Experimental Parasitology 147 (2014) 60–66
Contents lists available at ScienceDirect
Experimental Parasitology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r
Research Brief
Characterization of the pattern of ribosomal protein L19 production
during the lifecycle of Leishmania spp.
Janayna Hammes de Almeida-Bizzo a,1, Lysangela Ronalte Alves b,1, Felipe F. Castro a,
Juliana Bório Ferreira Garcia a, Samuel Goldenberg b, Angela Kaysel Cruz a,*
a Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade Medicina de Ribeirão Preto, Universidade de São Paulo, Av.
Bandeirantes, no. 3900, CEP 14049-900 Ribeirão Preto, São Paulo, Brazil
b
Laboratório de Regulação da Expressão Gênica, Instituto Carlos Chagas, Fiocruz-Paraná, Curitiba, Paraná, Brazil
H I G H L I G H T S
•
•
•
•
•
L19 levels are higher in log than in
stationary phase promastigotes in
all species tested.
L. major L19 protein is
undetectable in amastigotes
despite high transcript levels.
L19-overexpressor promastigotes
show impaired growth in axenic
culture.
L19-overexpressor accrues
polysome peaks contrasting with
low translation activity.
L19 overexpression may induce
translation arrest by blocking
elongation/termination.
G R A P H I C A L
A B S T R A C T
Ribosomal protein L19 in Leishmania spp.
L19 expression
pattern in
Leishmania spp
L19 transcript has
no correlation with
product levels
L19 overexpression
in L. major
L19 protein levels:
high in procyclics
low in stationary phase
Impaired growth in
axenic culture
Higher polysome peaks
in log-phase promastigotes
L19 mutant
Lower translation rates in L19
overexpressor
L. major – wild
type
* Corresponding author. Fax: 55-16-36020728.
E-mail address: [email protected] (A.K. Cruz).
1 Authors contributed equally to this work.
http://dx.doi.org/10.1016/j.exppara.2014.08.015
0014-4894/© 2014 Elsevier Inc. All rights reserved.
J.H. Almeida-Bizzo et al./Experimental Parasitology 147 (2014) 60–66
A R T I C L E
I N F O
Article history:
Received 19 October 2013
Received in revised form 16 August 2014
Accepted 26 August 2014
Available online 5 October 2014
Keywords:
Leishmania
Gene expression
Ribosomal protein
L19
61
A B S T R A C T
Leishmania is a genus of protozoan parasites causing a wide clinical spectrum of diseases in humans. Analysis of a region of chromosome 6 from Leishmania major (Iribar et al.) showed that the transcript of a
putative L19 ribosomal protein (RPL19) was most abundant at the amastigote stage. We therefore decided
to characterize L19 protein abundance throughout the lifecycle of Leishmania. Differential expression of
the L19 gene during development has been observed for all Leishmania species studied to date (L. major,
L. braziliensis, L. donovani, and L. amazonensis). Immunoblotting with polyclonal antibodies against L. major
RPL19 revealed that changes to L19 protein abundance follow a similar pattern in various species. The
amount of L19 protein was higher in exponentially growing promastigotes than in stationary phase
promastigotes. The L19 protein was barely detectable in amastigotes, despite the abundance of L19 transcripts observed in L. major at this stage. Immunofluorescence assays showed a granular, dispersed
distribution of RPL19 throughout the cytoplasm. Subcellular fractionation confirmed the presence of the
protein in the ribosomal fraction, but not in the cytosol of L. major. We generated a L. major transfectant
bearing a plasmid-borne L19 gene. Overproduction of the L19 transcript and protein resulted in impaired growth of the transfectants in association with high polysome peaks. We also showed by metabolic
labeling that L19 overexpressing clones display low rates of translation. These data suggest that L19
overexpression affects negatively translation elongation or termination. The lack of correlation between
L19 transcript and protein abundances suggest that the translation of L19 is differentially controlled during
development in the various species investigated.
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
Protozoan parasites of the genus Leishmania cause a spectrum
of human diseases ranging from self-healing cutaneous ulcers to
potentially fatal visceral infection. Clinical presentation depends primarily on the species of parasite involved (Desjeux, 2004). Leishmania
has a digenetic lifecycle alternating between two major developmental stages: extracellular, flagellated promastigotes in the
alimentary tract of the sandfly vector and intracellular amastigotes
residing in the macrophage phagolysosomes of the mammalian host.
The parasite undergoes multiple morphological and biochemical
changes during its lifecycle, ensuring its survival and proliferation
within the hostile environments of the insect gut and macrophage phagolysosomes.
Leishmania genes are organized into long, polycistronic units
called directional gene clusters, which are constitutively transcribed during both stages of the parasite’s lifecycle (Leifso et al.,
2007). Gene expression is regulated almost exclusively by posttranscriptional mechanisms (Clayton and Shapira, 2007).
Investigation of genes that are differentially expressed during the
parasite’s lifecycle has been conducted by directed strategies examining individual genes or, in more recent years, by global
proteomic approaches (Lynn et al., 2013; Pereira et al., 2013; Wu
et al., 2000, tritrypDB). In a previous study aimed to characterize
genes of unknown function in Leishmania major, we observed that
several transcripts were more abundant in promastigotes than in
amastigotes (Iribar et al., 2003). In this genomic region there were
two transcripts present in higher amounts in amastigotes; they
encode the putative L19 ribosomal protein (L19).
Ribosomal proteins are conserved throughout evolution, due to
the importance of their biological function (Requena et al., 2000).
Ribosomal protein L19 is a component of the large ribosomal subunit
and is involved in the formation of one of the intersubunit bridges
(Yusupov et al., 2001). In addition to their canonical function in translation, several ribosomal proteins have diverse extra-ribosomal
functions including roles in apoptosis, DNA repair, and transcriptspecific translational control (Lindström, 2009; Naora, 1999; Warner
and McIntosh, 2009; Wool, 1996). In addition, the ribosomal protein
S3a from L. major has an additional role in the regulation of T- and
B-cell reactivity (Cordeiro-Da-Silva et al., 2001). Nonetheless, little
is known about ribosomal proteins in trypanosomatids. The genome
of L. major contains two copies of the L19 gene, positioned sideby-side on chromosome 6 (LmjF06.0410 and LmjF06.0415). The
predicted isoforms are basic (pI 11.95) and have molecular masses
of 28.8 and 30.5 kDa, with 97% sequence identity. We describe herein
the characterization of L19 expression throughout the lifecycle of
Leishmania spp. and the effects of its overexpression in L. major.
2. Materials and methods
2.1. Parasites and promastigote culture
Promastigote forms of L. major (RHO/SU/59/P/LV39), Leishmania (Viannia) braziliensis (MHOM/BR/75/M2904), L. amazonensis
(MHOM/BR/1973/M2269) and L. donovani (MHOM/ET/67/HU3) were
grown at 26 °C in M199 medium supplemented as previously described (Kapler et al., 1990). We generated a recombinant plasmid
for the ectopic expression of RPL19 (pNeo-L19) in Leishmania, which
we used to transfect L. major (LV39) and L. braziliensis (Lb2904). L.
major L19-overproducing clones were cultured with G418 (Sigma)
at a concentration 20 times the IC50, for the selection of cells containing the plasmid. Cured (plasmid free) clones were obtained after
30 passages in drug-free medium. Loss of the plasmid was confirmed by Southern blotting with a probe for the L19 gene.
2.2. Cloning and expression vectors
The plasmid ET28a (Novagen, Darmstadt, Germany) was used for
the heterologous expression of L19 tagged with a histidine tail at
the N-terminus. For the induction of L19 expression in E. coli, BL21
(DE3) pLyS were used.
The Leishmania expression vector, pNEO-L19, is similar to the pX
series (Kapler et al., 1990). The plasmid contains a pUC moiety to
work as a shuttle vector, a neomycin phosphotransferase gene (which
confers G418 resistance) for selection, and a copy of LmjF.06.0415
which codes for L19. Both coding sequences are flanked by Leishmania 5’ and 3’UTRs to allow proper transcript processing (a
schematic representation of pNEO-L19 is presented, Supplementary Fig. S1).
2.3. Amastigote isolation
Amastigotes were isolated from infected female BALB/c mice (6–8
weeks old). Infection was conducted with late stationary phase
promastigotes (1 × 105 cells/mL) by subcutaneous injection in the
hind footpad. Amastigotes were isolated from the lesion and purified by rounds of fractionation and washing procedures, modified
from the original protocol (Hart et al., 1981).
62
J.H. Almeida-Bizzo et al./Experimental Parasitology 147 (2014) 60–66
2.4. RNA sample preparation, labeling and hybridization
Cells of various Leishmania spp. were isolated on the 3rd and 7th
days of culture and total RNA was prepared with Trizol (Invitrogen),
according to the manufacturer’s instructions. Samples were fractionated by electrophoresis in denaturing 1.8% agarose gels, and the
bands obtained were blotted onto nylon membranes and hybridized with the probe according to standard methods (Sambrook et al.,
1989). To generate probes for northern blotting, LmjF06.0415 (L19)
and the α-tubulin gene (control) were used as templates for a
random priming labeling reaction (Feinberg and Vogelstein, 1984)
with [α-32P] dCTP (GE Healthcare, Piscataway, PA, USA). The intensity of the L19 hybridization signal was quantified with Image J
(http://rsb.info.nih.gov/ij/), using the ethidium bromide-ribosomal
band signal as the RNA loading control.
2.5. Antibody production and western blotting
Polyclonal antibodies directed against L19 were generated with
a His-tagged protein.
Female rabbits (5–7 weeks old) were immunized with refolded His-L19 in Freund’s complete adjuvant (Sigma Immuno
Chemicals) and received two booster injections, on days 30 and 50,
of protein homogenized in Freund’s incomplete adjuvant (Sigma
Immuno Chemicals). Specific antibodies against L19 were affinitypurified from antiserum with His-L19 proteins immobilized on a
nitrocellulose membrane. Anti-GAPDH antibody was kindly provided by Professor Paul Michels (University of Louvain, Belgium).
For western blots, 1 × 107 cells were centrifuged (10 min, 2000×
g, 4 °C), washed and resuspended in protein electrophoresis buffer
(313 mM Tris, pH 6.8, 10% SDS, 10% glycerol, 0.6 mM beta
mercaptoethanol, 0.1% bromophenol blue). Samples were heated at
95 °C for 5 min and run on 12% acrylamide SDS-PAGE gels (Laemmli,
1970). The protein bands were transferred onto nitrocellulose membranes, which were then blocked by incubation with 5% skimmed
milk in TBS–Tween-20 (10 mM Tris-HCl, 100 mM NaCl, 0.05% Tween
20) for 2 h, washed, and probed with anti-L19 (1:60) or antiGAPDH (1:1000) primary antibody for 1 h. The membrane was
washed five times and incubated with a horseradish peroxidaseconjugated anti-rabbit secondary antibody for 1 h, after which it was
washed again, as before. Bound secondary antibodies were detected with the ECL system (GEHealthCare). We used Image J to
normalize the L19 signal with respect to that for GAPDH.
2.6. Immunofluorescence assay
Briefly, parasites were fixed by incubation in 2% paraformaldehyde in PBS (137 mM NaCl; 8 mM Na2HPO4; 2.7 mM KCl; 1.5 mM
KH2PO4; pH 7.0) for 20 min and permeabilized by incubation with
0.1% Triton X-100 in PBS for 30 min, at room temperature. Coverslips were incubated for 1 h with a primary antibody against L19
(1:20) in PBS supplemented with 1% BSA and then with CYTM3conjugated AffiniPure goat anti-rabbit secondary antibodies (Jackson
Immunoresearch Laboratories). The coverslips were washed in PBS
between steps. DNA was then stained with 0.6 μM DAPI (Sigma).
Coverslips were mounted and sealed on microscope slides for confocal microscopy observation (Leica TCS SP5).
a cocktail of protease inhibitors (Complete-Roche). Cells were lysed
by five freeze–thaw cycles in liquid N2 and a water bath at 30 °C,
and whole-cell extracts were centrifuged at 10,000× g for 15 min,
to remove mitochondria and debris. The supernatant was layered
over a sucrose (20% w/v) cushion and centrifuged at 149,000 × g for
2 h. This step was repeated with the nonribosomal supernatant, to
ensure that there was no ribosomal contamination. As a control of
fractionation quality, we extracted RNA from both fractions and subjected it to electrophoresis in a denaturing gel.
2.8. Polysome isolation
L. major polysomes were isolated on sucrose gradients. Cells
(7.5 × 108) were incubated with 200 μg mL−1 cycloheximide, for
10 min, or with 2 mM puromycin before polysome preparation. Cells
were kept on ice for 5 min and then pelleted by centrifugation and
washed with cold TKM buffer (10 mM Tris, pH 7.4, 300 mM KCl and
10 mM MgCl2) supplemented with 200 μg mL−1 cycloheximide or
with 2 mM puromycin. Cell pellets were then resuspended in 900 μL
TKM supplemented with 200 μg mL−1 cycloheximide or 2 mM puromycin, 10 μg mL−1 heparin, 10 μM E-64, and 1:100 EDTA-free
protease inhibitor cocktail (Roche). The suspension was transferred to a new tube containing 100 μL of lysis buffer (TKM
supplemented with 10% (v/v) NP-40 and 2 M sucrose), and was homogenized by repeated passages through a pipette. Lysis was
monitored by phase-contrast microscopy. The lysate was centrifuged at 18,000 × g, 4 °C, for 5 min. The cleared supernatant (500 μL;
equivalent to 5 × 108 cells) was layered onto linear 15–55% sucrose
density gradients prepared in TKM buffer supplemented with inhibitors (10 μM E-64, 1 mM PMSF, and 1 mg mL−1 heparin) and was
centrifuged at 4 °C for 2 h, at 365,000 × g in a Beckman SW41 rotor.
After centrifugation, 500 μL fractions were collected with the ISCO
gradient fractionation system.
2.9. Metabolic labeling
L19-overexpressing and cured clones (5 × 107 parasites) were collected by centrifugation and resuspended in 1 mL of M199 medium
without fetal bovine serum. For cycloheximide treatment, cells were
first incubated with 200 μg mL−1 of cycloheximide for 20 min at 25 °C.
We then added 100 μCi mL−1 [35S]-methionine (Redivue Pro-mix
L-[35S], >1000 Ci mmol−1; Amersham, Aylesbury, UK) and incubated the cells for 2 h at 25 °C, collecting a 10 μL: aliquot every
30 min during this period and spotting onto 3 mm paper slots. The
proteins were precipitated by incubation with 10% TCA for 10 min
at room temperature followed by 5% TCA heated at 100 °C for 10 min,
and air-dried. We then assessed 35S incorporation with a liquid scintillation counter.
2.10. Quantification by ImageJ
A straight line was selected from ImageJ and used to determine the number of pixels per band with the tool “plot profile”
(http://rsb.info.nih.gov/ij/). The number of pixels obtained per band
was subtracted from background pixels. Normalization of the values
was conducted using the corresponding RNA or protein control
signals (ethidium bromide rRNA upper band and GAPDH, respectively), which were obtained using the same plot profile tool.
2.7. Ribosome fractionation
3. Results and discussion
Ribosome fractionation was conducted in triplicate, essentially
as previously described (Mazumder et al., 2003). Briefly, 5 × 108 cells
in the late exponential growth phase were harvested, washed twice
in PBS and resuspended in 500 μL of fractionation buffer (20 mM
Tris pH 7.4, 10 mM MgCl2, 300 mM KCl, 10 mM dithiothreitol, 100
units/mL RNasin, and 100 μg/mL cycloheximide) supplemented with
We previously mapped the transcripts from a 40 kb region of L.
major chromosome 6; two of the mapped transcripts were more
abundant in amastigotes than in promastigotes (Iribar et al., 2003).
L. major genome sequencing and annotation identified genes
LmjF.06.0415 and LmjF.06.0410, both coding for the L19
J.H. Almeida-Bizzo et al./Experimental Parasitology 147 (2014) 60–66
a
kb
3
7
3
Days
7
1.5
3
ama
1.5
3
kb
2
2
Lb
Ldo
La
Lmj
Lmj Lmj+L19
7
3
7
3
7
3
2
2
1.5
1,0 1,09
1,0 0,94 1,19 1,19
Days
:kDa
Lmj
3
7
RNA
loading
2
1.5
1.5
b
Days
L19
1.5
3
63
Lmj
1,0
kDa
35
3
7 10
kDa
35
3
7 10
1,0 0,72
Ldo
Lbr
Lmj+L19
10
1,7 1,0 1,04
kDa
35
3
7
La
10
kDa
3
7
10
29
anti- L19
29
25
25
25
45
45
45
35
35
35
45
45
anti-GAPDH
1.1
0.1
0.1
0.02
1.2
0.6
0.7
0.5 0.3 0.2
1.9
0.8
1.2
3.1
1.3
0.6
Fig. 1. Expression profile of the L19 gene throughout the lifecycle of Leishmania spp. (A) Northern blot analysis of L19 transcript abundance at the start of the exponential
growth phase (day 3) and during the stationary phase (day 7). The membrane was hybridized with an L19 probe, as indicated. Ethidium bromide-stained gels are presented in the bottom panels, as control for RNA sample loading and integrity and for normalization of L19 transcript levels. (B) An immunoblotting analysis of L19 protein
abundance in various growth phases is presented, with the number of days in culture indicated (3, 7, or 10). Membranes were probed with anti-L19 and anti-GAPDH antibodies (as indicated on the right). Numbers depicted below the bottom panels (in A and B) correspond to the mean value of three independent experiments and represent
the intensity of the L19 signal relative to that of the corresponding controls; RNA (alpha-tubulin) and protein (GAPDH). Procedure for quantification using Image J program
is described in Section 2.10. Lmj, L. major; Lbr, L. braziliensis; Ldo, L. donovani; La, L. amazonensis; Lmj+L19, L. major L19-overexpressor. RNA marker sizes are indicated in kb.
Protein marker sizes are indicated in kDa.
ribosomal protein, as the origins of those transcripts (http://
tritrypdb.org). We therefore characterized the expression profile of
this ribosomal protein in L. major, L. braziliensis, L. donovani and L.
amazonensis.
We evaluated the profile of L19 expression in various species of
Leishmania, by performing northern blots with total RNA extracted from early exponential or late stationary phase promastigotes
of L. major, L. braziliensis, L. donovani, or L. amazonensis. We observed similar levels of L19 transcripts in exponential and stationary
phase promastigotes in all species tested and previous results that
indicated higher levels of the L19 transcripts in amastigotes of L.
major were not confirmed (Fig. 1A). In L. braziliensis the two bands
observed in the northern blots correspond to the different sizes of
L19 transcripts, probably due to the fact that three copies of the gene
seem to be present in the genome, and one coding sequence is predicted to be shorter than the other two (Peacock et al., 2007).
We evaluated the production dynamics and subcellular distribution of the L19 protein by generating polyclonal antibodies against
L. major L19 generated by heterologous expression from a pET vector
in E. coli. The recombinant L19 protein, with six histidine residues
(pET-LmL19His) fused to its N-terminus, was produced in E. coli, purified, and injected into rabbits to raise polyclonal antibodies against
L19. We used the resulting antibody for immunoblotting experiments. Despite the steady L19 transcript levels observed in
promastigote and amastigote phases in all Leishmania species, L19
protein abundance was always high in the early phases of axenic
promastigote culture, and decayed markedly toward the stationary phase (Fig. 1B). The same lack of correlation between the
abundance of mRNA and protein was observed in amastigote forms
of L. major: despite the abundant L19 transcript in the intracellular form of the parasite, we were unable to detect L19 protein on
immunoblots (Fig. 2B). Accordingly, a lack of correspondence
between the abundance of protein and mRNA has been reported
before in Leishmania (McNicoll et al., 2006). The biological
significance of the paradoxical divergence between the amount of
transcript and protein is unclear; however it is possible that the
Fig. 2. Subcellular localization of L19. (A) Confocal microscopy analysis of L. major
promastigotes with anti-L19 antibody (red). DNA (nuclei and kinetoplast) is stained
with DAPI (blue). Bars, 10 μm. (B) Immunoblotting analysis (antibodies indicated
on the right) of wild-type (Lmj) and transfectant (Lmj+L19) L. major. Ribosome fractionation. In the bottom panel, the ethidium bromide-stained agarose gel is shown,
to control for RNA loading and integrity. Cyt, ribosome-free cytosol fraction; Rib, ribosomal fraction. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
64
J.H. Almeida-Bizzo et al./Experimental Parasitology 147 (2014) 60–66
L19 transcript is stored in RNA granules and is therefore not
translated.
An analysis of the T. cruzi metacyclic proteome similarly revealed that the abundance of ribosomal proteins (Atwood et al., 2005)
was low in metacyclic trypomastigotes, consistent with their nonreplicating status. A decrease in protein production capacity would
be expected during the stationary phase, but the developmental regulation of ribosomal protein production has not been previously
investigated in Leishmania.
We used immunofluorescence assays to evaluate the subcellular
distribution of L19 (Fig. 2A). RPL19 was dispersed throughout the cytoplasm and was almost entirely absent from the nucleoplasm; a similar
distribution has been described for ribosomal proteins in other organisms (Krüger et al., 2007). The L19 signal intensity in axenic culture
was also higher during the exponential growth phase than during
the stationary phase, consistent with western blot results. In addition, we investigated the distribution of L19 by subcellular fractionation,
with the recuperation of ribosomal and cytosolic fractions (Fig. 2B).
L19 was present exclusively in the ribosomal fraction (Fig. 2B). The
absence of three ribosomal RNA bands from the cytosol fraction indicates that the experimental procedure efficiently removed all intact
ribosome subunits (Fig 2B).
We generated L19-overexpressing L. major clones to investigate
the effect of L19 overexpression on phenotype. The high abundance
of L19 mRNA and protein in these clones was confirmed by northern
and western blotting respectively (Fig. 1). Both endogenous and ectopically expressed L19 isoforms were associated with the ribosomal
fraction (Fig. 2B). The consequences of L19 overexpression included
impairment to growth (Fig. 3A), which was also observed in a L.
braziliensis clone overexpressing L19 (Fig. 3B). We investigated whether
this growth defect was due to the presence of excess L19 by curing
the L. major overexpressor of the episome by culture serial passages
in the absence of drug selection. Growth features of the parental strain
were partially rescued in the cured clone (Fig. 3) and levels of L19
protein returned to WT levels in the cured L. major clone (Supplementary Fig. S2). This suggests that excess L19 is deleterious to
promastigotes, and leads to growth defects.
We then investigated whether the excess L19 had deleterious
effects on polysome organization. Polysome profile analysis showed
that L19-overexpressors displayed higher peaks than control cells
(Fig. 4A–C), despite the fact that L. major L19-overexpressors displayed impaired growth. An excess of L19 may promote an increase
in mRNA sequestration to polysomes or decreased rates of mRNA
translation elongation or termination. This change in growth profile
was observed only in exponential-phase promastigotes, in which
the rate of transcription and translation are higher than in stationaryphase cells. On the other hand, the polysome profiles of L19overexpressors and control cells were similar in stationary-phase
parasites (Fig. 4D–F). A reversion of the phenotype to normality was
consistently observed in cured transfectants (Fig. 4G and H). The
growth impairment and the accumulation of polysome peaks in L19overexpressor log-phase promastigotes may be associated with low
rates of protein synthesis due to a decrease in ribosome run-off promoted by the presence of L19 excess. We tested this hypothesis by
evaluating the translation rate in the L19-overexpressing and cured
clones by following metabolic labeling with 35S-methionine (Fig. 5).
Translation rate was significantly lower in the L19-overexpressor than
in cured clones (Fig. 5). As a control, the parasites were treated with
cycloheximide to inhibit translation at the elongation step, by trapping mRNAs in polysomes. The translation rate of the L19overexpressor and the cured clones was similar after cycloheximide
treatment, and was comparable with that for the L19-overexpressor
in the absence of treatment (Fig. 5). We can hypothesize that the
L19 protein induces translation arrest by blocking the elongation
and termination of translation. The yeast L19 protein is involved in
the formation of the interribosomal subunit bridge (Ben-Shem et al.,
Fig. 3. Effects of RPL19 overexpression on L. major and L. braziliensis promastigote
growth. A comparative analysis of parasite growth was carried out. (A) transfected
L. major cells – L19 overexpressor (■), cured clones (▲) and wild type. (●). (B) transfected L. braziliensis cells – L19 overexpressor (■) and wild type (●). Parasites were
counted daily in a Neubauer chamber. The transfectants were maintained in media
containing G418 at 20 times the IC50.
2011). This bridge is the binding site for eIF4G, a protein that plays
a key role in the formation of the pre-initiation complex (Lomakin
et al., 2000). Thus, L19 may be involved in the final stages of translation initiation (Ben-Shem et al., 2011).
Other experimental approaches should be pursued to study ribosomal protein transcripts and the ribosomal protein themselves
and the mechanisms of expression control. The divergence of the
3’UTR sequence is high in orthologous copies of L19 transcripts from
different species of Leishmania, as shown by the alignment of these
untranslated regions (Supplementary Fig. S3). It will be of interest
to explore such divergence as well as the conserved elements within
the 3’UTRs to investigate putative regulatory cis-elements that may
be involved in the control of L19 translation rate and protein abundance in promastigote and amastigote stages. This is a relevant issue
in an organism in which gene expression is regulated principally
by posttranscriptional control mechanisms and should be investigated further.
J.H. Almeida-Bizzo et al./Experimental Parasitology 147 (2014) 60–66
65
Fig. 4. Polysome sedimentation profiles for L. major treated with cycloheximide in various growth conditions. Polysome sedimentation profile of the wild-type strain during
exponential growth (A), of the L19-overexpressor during exponential growth (B), and the merged polysome profile of the wild-type strain (in light gray) and the L19overexpressor (in dark gray) (C). Polysome sedimentation profile of the wild-type strain in the stationary phase (D), of the L19-overexpressor in the stationary phase (E),
and the merged polysome profile of the wild-type strain (in light gray) and the L19-overexpressor (in dark grey) (F). Polysome sedimentation profile of the cured transfectant
during exponential growth (G) and the merged polysome profile of the wild-type strain (in dark gray) and the cured transfectant (in light gray) (H).
Fig. 5. Incorporation of [S-35] L-methionine by the L. major L19-overexpressor and the cured clone. Cells were collected at various time points (X axis) and the amount of
radioactivity incorporated was determined in CPM (Y axis). The solid lines correspond to the overexpressor (Lmj+L19), the dotted lines correspond to the cured clone and
cycloheximide treatment is indicated as Cyclo. Two-way ANOVA refers to the comparison of Lmj+L19 and the cured clone, ***P < 0.001.
66
J.H. Almeida-Bizzo et al./Experimental Parasitology 147 (2014) 60–66
Acknowledgments
We thank Paul Michels for providing the GAPDH antibody and
Viviane Ambrosio and Tânia Defina for technical assistance. This work
was supported by FAPESP (06/50323-7) (JAB held a FAPESP fellowship 05/59546-6). SG and AKC hold research fellowships from
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq, Brazil) (AKC - 304025/2009-7 and SG - 301730/2011-3).
Appendix: Supplementary Material
Supplementary data to this article can be found online at
doi:10.1016/j.exppara.2014.08.015.
References
Atwood, J.A., 3rd, Weatherly, D.B., Minning, T.A., Bundy, B., Cavola, C., Opperdoes,
F.R., et al., 2005. The Trypanosoma cruzi proteome. Science 309 (5733), 473–476.
Ben-Shem, A., Garreau de Loubresse, N., Melnikov, S., Jenner, L., Yusupova, G., Yusupov,
M., 2011. The structure of the eukaryotic ribosome at 3.0 Å resolution. Science
334 (6062), 1524–1529.
Clayton, C., Shapira, M., 2007. Post-transcriptional regulation of gene expression in
trypanosomes and leishmanias. Mol. Biochem. Parasitol. 156 (2), 93–101.
Cordeiro-Da-Silva, A., Borges, M.C., Guilvard, E., Ouaissi, A., 2001. Dual role of the
Leishmania major ribosomal protein S3a homologue in regulation of T- and B-cell
activation. Infect. Immun. 69 (11), 6588–6596.
Desjeux, P., 2004. Leishmaniasis: current situation and new perspectives. Comp.
Immunol. Microbiol. Infect. Dis. 27 (5), 305–318.
Feinberg, A.P., Vogelstein, B., 1984. “A technique for radiolabeling DNA restriction
endonuclease fragments to high specific activity”. Addendum. Anal. Biochem.
137 (1), 266–267.
Hart, D.T., Vickerman, K., Coombs, G.H., 1981. A quick, simple method for purifying
Leishmania mexicana amastigotes in large numbers. Parasitology 82 (3), 345–355.
Iribar, M.P., Tosi, L.R., Cruz, A.K., 2003. A processed short transcript of Leishmania,
ODD1. Mol. Biochem. Parasitol. 127 (2), 205–208.
Kapler, G.M., Coburn, C.M., Beverley, S.M., 1990. Stable transfection of the human
parasite Leishmania major delineates a 30-kilobase region sufficient for
extrachromosomal replication and expression. Mol. Cell. Biol. 10 (3), 1084–1094.
Krüger, T., Zentgraf, H., Scheer, U., 2007. Intranucleolar sites of ribosome biogenesis
defined by the localization of early binding ribosomal proteins. J. Cell Biol. 177
(4), 573–578.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 227 (5259), 680–685.
Leifso, K., Cohen-Freue, G., Dogra, N., Murray, A., McMaster, W.R., 2007. Genomic and
proteomic expression analysis of Leishmania promastigote and amastigote life
stages: the Leishmania genome is constitutively expressed. Mol. Biochem.
Parasitol. 152 (1), 35–46.
Lindström, M.S., 2009. Emerging functions of ribosomal proteins in gene-specific
transcription and translation. Biochem. Biophys. Res. Commun. 379 (2), 167–170.
Lomakin, I.B., Hellen, C.U., Pestova, T.V., 2000. Physical association of eukaryotic
initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to
the internal ribosomal entry site of encephalomyocarditis virus and is required
for internal initiation of translation. Mol. Cell. Biol. 20 (16), 6019–6029.
Lynn, M.A., Marr, A.K., McMaster, W.R., 2013. Differential quantitative proteomic
profiling of Leishmania infantum and Leishmania mexicana density gradient
separated membranous fractions. J. Proteomics 82, 179–192.
Mazumder, B., Sampath, P., Seshadri, V., Maitra, R.K., DiCorleto, P.E., Fox, P.L., 2003.
Regulated release of L13a from the 60S ribosomal subunit as a mechanism of
transcript-specific translational control. Cell 115 (2), 187–198.
McNicoll, F., Drummelsmith, J., Müller, M., Madore, E., Boilard, N., Ouellette, M., et al.,
2006. A combined proteomic and transcriptomic approach to the study of stage
differentiation in Leishmania infantum. Proteomics 6 (12), 3567–3581.
Naora, H., 1999. Involvement of ribosomal proteins in regulating cell growth and
apoptosis: translational modulation or recruitment for extraribosomal activity?
Immunol. Cell Biol. 77 (3), 197–205.
Peacock, C.S., Seeger, K., Harris, D., Murphy, L., Ruiz, J.C., Quail, M.A., et al., 2007.
Comparative genomic analysis of three Leishmania species that cause diverse
human disease. Nat. Genet. 39 (7), 839–847.
Pereira, M.M., Malvezzi, A.M., Nascimento, L.M., Lima, T.D., Alves, V.S., Palma, M.L.,
et al., 2013. The eIF4E subunits of two distinct trypanosomatid eIF4F complexes
are subjected to differential post-translational modifications associated to distinct
growth phases in culture. Mol. Biochem. Parasitol. 190 (2), 82–86.
Requena, J.M., Alonso, C., Soto, M., 2000. Evolutionarily conserved proteins as
prominent immunogens during Leishmania infections. Parasitol. Today 16 (6),
246–250.
Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning: a laboratory manual,
second ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Warner, J.R., McIntosh, K.B., 2009. How common are extraribosomal functions of
ribosomal proteins? Mol. Cell 34 (1), 3–11.
Wool, I.G., 1996. Extraribosomal functions of ribosomal proteins. Trends Biochem.
Sci. 21 (5), 164–165.
Wu, Y., El Fakhry, Y., Sereno, D., Tamar, S., Papadopoulou, B., 2000. A new
developmentally regulated gene family in Leishmania amastigotes encoding a
homolog of amastin surface proteins. Mol. Biochem. Parasitol. 110 (2), 345–357.
Yusupov, M.M., Yusupova, G.Z., Baucom, A., Lieberman, K., Earnest, T.N., Cate, J.H.,
et al., 2001. Crystal structure of the ribosome at 5.5 Å resolution. Science 292
(5518), 883–896.