Lipoic acid metabolism in Trypanosoma cruzi as putative target for chemotherapy

Article abstract of DOI:10.1016/j.exppara.2018.01.017

Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic deleterious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC₅₀ values one order of magnitude lower (62–66 μM). LA did not bypass the toxic effect of its analogues. Small monocarboxylic acids appear to be poorly internalized by T. cruzi: [¹⁴C]-octanoic acid was taken up 12 fold less efficiently than [¹⁴C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydrogenase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with BrO (1 mM) and MBrO (0.1 mM) completely inhibited E2 lipoylation and all three dehydrogenases activities. These observations indicate the lack of specific transporters for octanoic acid and most probably also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target for drug intervention. Moreover, the free LA available in the host would not interfere with such chemotherapeutic treatments.

Full text of DOI:10.1016/j.exppara.2018.01.017

Experimental Parasitology 186 (2018) 17e23
Contents lists available at ScienceDirect
Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr
Lipoic acid metabolism in Trypanosoma cruzi as putative target for
chemotherapy
*
Paola Vacchina, Daniel A. Lambruschi, Antonio D. Uttaro
Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquímicas y Farmac ꢀe uticas, Universidad Nacional de Rosario,
Rosario, Argentina
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
ꢀ
ꢀ
ꢀ
Trypanosoma cruzi protein lipoylation
is dependent of glucose availability.
Protein lipoylation is inhibited by
lipoic acid analogues.
Lipoic acid, octanoic acid and 8-
bromo-octanoic acid are poorly
taken up by T. cruzi.
ꢀ
ꢀ
Methyl-8-bromo-octanoic
MBrO) is efficiently incorporated by
T. cruzi.
MBrO is an effective growth inhibitor
and inhibition is not bypassed by
lipoc acid.
acid
(
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 December 2016
Received in revised form
Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-
ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic dele-
terious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma
cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth
of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically
irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC50 values
12 January 2018
Accepted 30 January 2018
Available online 1 February 2018
one order of magnitude lower (62e66 mM). LA did not bypass the toxic effect of its analogues. Small
Keywords:
14
monocarboxylic acids appear to be poorly internalized by T. cruzi: [ C]-octanoic acid was taken up 12
fold less efficiently than [14C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the
detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydroge-
nase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower
glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with
BrO (1 mM) and MBrO (0.1 mM) completely inhibited E2 lipoylation and all three dehydrogenases ac-
tivities. These observations indicate the lack of specific transporters for octanoic acid and most probably
also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested
for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target
for drug intervention. Moreover, the free LA available in the host would not interfere with such
chemotherapeutic treatments.
Lipoic acid
Protein lipoylation
Drug target
Chagas disease
Trypanosoma
©
2018 Elsevier Inc. All rights reserved.
*
Corresponding author. Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farm-
ac eꢀ uticas, Universidad Nacional de Rosario. Ocampo y Esmeralda, 2000 Rosario, Santa Fe, Argentina.
E-mail address: toniuttaro@yahoo.com.ar (A.D. Uttaro).
https://doi.org/10.1016/j.exppara.2018.01.017
0
014-4894/© 2018 Elsevier Inc. All rights reserved.

18
P. Vacchina et al. / Experimental Parasitology 186 (2018) 17e23
1. Introduction
subtilis lacks a LipB homologue, but encodes one LipA and three LplA
homologues (LplJ, LipM and LipL). One of them, LplJ has the ligase
function. This Gram-positive bacterium presents a variation in the
synthesis/lipoylation pathway: LipM is an octanoyl transferase that
specifically transfers octanoate from ACP to the H protein. LipL is an
amidotransferase that transfers the octanoyl moiety from H protein
to the E2 subunits of dehydrogenases (Cronan, 2016). Saccharomyces
cerevisiae encodes Lip2, Lip5 and Lip3, structural homologues of LipB,
LipA and LplA respectively. Lipoylation of H protein is required for
lipoylation of E2 and it has been suggested that the yeast would be
unable to scavenge lipoate, indicating that Lip3 could have amido-
transferase activity, like B. subtilis LipL (Schonauer et al., 2009).
However, it was recently shown that whereas Lip2 is an octanoyl-
ACP:protein transferase, apparently specific for H protein, Lip3 is an
octanoyl-CoA:protein transferase involved in the acylation of E2
subunits (Hermes and Cronan, 2013).
A survey of trypanosomatid genomes showed the presence of
genes encoding subunits of PDH (E1p, E3Bp, E2p), KGDH (E1k, E2k),
BCDH (E1b, E2b) and GCS (P, H and T proteins), and a sole DHLDH
probably shared by the four complexes. It also revealed the pres-
ence of enzymes putatively involved in lipoate synthesis/lip-
oylation, including orthologs of LipA (or Lip5), LipB (Lip2) and LplA
(lip3) (Spalding and Prigge, 2010).
Indirect experimental evidence indicated the absence of lipoate
salvage in these protists (Stephens et al., 2007), suggesting that as
observed in yeast, LplA or Lip3 homologues could not be true li-
gases. The lack of salvage however, should facilitate the use of in-
hibitors of lipoate metabolism in chemotherapy, as the only source
of LA for the parasite would be the one produced by the de novo
pathway. The fact that mammals are highly dependent on LA
derived from food and intestinal bacteria makes this feature even
more attractive (Bustamante et al., 1998).
Several enzyme complexes widely distributed in nature require
lipoic acid (6,8-dithiooctanoic acid;LA) as prostheticgroup. The most
relevant of them are pyruvate dehydrogenase (PDH), 2-ketoglutarate
dehydrogenase (KGDH), branched-chain 2-ketoacid dehydrogenase
(
BCDH) and the glycine cleavage system (GCS) (Perham, 2000; Douce
et al., 2001; Spalding and Prigge, 2010). These are involved in
important metabolic pathways needed to sustain cell viability. PDH
produces acetyl-CoA, driving the pyruvate generated in glycolysis to
the tricarboxylic acid (TCA) cycle (Tielens and van Hellemond, 2009)
and to the synthesis of fatty acids (Ramakrishnan et al., 2013; Uttaro,
2
014), sterols (de Souza and Rodrigues, 2009) and other cell com-
ponents. Succinyl-CoA produced by KGDH is an intermediate of the
TCA cycle, precursor of several amino acids and substrate in the
synthesis of porphyrins. Ketoacids derived from the deamination of
valine, leucine and isoleucine are decarboxylated by BCDH gener-
ating CoA-activated primers used in the synthesis of branched-chain
fatty acids (Perham, 2000). GCS catalyses the reversible decarbox-
ylation of glycine with release of ammonia and methylene groups.
Methylene groups are transferred to tetrahydrofolate, generating
5
,10-methylene-tetrahydrofolate involved in the synthesis of amino
acids and nucleotides (Douce et al., 2001). In addition, the de-
carboxylations described above generate NADH, which can be used
in oxidative phosphorylation.
The eukaryotic dehydrogenase complexes are localized in the
mitochondrial matrix and are composed of multiple copies of each
of three enzymatic subunits referred to as E1 (decarboxylase), E2
(
acyl transferase), and E3 (dihydrolipoamide dehydrogenase;
DHLDH). Most eukaryotic PDHs contain a fourth subunit (E3Bp),
that acts as a linker between E3 and the E2 multimer. LA is cova-
6
lently attached to the N amino group of a lysyl residue in the lipoyl
domain of E2 and to conserved lysyl moieties of E3Bp (Perham,
It was recently found that the interference of DHLDH expression,
which affects the recycling of LA, had a strong proliferation defect
in T. brucei followed by rapid cell death (Roldan et al., 2011).
However, no data about the effect of inhibition on LA synthesis or
protein lipoylation are available.
The aim of this work is to validate LA metabolism as a drug
target against T. cruzi. We show here a chemical approach, by using
LA analogues, to study the relative importance of LA biosynthesis
and LA salvage in this organism.
2
000). The subunits of GCS are named P (glycine decarboxylase),
H, T, and L proteins. LA is covalently bound to a lysyl moiety of the H
protein, which does not have catalytic activity but acts as a scaffold
to protect the unstable intermediate during transfer to the T pro-
tein, which catalyses the release of ammonia from methyleneamine
and the transfer of the methylene group to tetrahydrofolate (Douce
et al., 2001). L protein is the DHLDH that regenerates lipoamide;
most organisms share the same DHLDH as E3 or L protein in both 2-
ketoacid dehydrogenase complexes and GCS.
LA plays an essential role in the catalytic activity of these
complexes and any intervention with its synthesis or regeneration
would probably lead to a general deleterious effect in the cell. This
feature heightens its value as a chemotherapeutic target. As LA
metabolism has not been studied in detail in parasitic organisms
like trypanosomatids (Spalding and Prigge, 2010), it is our interest
to evaluate and validate it as a putative target for drug discovery.
Trypanosomatids are flagellated protists belonging to the Kineto-
plastida, grouping species like Trypanosoma brucei and Trypano-
soma cruzi, the causative agents of sleeping sickness and Chagas
disease, respectively (Barrett et al., 2003). These are considered
neglected diseases with elevated morbidity and mortality if not
treated. The repertoire of available treatments is limited and most
of the drugs used are toxic and, in some cases, ineffective, requiring
urgent development of new chemotherapies.
2. Materials and methods
2.1. Parasite culture and growth inhibition assays
Epimastigotes of T. cruzi CL Brener and Dm28c strains were
grown in LIT medium at 28 C and the culture medium was sup-
plemented with 10% fetal bovine serum. When indicated, modified
LIT medium (mLIT) was used, containing 0.4 g/l of glucose instead
of the regular amount (4 g/l) (Camargo, 1964). Growth curves were
obtained by direct observation and cell counting in a Neubauer
ꢁ
6
haemocytometer, starting from a parasite density of 1 ꢂ 10 cells/
ml. To study the effect of the LA analogues 8-bromo-octanoic acid
(BrO) and methyl-8-bromo-octanoic acid (MBrO) (Fig. 1), parasites
6
were seeded in 24 well plates at 1 ꢂ 10 cells/ml and compounds
were added at increasing concentrations. Solvent (DMSO) was al-
LA metabolism has been described in detail only in some model
organisms (Cronan, 2016). Escherichia coli for instance, exhibits the
simplest route involving the transfer of an octanoyl moiety from
octanoyl-acyl carrier protein (octanoyl-ACP) to E2 and H protein by
LipB (octanoyl transferase). Subsequently, LipA (lipoate synthase)
inserts two sulfur atoms into the octanoyl moiety giving the dithio-
lane ring of the lipoyl moiety. E. coli is also able to scavenge LA; the
lipoate ligase LplA uses free LA to acylate E2 and H protein. Bacillus
ways at a final concentration of 0.5% (v/v), including the control.
Plates were incubated at 28 C. EC50 values were calculated by non-
linear regression analysis using SigmaPlot (v 11.0). All experiments
were done in triplicate with appropriate controls in each case.
ꢁ
2.2. Synthesis of methyl-8-bromo-octanoic acid
Fifty
mg of BrO (Sigma) were dissolved in 2 ml of freshly

P. Vacchina et al. / Experimental Parasitology 186 (2018) 17e23
19
mitochondrial fraction, cells were pelleted by centrifugation at
ꢁ
3
000 ꢂ g for 5 min at 4 C, washed and resuspended in 50 mM Tris-
HCl, pH 7.4 in the presence of a cocktail of protease inhibitors. After
cycles of freezing-thawing and vortexing, the lysate was ho-
3
mogenized with a 21G syringe and subsequently centrifuged at
ꢁ
1
000 ꢂ g for 5 min at 4 C. The mitochondrial fractions recovered in
the supernatants were next lysed by the addition of 0.1% Triton X-
00. Protein concentration was determined using BSA as standard.
1
PDH, KGDH and BCDH activities were measured by following the
ꢁ
production of NADH at 340 nm at 25 C and calculations were based
on the initial rates. The reaction mixture contained 150 mM Tris/
þ
HCl, pH 7.4, 3 mM cysteine, 0.2 mM CoASH, 4 mM NAD , 1 mM
MgCl
00
substrate (sodium pyruvate, sodium
2
m
and 140
l. The reactions were initiated by the addition of 10 mM of
-ketoglutarate or sodium a-
mg of mitochondrial extract in a final volume of
2
a
ketoisovalerate, respectively). Assays were done in triplicate. Ma-
late dehydrogenase, as a control of cell viability, was assayed as
previously described (Hunter et al., 2000).
Fig. 1. Structures of lipoic acid and its analogues.
prepared (2%) H
2
SO
4
in anhydrous methanol and incubated during
3. Results and discussion
ꢁ
2
h at 80 C. The resultant methyl ester derivative (MBrO) was
extracted twice with hexane. After drying under a N
was dissolved in DMSO.
2
stream, MBrO
3.1. LA analogues are inhibitors of T. cruzi growth
We first evaluated the effect of an LA analogue on T. cruzi growth
2
.3. Proteins extraction and Western blots
and viability. Growth inhibition assays were carried out using
T. cruzi epimastigotes of CL Brener and Dm28c strains and parasite
viability was assessed by direct microscopic observation and cell
counting. Both strains were susceptible to BrO (Fig. 1), showing
EC50s of 0.470 ± 0.010 mM (CL Brener) and 0.885 ± 0.015 mM
Dm28c) (Fig. 2). The effect of BrO on cell growth and lipoylation
profile has been previously studied in T. gondii (Crawford et al.,
006) and P. falciparum (Allary et al., 2007). The addition of this
compound to cultures of these organisms drastically inhibited
parasite replication and LA supplementation was able to partially
rescue the death phenotype. Although the effective BrO concen-
tration was different for each of these parasites (approximately
For the preparation of crude cell lysates, parasites were grown in
the corresponding medium and proteins extracted after cell lysis.
Briefly, after harvesting, cells were washed once by centrifugation
with ice-cold phosphate saline buffer (PBS) and lysed in buffer
(
(
150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl, pH 8) in the pres-
ence ofa cocktailofprotease inhibitors(Complete Mini tablets, Roche
Applied Science). For mitochondrial enrichment, digitonin mem-
brane permeabilization was done as previously described (Foucher
et al., 2006). The protein concentration was determined using BSA
as standard. Samples were run in 12% SDS-PAGE gelsandblotted onto
polyvinylidene difluoride (PVDF) membranes. Proteins were
2
10 mM and 100 mM, respectively), the ability of LA to revert the
revealed using anti-lipoic acid antibody (Abcam) or anti-a-tubulin
growth phenotype indicated that apicomplexan parasites have an
active LA scavenging route. The mechanism of BrO inhibition was
shown to require the action of LipB, which catalyses the ligation of
the analogue to the apo-protein and produces an inactive inter-
mediate that cannot be used as substrate for LipA and hence cannot
produce the active lipoyl-group.
antibody (Sigma-Aldrich, St. Louis, USA) and anti-rabbit HRP-conju-
gated antibodies (Pierce). After washing, blots were processed using
the SuperSignal chemiluminescent detection system (Pierce).
2.4. Monocarboxylic acids uptake assay
We also tested whether LA addition to a T. cruzi culture would be
able to influence the parasite's response to 1 mM of the inhibitor.
Interestingly, 1 mM of LA appears to be toxic, with evident growth
inhibition after 48 h of treatment. Simultaneous supplementation
of LA and BrO to the Dm28c strain showed an additive deleterious
effect (Fig. 3A). A similar result was obtained by using the most
susceptible CL Brener strain and 0.5 mM of each, LA and BrO
The protocol was adapted from Voorheis (1980). Briefly, to 500 ml
of a T. cruzi epimastigote culture in LIT medium in its logarithmic
8
growth phase and enriched to a cell density of 2 ꢂ 10 parasites/ml,
5
5
00 ml of LIT medium without foetal bovine serum but containing
1
4
m
M fatty acid free bovine serum albumin (Sigma), 1
m
l [1e C]
octanoic acid (396 290 dpm; New England Nuclear) and 60
mM so-
dium octanoate were added. The cultures were incubated for
(Fig. 3B). Lower concentrations of LA were not toxic, but did not
different periods of time. Incubations were terminated by adding
bypass the effect of BrO.
ꢁ
1
0 ml PBS at 0 C and cooled on ice. Cells were harvested, resus-
pended in 500
porated octanoate (
positive control, a similar experiment was carried out by using the
same specific activity of [1e C] palmitic acid (13 200 dpm/nmole,
New England Nuclear). The experiment was done in triplicate.
m
l of PBS and transferred to scintillation vials. Incor-
3
.2. T. cruzi epimastigotes cannot efficiently incorporate small
mmols) were calculated from total cpms. As a
monocarboxylic acids
14
The relative high EC50s determined for both T. cruzi strains may
be an indication that BrO is not effectively incorporated into the
cells. Due to the unavailability of radioactive LA or BrO, we used the
commercially available [ C] octanoate, in order to evaluate the
uptake of related small monocarboxylic acids by T. cruzi (Fig. 4). We
could not detect significant amounts of radioactive label associated
to cells, which indicated that free octanoate was not actively
transported across the plasma membrane. As a positive control, the
14
2.5. Mitochondria isolation and enzyme activity assays
Parasites were grown in LIT and mLIT for 72 h. Additionally, a
subset of parasites cultured in mLIT plus 100 mM MBrO was also
included in the study. A modification of the method described by
Schonauer et al. (2009) was used. Briefly, for the isolation of a crude
14
uptake of [ C] palmitate was also assayed, as this fatty acid was

20
P. Vacchina et al. / Experimental Parasitology 186 (2018) 17e23
Fig. 2. Growth curves of T. cruzi epimastigotes cultured with increased concentrations of 8-bromo-octanoic acid (BrO). The CL Brener (A) and Dm28c (B) strains were tested. Closed
and open circles in the right hand panels represent the values of each duplicate at 48 h incubation.
previously shown to be readily incorporated by trypanosomatids,
100 mM MBrO for 72 h. Bands corresponding to the E2 subunits of
presumably using specific transporters (Voorheis, 1980; Uttaro,
PDH (E2p), BCDH (E2b) and KGDH (E2k) were revealed in a Western
blot with a polyclonal antibody that detects lipoyl moieties. Sub-
lethal concentrations of both compounds were able to fully
inhibit protein lipoylation, indicating that BrO and MBrO could
inhibit the acyl-transfer step or the introduction of sulfydryl groups
catalyzed by a lipoyl synthase.
2
014).
3.3. MBrO has increased membrane permeability and exhibits
higher toxicity to trypanosomes
Our data have shown that T. cruzi cannot avidly incorporate
octanoate and, most probably, neither LA and BrO from the external
medium, which is in agreement with the lack of a LA scavenging
route. This result validates the high EC50 values obtained when
cultures were exposed to BrO. This also suggests that LA and LA
analogues could only be partially incorporated into the cell as a
result of very inefficient passive diffusion. Under these circum-
stances, the chemotherapeutic relevance of the inhibitor is clearly
reduced as the amount of it needed to meaningfully reduce parasite
viability is considerably high (millimolar range). Therefore, we
modified the BrO molecule to its methyl ester derivative MBrO
3
.5. T. cruzi lipoylation pattern is regulated by growth conditions
A curious result of the previous section (Fig. 7) was the weak
signal for the lipoylated E2 subunits of BCDH (E2b) and PDH (E2p,
most notable). Although the insect stages of T. cruzi uses L-proline
as the main carbon and energy source, the parasite favors the use of
glucose, if it is available, over the amino acid (Bringaud et al., 2012).
The sugar is metabolized via the glycolytic pathway that takes place
mainly inside glycosomes, leading to the production of succinate
that is excreted into the medium (Maugeri et al., 2011). A significant
fraction of the glycolytic flux, however, goes to the synthesis of
pyruvate by the action of cytosolic pyruvate kinase; pyruvate is
transported into the mitochondria and converted into acetyl-CoA
by PDH. In T. brucei, acetyl-CoA is used for substrate level phos-
phorylation, producing ATP by two coupled reactions catalyzed by
the acetyl:succinate CoA transferase and succinyl CoA synthetase.
Genes encoding both enzymes are also present in the T. cruzi
genome. When glucose is not present, proline can be metabolized
to 2-ketoglutarate and subsequently converted into succinyl-CoA
by the action of KGDH (Bringaud et al., 2012). It can be concluded
that both PDH and KGDH are relevant enzymatic complexes to
support growth of T. cruzi epimastigotes in standard culture media
like LIT (rich in glucose and aminoacids) and therefore a robust
expression of subunit components of both dehydrogenases is ex-
pected. We decided to evaluate the effect of glucose content on
(Fig. 1) to increase its permeation across the parasite cell mem-
brane. Both parasite strains were significantly more sensitive to
treatment with this derivative when compared to BrO (Fig. 5), with
EC50 values more than one order of magnitude lower being
6
6.2 ± 10.4 and 62.2 ± 7.5
respectively. The toxic effect of MBrO on CL Brener strain was not
bypassed by LA addition when assayed at 100 M (Fig. 6), a con-
mM for CL Brener and Dm28c strains,
m
centration at which it did not show significant deleterious effect
when administered alone.
3.4. LA analogues block protein lipoylation
LA metabolism and protein lipoylation were also altered by BrO
and MBrO. Fig. 7 shows the lipoylation profile of T. cruzi parasites
Dm28c strain) grown in the absence and presence of 1 mM BrO or
(

P. Vacchina et al. / Experimental Parasitology 186 (2018) 17e23
21
Fig. 4. Monocarboxylic acid incorporation into T. cruzi epimastigote cells. Cells from
ꢁ
14
Dm28c strain were incubated at 30 C during different periods with 30
octanoic acid or 30
dpm/nmole).
m
M [1e C]
14
mM [1e C] palmitic acid, both at the same specific activity (13200
Fig. 3. Effect of lipoic acid on cultures of T. cruzi. (A) Epimastigotes of T. cruzi Dm28c
strain were grown in the absence or presence of 1 mM BrO, in LIT medium supple-
mented with lipoic acid (LA, 1 mM). (B) Epimastigotes of CL Brener strain grown in the
absence or presence of 0.5 mM BrO and/or 0.5 mM LA.
parasite protein lipoylation. Cells were cultivated in media con-
taining regular (4 g/l) or low (0.4 g/l) glucose concentration (LIT and
mLIT media, respectively) and growth rates were followed over
time. In mLIT medium parasite growth reached stationary phase
faster (96 h) than parasites grown in LIT (Fig. 8A). Culture lysates
taken at 72 h (logarithmic phase) were run on SDS-PAGE and
analyzed by Western blot to detect lipoylated proteins. Notably,
parasites grown at low glucose concentration presented increased
E2p lipoylation when compared with control cultures (Fig. 8B, lanes
mLIT and LIT, respectively). These data indicate that at least T. cruzi
E2p expression and/or lipoylation can be modified by growth
conditions. Additionally, protein lipoylation was still significantly
inhibited when cells cultivated in mLIT were treated with 100 mM
MBrO (Fig. 8B, lane mLIT þ MBrO). Mitochondria-enriched fractions
were obtained using digitonin, a detergent that based on choles-
terol content allows progressive membrane permeabilization.
Mitochondria-enriched fractions were analyzed for the presence of
lipoylated proteins (Fig. 8C). Similar to our previous observations,
lipoylated E2p was increased in T. cruzi grown in mLIT and a
Fig. 5. Growth curves of T. cruzi epimastigotes cultured with increased concentrations
of methyl-8-bromo-octanoic acid (MBrO). The CL Brener (A) and Dm28c (B) strains
were tested.

22
P. Vacchina et al. / Experimental Parasitology 186 (2018) 17e23
Fig. 6. Effect of lipoic acid on cultures of T. cruzi grown in the presence of methyl-8-
bromo-octanoic acid (MBrO). Epimastigotes of T. cruzi CL Brener strain were grown
in the absence or presence of 66
lipoic acid (LA, 100 M).
mM MBrO, in LIT medium supplemented or not with
m
Fig. 8. Effects of low glucose availability on growth (A) and protein lipoylation (B, C) of
T. cruzi epimastigotes. LIT, normal LIT medium (4 g/l glucose); mLIT, modified LIT
medium (0.4 g/l glucose). (A) Growth curves of T. cruzi epimastigotes in LIT and mLIT
medium. (B) Western blot of cells grown in mLIT in the absence or presence of MBrO,
compared to cells grown in LIT. aTub: a-tubulin, as loading control. (C) Mitochondrial
proteins enrichment after digitonine titration; the last two fractions for each treatment
are shown.
Fig. 7. Western blot analysis of lipoylated proteins in T. cruzi epimastigotes (Dm28c
strain) grown in LIT medium with or without LA analogues. BrO, 8-bromo-octanoic
acid; MBrO, methyl-8-bromo-octanoic acid; E2p, E2b and E2k indicate the lipoylated
E2 subunits of PDH, BCDH and KGDH complexes, respectively (their theoretical mo-
lecular masses are indicated).
dehydrogenase activities (Table 1). At low glucose concentration,
PDH activity was 5 times higher than the one measured in regular
LIT medium. These data nicely correlate with the lipoylation
pattern detected under the same growth conditions (Fig. 8). In
accordance with the Western blot results, the KGDH specific ac-
tivity was unaltered by the medium composition while BCDH
exhibited very low values under all tested conditions, indicating
that both their activities and lipoylation patterns are independent
of the glucose concentration. MBrO abolished completely the three
enzyme activities, no matter the medium composition. Malate
dehydrogenase specific activity was not significantly altered by any
of the treatments described above (data not shown), indicating that
only lipoylated enzymes were affected.
substantial decrease in protein lipoylation was seen after supple-
mentation with sublethal MBrO concentration.
The effect of BrO on T. cruzi grown in low glucose media was also
tested. This was achieved by challenging cultures with increasing
concentrations of the compound (Fig. 9). Two types of parasite
populations were tested: the first one derived from epimastigotes
grown in LIT medium, which were washed and cultivated in mLIT
(
LIT/mLIT), and a second one obtained by successive passages of cell
populations grown in mLIT (71 passages, during ca. 9 months). In
both cases similar EC50 were obtained: 1.26 ± 0.04 and
.33 ± 0.11 mM, respectively. This represents an increase of 1.5e1.6
s
1
fold compared to the EC50s shown in section 3.1, suggesting an
increased amount of the compound's target once cells were
adapted to growth in low glucose medium.
3.7. Conclusions
Protein lipoylation in trypanosomes has been poorly studied to
date, although the great number of metabolic pathways involved
and/or associated to LA metabolism underscores its relevance in
cell biology and highlights it as a promising chemotherapeutic
target. Results compiled in this work support the hypothesis that LA
metabolism is essential for T. cruzi survival and validates the
3
.6. Oxoacid dehydrogenase enzyme activities were completely
inhibited by MBrO
T. cruzi parasites grown in LIT, mLIT and mLIT plus the inhibitor
MBrO were assayed for the three LA-dependent oxoacid

P. Vacchina et al. / Experimental Parasitology 186 (2018) 17e23
23
suggestions on the manuscript and Vanesa Frea for technical
assistance. ADU and PV are members of the Carrera del Investigador
Científico, CONICET, Argentina. DAL had a doctoral fellowship from
CONICET, Argentina. This work was supported by FONCyT, ANPCyT,
ꢁ
Argentina, through grant PICT 2012 N 1301 and by CONICET,
Argentina, through grant P-UE 2016-IBR.
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Table 1
Enzymatic activities of mitochondrial dehydrogenase complexes of T. cruzi
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Substrate
Growth condition
LIT medium
mLIT medium
mLIT medium, MBrO
pyruvate
0.61 ± 0.11a
0.58 ± 0.08
0.15 ± 0.05
3.01 ± 0.23
0.60 ± 0.10
0.21 ± 0.06
ND b
ND
ND
2
2
-keto-glutarate
-keto-isovalerate
a
mU, nmoles/min/mg of protein.
Not detected, less than 0.1 mU.
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We wish to thank Paul A. M. Michels for comments and