6-phosphogluconolactonase; R5PI, ribose5-phosphate isomerase; Ru5PE, ribulose-5-phosphate epimerase;
TKT, transketolase; TA, transaldolase; GPI, glucose phosphate
isomerase; PK, pyruvate kinase; HK, hexokinase; ICDH, isocitrate
dehydrogenase; PEPCK, phosphoenolpyruvate carboxykinase; ROS,
reactive oxygen species; E-64, trans-epoxy succinyl leucylamido 4guanidinobutane; N, LG, SG, M and S, nuclear, large granule, small
granule, microsomal and soluble subcellular fractions, respectively.
*
Corresponding author. Tel.: +54 11-4580-7255; fax: +54 11-47529639.
E-mail address: [email protected] (J.J. Cazzulo).
the glycosome. On the other hand, phosphoglycerate kinase, phosphoglyceromutase, enolase and pyruvate kinase
(PK) are cytosolic [1]. Glucose phosphate isomerase (GPI),
is present in both compartments in T. cruzi [2].
The other important pathway for glucose utilization
in most organisms, the pentose phosphate pathway
(PPP) has been the subject of fewer studies. The PPP
usually has two major roles, namely the reduction of
NADP to NADPH, necessary for biosynthetic reactions
and also for the protection of cells against oxidative
stress imposed by reactive oxygen species (ROS), as well
as for the production of ribose-5-phosphate to be used
in nucleic acid synthesis [3]. In most organisms the PPP
enzymes are located essentially in the cytosol, with the
exception of plants, where they are also present in
plastids [4]. In rat liver a minor fraction of all the PPP
enzyme activities has been localized in the endoplasmic
reticulum [5], and the two dehydrogenases are also
present in peroxisomes [6].
Over the last 10 years, the PPP has been the subject of a
number of studies in Trypanosomatids. All the enzymes
of the pathway have been detected in procyclics, and most
of them in bloodstream trypomastigotes, of T. brucei [7]
and some of the genes encoding these enzymes have been
cloned, sequenced and characterized [8,9]. We have
0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsle.2004.03.018
118
D.A. Maugeri, J.J. Cazzulo / FEMS Microbiology Letters 234 (2004) 117–123
recently shown the presence of all the enzymes of the PPP
in promastigotes of Leishmania mexicana, as well as the
functioning of the pathway in vivo [10]. In the case of T.
cruzi, early studies showed that the two dehydrogenases
of the oxidative branch of the pathway [3] were present
[11], and one of them, glucose-6-phosphate dehydrogenase (G6PDH), was partially purified and some of its
properties were determined [12]. Recently we have cloned,
expressed and characterized the 6-phosphogluconate dehydrogenase (6PGDH) of T. cruzi [13]. Moreover, studies
with labelled glucose suggested that the PPP was functional in some strains of the parasite [14,15]. Most of the
enzymes of the pathway, however, had not been detected
up to now, and nothing was known about their subcellular localisation and properties.
We show in this communication that all the enzymes of
the PPP are present in the four major developmental
stages of T. cruzi; that their subcellular localisation is essentially cytosolic, with the exception of ribulose-5phosphate epimerase (Ru5PE), although particulate
compartments are found for all of them, and that the PPP
is functional in the parasite and increases its activity in the
presence of a chemical that mimics oxidative stress.
2. Materials and methods
2.3. Enzyme assays
All enzyme assays were performed at 30 °C; the reaction mixtures were equilibrated for 3 min at this
temperature, and the reactions were usually started by
addition of the cell-free extract.
G6PDH (EC 1.1.1.49), 6-phosphogluconolactonase
(Lactonase, EC 3.1.1.31), 6PGDH (EC 1.1.1.44), ribose5-phosphate isomerase (R5PI, EC 5.3.1.6), Ru5PE (EC
5.1.3.1), transaldolase (TA, EC 2.2.1.2) and transketolase (TKT, EC 2.2.1.1) (using as substrate either D -ribose-5-phosphate or D -erythrose-4-phosphate) were
assayed as described in [10].
Citrate synthase (CS, mitochondrial marker, [20]),
PK (cytosolic marker, [21]) and phosphoenolpyruvate
carboxykinase (PEPCK, glycosomal marker, [22]) were
assayed as previously described. HK, another glycosomal marker, was assayed in the presence of 2 mM 6phosphogluconate, in a reaction mixture containing 0.05
M triethanolamine, pH 7.5, 5 mM MgCl2 , 0.5 mM
NADP, 2 mM glucose, 0.25 mM ATP and 1 U of
G6PDH. The activity measured in the absence of 6phosphogluconate was subtracted from that attained in
its presence. GPI, which is a cytosolic and glycosomal
marker, was assayed in a reaction mixture containing
triethanolamine, MgCl2 , NADP and G6PDH at the
same concentrations, plus 2 mM fructose 6-phosphate.
2.1. Parasites and culture
2.4. Subcellular localisation experiments
Epimastigotes of the CL Brener clone were grown in
axenic medium, harvested and washed as previously
described [16]. Metacyclic trypomastigotes, amastigotes
and cell-culture trypomastigotes were obtained as before
[17]. Cell-free homogenates were obtained by sonication
[10]. The suspensions were used immediately for activity
determinations in hypotonic reaction mixtures, in order
to prevent the inactivation of some of the enzymes.
Protein concentration was determined by the Lowry
et al. method [18].
2.2. Production of 14 CO2 from [1-14 C] or [6-14 C]D glucose, in the absence or in the presence of methylene blue
Epimastigotes (5 108 ml1 ) were incubated with
shaking at 26 °C in Warburg flasks, with 10 mM glucose
(0.007–0.02 lCi lmol1 of either [1-14 C]D -glucose or [614
C]D -glucose), with or without 0.2 mM methylene blue.
The experiments were performed as previously described
for L. mexicana [10]. The specific yield of CO2 from [114
C]D -glucose and [6-14 C]D -glucose, G1CO2 and G6CO2 ,
respectively, was calculated as the ratio of 14 CO2 produced to radioactive glucose consumed, and used to
calculate the fraction of glucose used through the PPP
employing the formula [19]
G1CO2 G6CO2 =ð1 G6CO2 Þ ¼ 3PPP=ð1 þ 2PPPÞ:
A preliminary assessment of the subcellular localisation of the enzymes was made by digitonin treatment of
intact parasite cells. Epimastigotes of the CL Brener
clone were suspended in 25 mM Tris–HCl buffer, pH
7.6, containing 1 mM EDTA and 0.25 M sucrose, E-64
10 lM, with the addition of a freshly prepared digitonin
solution in dimethylformamide, at final concentrations
up to 3 mg ml1 . After incubation at 25 °C for 5 min, the
cells were separated by centrifugation in an Eppendorf
bench centrifuge at 18,000g for 2 min at room temperature, and the supernatants were kept for enzyme assays. The pellets were suspended in the same buffer and
sonicated (three pulses, 30 s each, at 60% of maximum
power). All the PPP enzyme activities were determined
in both fractions, together with the activities of marker
enzymes for mitochondrion, glycosomes and cytosol.
Addition of 0.2% Triton X-100 increased the activity of
HK, but not of the other PPP enzymes, by 30%.
Therefore HK activity was assayed in the presence of the
detergent in both fractions. 100% activity is taken as the
sum of the activities in both supernatants at a given
digitonin concentration [20].
Further experiments for subcellular localization included fractionation by differential centrifugation.
Epimastigotes of the CL Brener clone were disrupted in
a mortar using silicon carbide, in a ratio of 2 g per g of
D.A. Maugeri, J.J. Cazzulo / FEMS Microbiology Letters 234 (2004) 117–123
cells, wet weight. The cell paste was suspended in the
same buffer used for the digitonin experiments.
The suspension was centrifuged 3 min at 100g to remove
the abrasive, which was then washed and centrifuged
again. Both supernatants were mixed, to give the total
homogenate, and submitted to fractionation by differential centrifugation. The fractions obtained were: nuclear fraction (N, 1000g, 10 min), large granules (LG,
7600g, 10 min), small granules (SG, 27,000g, 20 min),
microsomal fraction (M, 200,000g, 1 h) and soluble
fraction (S). The latter contains the cytosol as well as
soluble proteins leaking out of damaged organelles. The
pellets were washed three times and suspended in 1.1 ml
of the same buffer. Latency of enzymes in the particulate
fractions was determined by assaying the enzyme activities in reaction mixtures made isotonic by the addition of sucrose (0.25 M final concentrations), in the
absence or in the presence of 0.2% Triton X-100.
119
0.25
A
Glucose utilization
0.20
0.15
0.10
0.05
0
0.10
B
0.08
CO2 evolution
2.5. Chemicals
0.06
0.04
14
Glucose labelled with 14 C at C1 or C6 was obtained
from American Radiolabeled Chemicals, St. Louis, MO,
USA. All substrates, coenzymes, coupled enzymes and
digitonin, were obtained from Sigma Chemical Co., St.
Louis, MO, USA. Salts and buffers were analytical reagents of the highest purity available.
0.02
3. Results and discussion
0
0
3.1. Production of 14 CO2 from [1-14 C] or [6-14 C]D glucose, in the absence or in the presence of methylene blue
The 6PGDH reaction liberates CO2 from the C1
position of D -glucose and the relative flux of glucose via
the PPP and the Embden–Meyerhof pathway has classically been determined by measuring the amount of
carbon dioxide liberated from glucose labelled at either
the C1 or the C6 position [19]. Since in T. cruzi
30
60
90
120
Time (min)
Fig. 1. Utilization of glucose and production of 14 CO2 from [1-14 C] or
[6-14 C]D -glucose, in the absence or in the presence of methylene blue.
The experiment was carried out as described under Materials and
Methods. (A) Glucose utilization (glucose utilized divided by initial
concentration) in the absence ðsÞ or in the presence ðdÞ of methylene
blue. (B) 14 CO2 evolution (14 CO2 divided by total initial radioactivity
in glucose) from [1-14 C]D -glucose, in the absence ðsÞ or in the presence
ðÞ of methylene blue; production of 14 CO2 from [6-14 C]D -glucose, in
the absence ðdÞ or in the presence ðjÞ of methylene blue.
Table 1
Activities of the pentose phosphate pathway enzymes in the four major developmental stages of Trypanosoma cruzi
Enzymea
Epimastigotes
Amastigotes
Metacyclic trypomastigotes
Cell-culture trypomastigotes
Glucose-6-phosphate dehydrogenase
6-Phosphogluconolactonase
6-Phosphogluconate dehydrogenase
Ribose-5-phosphate isomerase
Ribulose-5-phosphate epimerase
Transketolase (ribose-5-phosphate)
Transketolase (erythrose-4-phosphate)
Transaldolase
69 2
1020 10
289 8
93 4
527 24
166 4
194 3
45 2
767 81
1190 490
346 39
21 2
517 16
87 5
140 7
70 8
1030 150
2060 110
1130 80
108 4
255 18
376 27
529 9
204 18
541 42
793 81
289 5
76 8
385 11
168 6
225 6
135 6
a
The enzyme activities were assayed in parasite homogenates, as described under Materials and methods, and expressed as nmoles min1 mg of
protein1 . The protein concentrations in extracts from 109 epimastigotes, amastigotes, metacyclic trypomastigotes and cell-culture trypomastigotes
were 12.39 0.48; 1.43 0.18; 1.26 0.08 y 1.96 0.16 mg ml1 , respectively. Transketolase activity was assayed using as substrates ribose 5phosphate or erythrose 4-phosphate.
D.A. Maugeri, J.J. Cazzulo / FEMS Microbiology Letters 234 (2004) 117–123
3.2. Presence and activities of the PPP enzymes in the
four major developmental stages of T. cruzi
Table 1 shows that all seven enzymes of the classical
PPP could be detected in cell homogenates of the four
major developmental stages of T. cruzi, CL Brener
clone. With the exception of Ru5PE, which had its
highest specific activity in epimastigotes and amastig-
100
A
80
% Activity
epimastigotes a substantial proportion of glucose carbon is excreted as incompletely oxidized products
branching before the PK reaction [1], the determination
of glucose flux through the glycolytic pathway can not
be reliably made unless the incorporation of label in
these fermentative products is determined. However, the
utilization of glucose through the PPP may be estimated
from the specific yields of CO2 . G6CO2 is the fraction of
glucose liberated as CO2 in the tricarboxylic acid cycle
and G1CO2 measures the fraction of glucose utilized via
both, the cycle and the PPP. Therefore, glucose utilization through the PPP is proportional to the difference
between G1CO2 and G6CO2 . This approach was taken to
estimate the flux of glucose through the oxidative
branch of PPP in T. cruzi. As no nitrogen source was
used in these working conditions, accumulation of
pentose phosphate for nucleotide syntesis should be
negligible, allowing recycling of G6P through reactions
catalyzed by TA, TKT and GPI, and the operation of a
complete PPP [19]. This is possible, since all the enzymes
of the non-oxidative pathway are present (see below).
Cells were also incubated in the presence of methylene
blue (0.2 mM), which enters cells and oxidizes NADPH
to NADP [23]. Glucose utilization was linear over the
whole time interval and there was very little difference in
the presence or absence of methylene blue (1.92 vs. 2.06
lmol glucose h1 109 parasites, in the absence or in
the presence of the chemical, respectively) (Fig. 1A). The
liberation of CO2 from [1-14 C]D -glucose was clearly
higher than that from [6-14 C]D -glucose, showing that the
PPP was functional. In the presence of methylene blue,
the production of CO2 from [1-14 C]D -glucose increased,
whereas that from [6-14 C]D -glucose, representative of
the carbohydrate fraction completely oxidized, was not
significantly changed (Fig. 1B). Utilization of glucose
through the PPP, calculated as described under Materials and methods, was 0.19 lmol glucose h1 109
parasites in the absence, and 0.42 lmol glucose
h1 109 parasites in the presence of methylene blue,
corresponding to 9.9% and 20.4%, respectively, of the
glucose consumed. These results show that the PPP is
operative under the experimental conditions tested. In
addition, the regulatory character of the PPP is shown,
since in the presence of methylene blue the inhibition of
G6PDH by a high NADPH:NADP ratio is relieved,
and selective utilization of glucose through the PPP is
stimulated.
60
40
20
0
100
B
80
% Activity
120
60
40
20
0
0
1
2
3
Digitonin (mg.ml-1)
Fig. 2. Digitonin extraction of intact Trypanosoma cruzi epimastigotes.
The experiment was carried out as described under Materials and
Methods. 125 mg of epimastigotes were used per eppendorf tube, in the
presence of the digitonin concentrations stated on the abscissa. Panel
A: glucose-6-phosphate dehydrogenase ðdÞ, lactonase (X) and 6phosphogluconate dehydrogenase ðsÞ. Panel B: ribose 5 phosphate
isomerase ð.Þ, ribulose 5 phosphate epimerase ðOÞ, transketolase ð}Þ
and transaldolase ðrÞ. Panels A and B, marker enzymes: hexokinase
ðNÞ, citrate synthase ðÞ, glucose phosphate isomerase ðMÞ and pyruvate kinase ðjÞ. Transketolase activity was determined using erythrose-4-phosphate and xylulose-5-phosphate as substrates.
otes, all the other PPP enzymes presented their highest
specific activities in metacyclic trypomastigotes. It is
noteworthy that the activity of G6PDH, the regulatory
enzyme of the pathway in many systems, was about an
order of magnitude lower in epimastigotes than in the
other stages. This suggests that the PPP may be more
active in the stages present in the mammalian host, or
ready for invasion. Although cell disruption by freezing
and thawing yielded higher specific activities for all the
enzymes in the epimastigote extract (not shown)
sonication and enzyme assay of cell homogenates in
D.A. Maugeri, J.J. Cazzulo / FEMS Microbiology Letters 234 (2004) 117–123
hypotonic media was chosen to discard the possibility of
underestimation of some of the enzyme activities, if
placed in resistant subcellular compartments, which
might be the case for the glycosome.
3.3. Subcellular localisation experiments
Fig. 2 shows the results of a typical digitonin experiment. The pattern of digitonin extraction of the PPP
enzymes was compared with those of cytosolic (PK),
mitochondrial (CS) and glycosomal (HK) markers. GPI,
which is known to have a double localisation, cytosolic
121
and glycosomal [2], is also included. With the exception
of Ru5PE, all the PPP enzymes had a major cytosolic
component, since their extraction curves initially followed those of the cytosolic marker PK and the first
phase of extraction of GPI. At a digitonin concentration
of 1 mg ml1 , where almost all the cytosolic marker had
been extracted, 35% of the G6PDH and TA activities,
20% of the R5PI y TKT activities, 15% of the 6PGDH
activity and 10% of the lactonase activity still remained in
the pellet. Between 1 and 2 mg ml1 6PGDH, R5PI, TA y
TKT presented a second phase of extraction similar to
that of GPI. There was no further extraction of G6PDH
Fig. 3. Subcellular fractionation of Trypanosoma cruzi epimastigotes by differential centrifugation. The experiment was carried out as described under
Materials and Methods, using 3.3 g (wet weight) of parasites. Fractions are plotted in the order of their isolation, from left to right: nuclear (N), large
granule (G), small granule (SG), microsomal (M) and final supernatant (S). The ordinate represents relative specific activity (percentage of total
activity/percentage of total protein). The abcissa indicates the cumulative protein content. The percentage of recovery for the markers and the PPP
enzymes varied between 73.9 and 94.3, with the exception of ribulose-5-phosphate isomerase (55.5%), RNA (60.3) and isocitrate dehydrogenase
(61.8%). Transketolase activity was determined using erythrose-4-phosphate and xylulose-5-phosphate as substrates.
122
D.A. Maugeri, J.J. Cazzulo / FEMS Microbiology Letters 234 (2004) 117–123
up to 3 mg of digitonin per ml1 . Ru5PE consistently
showed, in different experiments, a very odd behaviour,
since most of the enzyme activity was extracted at very
low digitonin concentrations, below those required to
release the cytosolic marker. This suggests that Ru5PE is
present in a highly accessible compartment. Subcellular
fractionation by differential centrifugation (Fig. 3) confirmed that, with the only exception of Ru5PE, over 80%
of the total activity of each of the PPP enzymes was found
in the S fraction, which consists of the cytosol and of
material leaking out of damaged organelles. Latency of
the enzymes in the particulate fractions ranged from
about 50% to 100%. The presence of all the PPP enzymes
in the SG fraction suggests the possibility of a small
glycosomal component for most of them. It is noteworthy that both dehydrogenases presented their highest
particulate activities in the M fraction, suggesting a
possible minor localization in the endoplasmic reticulum
as reported for mammalian cells [5]. On the other hand,
R5PI, Ru5PE and TKT were not detected in the latter
fraction. Ru5PE was clearly present in membrane-bound
vesicles, despite its odd behaviour in the digitonin experiments (Figs. 2 and 3).
The experiments shown in this communication indicate that the PPP is operative in living epimastigotes,
and its activity increases in the presence of a chemical
which oxidizes NADPH, thus mimicking oxidative
stress. Trypanothione, which is maintained in a reduced
state by trypanothione reductase using NADPH, and
the enzymes involved in its metabolism [24] are the
chief mechanism to counteract oxidative stress. Although these experiments were performed, for practical
reasons, only with the easily available culture epimastigote stage, the presence of all the PPP enzymes in the
four major developmental stages of the parasite suggests that the protective role of the PPP against ROS is
probably operative throughout the parasite’s life cycle.
The enzyme levels shown in Table 1, indicate that all
the enzymes of the PPP have substantial levels in the
parasite stages present in the mammal, or ready to invade the mammalian host (the metacyclic trypomastigote). This fact may be related to a possibly higher
exposure of the parasite to oxidative stress inside the
mammal. This would be particularly significant for the
amastigotes of reticulotropic strains of T. cruzi inside
the macrophages.
As discussed for T. brucei [3] the fact that most of the
enzyme activities are cytosolic poses the question of
availability of the phosphorylated sugars for operation
of the PPP. However, it has been recently reported that
a fraction of HK is outside the glycosome, probably in
the cytosol, in T. cruzi epimastigotes [25]. The experiments reported here suggest the possibility of multiple
minor localisations for most of the PPP enzymes. Future
work will attempt to characterize these localisations in
more detail.
Acknowledgements
The present work was supported by Grant PICT2000
08149 from the ANPCyT, SECYT, Argentina. J.J.C. is a
member of the Research Career of the Argentinian
National Research Council (CONICET).
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