Expression of trans-Sialidase and 85-kDa Glycoprotein
Genes in Trypanosoma cruzi Is Differentially Regulated at
the Post-transcriptional Level by Labile Protein Factors*
Grace
Abuin
§,
Lúcio H. G.
Freitas-Junior
§,
Walter
Colli¶,
Maria Julia M.
Alves¶, and
Sergio
Schenkman
From the
Departamento de Microbiologia, Imunologia e
Parasitologia, Escola Paulista de Medicina, Universidade Federal de
São Paulo, R. Botucatu 862 8o A, 04023-062 São
Paulo, São Paulo, Brasil and the ¶ Departamento de
Bioquímica, Instituto de Química, Universidade de
São Paulo, 05599-970 São Paulo, Brasil
 |
ABSTRACT |
To adapt to different environments,
Trypanosoma cruzi, the protozoan parasite that causes
Chagas' disease, expresses a different set of proteins during
development. To begin to understand the mechanism that controls this
differential gene expression, we have analyzed the levels of
amastin and trans-sialidase mRNAs and the
mRNAs encoding members of the 85-kDa glycoprotein gene family,
which are differentially expressed in the T. cruzi stages found in the mammalian host. Amastin mRNA is expressed
predominantly in intracellular and proliferative amastigotes.
trans-Sialidase mRNAs are found mostly in forms
undergoing transformation from amastigotes to trypomastigotes inside
infected cells, whereas mRNAs encoding the 85-kDa glycoproteins
appear only in the infective trypomastigotes released from the cells.
The genes coding for these mRNA species are constitutively
transcribed in all stages of T. cruzi cells, suggesting
that expression is controlled post-transcriptionally during
differentiation. Inhibition of transcription by actinomycin D revealed
that each mRNA species has a relatively long half-life in stages
where it accumulates. In the case of the trans-sialidase and 85-kDa glycoprotein genes, mRNA accumulation was induced by treatment with the protein synthesis inhibitor cycloheximide at the
stages that preceded the normal accumulation. Therefore, mRNA stabilization may account for mRNA accumulation. mRNA
degradation could be promoted by proteins with high turnover, or
stabilization could be promoted by forming a complex with the
translational machinery at defined times in development. Identification
of the factors that induce mRNA degradation or stabilization is
essential to the understanding of control of gene expression in these organisms.
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INTRODUCTION |
Gene expression in Trypanosoma cruzi as well as in
other trypanosomes is largely controlled at the post-transcriptional
level (1-6), although there are a few exceptions where promoters and transcriptional activation have been described (7). Well defined RNA
polymerase II initiation sites have not been found, and most genes are
transcribed as part of polycistronic units and are processed by
trans-splicing (8, 9). During processing, capped spliced leader sequence is added to the 5'-end, and a polyadenosine tail is
added to the 3'-end of mRNA (10), probably ensuring mRNA stability and transport to the cytoplasm (11). In particular, correct
splicing and polyadenylation, as well as the existence of specific
3'-untranslated portions of the transcribed genes, have been shown to
promote either mRNA stability (12) or an increase in the
translational efficiency (13). However, the nature of controlling
factors and how environmental modifications induce differential
expression remain obscure.
T. cruzi provides an attractive model to study how
modifications in the environment induce differential gene expression.
In the insect host, the epimastigote form of the parasite proliferates in the gut. In the posterior gut, where the nutrients become scarce, the parasite transforms into metacyclic trypomastigote forms, which are
nonproliferative and highly infective to the mammalian host. Within the
mammalian host, the parasite proliferates only inside the cell
cytoplasm, as amastigote forms. Once the infected cell is full of
parasites, and probably deprived of nutrients, the amastigote form
transforms into nonproliferative and infective trypomastigote forms,
which rupture the cell, escaping to the bloodstream. The
differentiation between noninfective and infective forms is related to
the shutdown of the proliferative machinery and the expression of a set
of genes involved in cell invasion and induction of resistance to host
defenses (14, 15). These changes are correlated to the activation of
adenylate cyclase (16-21) via transducing pathways involving
G-proteins (22, 23), phospholipase C (20), and calcium internalization
(24). The molecules expressed during this phase of differentiation are
members of a large family of 85-kDa glycoproteins (14) that are
localized on the surface of infective forms and are thought to
participate in adhesion to the host cell surface during invasion
(25-27). The large family of 85-kDa glycoproteins, collectively
baptized as gp85 (14), encompasses a subfamily of several members,
earlier defined as Tc85, that are capable of binding to wheat germ
agglutinin (28) and are recognized by a specific monoclonal antibody
(29). The most acidic component of the Tc85 subfamily was characterized as a laminin ligand (30), which may be directly involved in the
interaction of trypomastigotes with the mammalian cell (25, 29).
Another component expressed in large amounts in differentiated trypomastigotes is the enzyme trans-sialidase
(TS).1 This enzyme transfers
host sialic acid to abundant surface molecules characterized as
mucin-like glycoproteins (31-34), which form a protective coat on the
trypomastigote surface.2
Several pieces of evidence suggest that TS is involved in cell invasion
(35) and escape from the phagosome (36) and is an important virulence
factor mediating the immune response to the parasite in the mammalian
host (37, 38). TS is encoded by a family of ~80 genes that have a
common amino-terminal domain, which includes the catalytic portion of
the enzyme, and a variable number of 12-amino acid repeats in the
carboxyl-terminal region (39).
To investigate how expression of the Tc85 and TS
genes is regulated during T. cruzi differentiation, the
steady-state levels of mRNA coding for these proteins have been
analyzed. This study revealed that the corresponding mRNAs
accumulate at different time periods during the differentiation of
intracellular amastigotes to extracellular trypomastigotes. The time of
accumulation depends on the number of infected parasites per cell,
suggesting that interaction with the host cell environment is involved
in the control of TS and Tc85 gene expression. To
initiate a systematic characterization of the controlling mechanism,
the rate of transcription in lysolecithin-permeabilized parasites and
the mRNA stability in the presence of transcription and translation
inhibitors have been studied.
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EXPERIMENTAL PROCEDURES |
Parasites--
The Y strain (40) of T. cruzi was used
in this study. Epimastigote forms were maintained in liver infusion
tryptose medium containing 10% fetal bovine serum at 28 °C (41).
Intracellular amastigotes, intracellular intermediate forms, and
trypomastigotes were obtained from infected L6E6 cells (American Type
Culture Collection, Rockville, MD) grown in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum at 37 °C and 5%
CO2 as described previously (42). Trypomastigotes were
collected from the extracellular medium 5-7 days after infection and
recovered after centrifugation at 2000 × g for 10 min.
The intracellular forms were obtained from the infected cells by
scraping the monolayer with a Teflon policeman in a buffer containing
27 mM K2HPO4, 8 mM
Na2HPO4, and 26 mM
KH2PO4, pH 7.2. The cells were broken in a
Potter-Elvehjem homogenizer, and free parasites were separated from
cell debris by centrifugation at 800 × g for 5 min.
The parasites were then collected by centrifugation at 2000 × g. Preliminary results showed that the intracellular forms
could be maintained in Grace's medium (Life Sciences, Inc.) containing
2% fetal bovine serum for at least 24 h without loss of viability
as checked by the maintenance of morphology and motility and by
incorporation of calcein/AM (Molecular Probes, Inc.). For this reason,
the experiments with intracellular parasites were done in Grace's
medium, whereas trypomastigotes were incubated in Dulbecco's modified
Eagle's medium containing 2% fetal bovine serum.
In some experiments, the parasites were maintained in the presence of
actinomycin D and cycloheximide. In these cases, intracellular parasites and trypomastigotes were resuspended at 5 × 107/ml of Grace's medium or at 1 × 108/ml of Dulbecco's modified Eagle's medium and
incubated with 100 µg/ml actinomycin D or 250 ng/ml cycloheximide at
37 °C. This concentration of cycloheximide inhibited protein
synthesis by >97% in T. cruzi (43). At the indicated time
points, 1.0 ml of the parasite suspension was removed; the parasites
were collected by centrifugation; and total RNA was extracted.
RNA Extraction--
The parasites were centrifuged and washed
with 0.1 M NaCl, 0.1 M Tris-HCl, pH 7.4, 2 mM MgCl2, and the final pellet was resuspended in acid phenol/guanidine isothiocyanate (44). Usually 3 × 108 parasites were lysed in 1 ml of solution. After
complete dissolution, 0.1 ml of chloroform was added, and the aqueous
phase was recovered by centrifugation (14,000 × g, 15 min). The total RNA was precipitated from the aqueous phase by adding
an equal volume of isopropyl alcohol and then washed with 70% ethanol
and resuspended in RNase-free water.
RNA Analysis and Hybridization--
5-10 µg of total RNA was
denatured in the presence of 50% formamide and 2.2 M
formaldehyde and fractionated by electrophoresis on a 1.0% agarose gel
containing formaldehyde (45). The RNA was stained with ethidium
bromide, blotted onto nylon membrane (Hybond-N, Amersham Pharmacia
Biotech) in 20× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate), and immobilized on the filter by UV
irradiation. The membranes were prehybridized in 50% formamide, 5×
SSC, 5× Denhardt's solution, 0.5% SDS, 5 mM EDTA, and
0.1 mg/ml tRNA at 42 °C for at least 1 h and then hybridized
overnight at the same temperature with labeled probes. The membranes
were washed with 2× SSC containing 0.1% SDS at 65 °C (two times,
30 min each) and exposed to x-ray film. Quantitative measurements were
obtained with a phosphor imaging apparatus (Bio-Rad). The following
probes were used: 1) a probe corresponding to the carboxyl-terminal
repeats of TS (this probe was obtained by EcoRI digestion of
a plasmid derived from pBluescript SK
containing a
PvuII fragment encoding the TS repeats derived from plasmid
154-0 (46)); 2) a probe corresponding to the catalytic domain of TS,
which consisted of an NcoI-PvuII fragment derived from plasmid pTScat7 (47); 3) a probe corresponding to the Tc85 glycoprotein subfamily (this was an EcoRI 390-base pair
fragment derived from plasmid pTc85 (28) (GB-U11500)); 4) a probe for
- and
-tubulin, which was a 2.3-kilobase BamHI
fragment derived from plasmid pDC1 (provided by Yara Traub-Cseko,
Instituto Oswaldo Cruz, Rio de Janeiro, Brazil) (48); and 5) a probe
for the amastin gene, which was a 1.8-kilobase
XbaI-XhoI fragment derived from plasmid TcA33
(provided by Dr. Santuza Teixeira) (49). The DNA fragments were
purified by elution after agarose gel electrophoresis and radiolabeled
by random hexanucleotide priming with DNA polymerase I (Klenow
fragment) and [
-32P]dCTP (3000 Ci/mmol) for 3 h
at room temperature. At the end of this period, the labeled probes were
purified from free nucleotides by Sephadex G-50 spin columns.
Transcription in Permeable Cells--
T. cruzi
parasites in different stages were collected and washed as described
(50). Briefly, parasites (2 × 108) were washed twice
at room temperature with 20 mM potassium glutamate, 3 mM MgCl2, 150 mM sucrose, 10 µg/ml leupeptin, and 1 mM dithiothreitol (transcription
buffer) and resuspended in 800 µl of the same buffer. The parasite
suspension was chilled on ice for 5 min, and
palmitoyl-L-
-lysophosphatidylcholine (Sigma) dissolved
in water at 10 mg/ml was added to obtain a final concentration of 500 µg/ml. After 1 min, the parasites were centrifuged; washed once with
transcription buffer; and resuspended in 200 µl of transcription
buffer containing 2 mM dATP, 1 mM dCTP, 1 mM dGTP, 06 mg/ml creatine kinase (Roche Molecular
Biochemicals), 25 mM creatine phosphate, and 100 µCi of
[
-32P]UTP (3000 Ci/mmol). After 30 min at 30 °C,
the parasites were centrifuged again; the pellets were lysed with 1 ml
of acid phenol/guanidine isothiocyanate; and the labeled RNA was
recovered as described above. The labeled RNA was then hybridized in
5× saline/sodium phosphate/EDTA, 5× Denhardt's solution, 0.1 mg/ml
yeast tRNA, and 0.1% SDS to dot blots containing 5-µg spots of the
indicated DNA. For preparation of the dot blots, plasmid DNA was
denatured in 0.3 N NaOH for 30 min at 55 °C, neutralized
by adding ammonium acetate to 2 N, and loaded onto
nitrocellulose membranes using a minifold filtration apparatus. The
following plasmids were linearized and adsorbed to the filters: 1)
pTc18S (18 S ribosomal gene) (51), 2) pTScat7 (TS catalytic domain), 3)
pTCTR (TS repeats), 4) TcA33 (amastin), 5) pDC1 (tubulin), 6) pTc85
(Tc85), 7) pBluescript SK
containing the region between
the open reading frame of two TS genes (TS intergenic; this was
generated by polymerase chain reaction amplification of a cosmid
containing TS genes (COS-7) (39) using 5'-GCGCGTCGACCCTGTGTGTCCTTCGGGTGT-3' and
5'-GCGCGTCGACTGATGTAGTGAGAGAGTCTC-3' as primers), and 8) a plasmid
generated by the removal of the internal KpnI fragments of
plasmid 154 (46) (this contains the 89 base pairs upstream of the
splice site of TS (TS 5'-end)). The filters were washed with 6× SSC,
incubated 2 h at 80 °C, and prehybridized with the
hybridization solution described above. Hybridization was carried out
for 48 h at 65 °C, and then the filters were washed three times
at 65 °C with 2× SSC and 0.1% SDS and once with 2× SSC and
treated with 10 µg/ml RNase A to reduce nonspecific hybridization.
The membranes were then exposed to x-ray films.
 |
RESULTS |
The morphology of T. cruzi stages found during
differentiation of intracellular amastigotes to extracellular
trypomastigotes is shown in Fig. 1.
As the infected cell becomes full of amastigotes (shown in Fig.
1a), intracellular parasites start to elongate, and a clear
flagellum can be recognized (Fig. 1b). However, these intermediate forms are morphologically distinct from the released trypomastigotes. They show a migration of the flagellum insertion point
(flagellar pocket) to the anterior end of the parasite, as is typical
of trypomastigote forms (Fig. 1c). The kinetoplast (mitochondrial DNA) follows the apparent migration of the flagellar pocket and is found at several orientations with respect to the nucleus. Mechanically released intermediate forms do not swim and are
clearly different from the other stages. Intermediate forms are also
distinct from epimastigotes, which have the kinetoplast in a position
similar to intracellular amastigotes (Fig. 1d). To follow
the molecular events occurring during this differentiation process,
expression of the amastin, TS, and Tc85 genes was
studied in typical amastigotes, in intermediate forms, and in
trypomastigotes released from infected cells. Trypomastigotes released
from over-infected cells were discarded, as they contained round forms,
morphologically similar to amastigotes.

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Fig. 1.
Morphological differences of T. cruzi developmental stages. Intracellular amastigotes
(a), intermediate forms (b), released
trypomastigotes (c), and cultured epimastigotes
(d) were fixed with formaldehyde, stained with
4,6-diamidino-2-phenylindole, and observed under fluorescent light
(left) and phase contrast (right). The
arrows indicate the nuclei, and the arrowheads
indicate the kinetoplast in selected images. Bar = 2 µm.
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By measuring the steady-state levels of the transcripts corresponding
to amastin, TS, and Tc85 mRNAs in
these different stages, we found that amastin mRNA is mainly
expressed in intracellular amastigotes, as shown previously (5).
TS mRNA is mainly expressed in the intermediate forms,
and Tc85 mRNA is detected only in extracellular trypomastigotes (Fig. 2). All these
mRNAs are weakly expressed in epimastigotes. Tubulin
mRNA levels are approximately constant in epimastigotes and
intermediate forms and slightly reduced in amastigotes and
trypomastigotes. The reduced levels of tubulin mRNA in
amastigotes may be related to this being a spherical stage; thus,
amastigotes contain less tubulin. In released trypomastigotes, the
reduced levels of tubulin mRNA may be related to the
decrease in the overall amount of mRNA in this differentiated
nondividing stage. In fact, large numbers of trypomastigotes have been
used to obtain equivalent amounts of total RNA compared with the other stages. When the same number of parasites, instead of the same amount
of total RNA or poly(A)+ RNA of each stage, were used in
the blot, similar qualitative results were obtained (data not shown).
In this case, TS mRNA was found also mainly in
intracellular trypomastigotes, and Tc85 mRNA only in
extracellular trypomastigotes.

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Fig. 2.
Steady-state levels of amastin, TS,
and Tc85 mRNAs during T. cruzi development. 5 µg of total RNA from
epimastigotes (E; equivalent to 1 × 107
parasites), intracellular amastigotes (A; equivalent to
2 × 107 parasites), intracellular intermediate forms
(IT, equivalent to 1 × 107 parasites), and
trypomastigotes (T; equivalent to 4 × 107
parasites) was fractionated on formaldehyde-containing 1% agarose
gels; transferred to nylon membranes; and probed successively with an
/ -tubulin coding sequence, a DNA probe coding for
amastin, and a probe coding for the carboxy-terminus repeats
repeats of TS. A similar membrane was probed with the catalytic domain
of TS and a probe for the Tc85 glycoprotein subfamily. The numbers on
left represent the sizes of RNA markers (in kilobases
(Kb)).
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To investigate at which level the expression of the TS and
Tc85 genes is regulated, we have analyzed transcription of
these genes in lysolecithin-permeable parasites. Under these
conditions, nascent RNAs are elongated and processed by
trans-splicing and polyadenylation (50). As shown in Fig.
3a, transcription occurred intensely in all stages for the TS, amastin, and
Tc85 genes. There was no decrease in transcription of the
genes that showed diminished steady-state levels, suggesting that these
genes are constitutively transcribed to similar extents in all
developmental stages. As TS genes are organized in long tandem repeats,
with each open reading frame separated by ~2 kilobases (Fig.
3c), the possibility of transcription along the intergenic
region was also examined. As shown in Fig. 3b, comparable
levels of transcription were found in coding and noncoding regions of
TS genes. Moreover, no mature mRNA was detected at any
stage using the intergenic region to probe Northern blots (data not
shown), suggesting that this region probably does not produce stable
mRNA.

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Fig. 3.
TS, Tc85, and
amastin genes are constitutively transcribed during
T. cruzi development. Epimastigotes
(E), intracellular amastigotes (A), intermediate
forms (IT), and trypomastigotes (T) were
permeabilized with lysolecithin and incubated with
[ -32P]UTP for 30 min as described under
"Experimental Procedures." Total RNA was extracted and used to
hybridize to membranes containing 5 µg of the indicated plasmids.
a and b show the results of two independent
experiments, and c shows the positions of the TS probes
relative to the gene. kb, kilobase.
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Our results indicate that TS genes are highly expressed in
the transition forms arising from differentiation between amastigotes to trypomastigotes, just before parasites are released from the infected cell. This finding suggests that the TS gene
expression mechanism is associated with the differentiation process.
Two possibilities could explain this association: 1) TS gene
expression is a programmed event and occurs after a given number of
amastigote divisions; and 2) TS gene expression is activated
by a mechanism that depends on factors produced by the infected cell.
Such factors would become available when a certain degree of infection
is reached, and this would induce the accumulation of TS
mRNA. To distinguish between these two possibilities, L6E6 cells
were infected with increasing numbers of parasites, and after different
periods of time, the percentage of intermediate forms and the level of
TS mRNA were measured in the same amount of
intracellular parasites. The time of appearance and the prevalence of
intermediate forms were dependent on the size of the initial inoculum.
The larger the infective load, the sooner intermediate forms were
detected, at least 48 h after infection (Fig.
4). Furthermore, the accumulation of
TS mRNA paralleled the accumulation of intermediate
forms. This suggests that the switch from amastigote to
trypomastigote depends on the parasite burden within the infected cells
and does not seem to be programmed by the number of cell divisions of
the amastigote forms.

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Fig. 4.
The time of TS gene
expression depends on the degree of cell infection. Subconfluent
L6E6 cells in 75-cm2 flasks were infected with 0.5 × 107, 1.0 × 107, and 3.0 × 107 parasites. After 48, 64, and 72 h, intracellular
parasites were collected; the total RNA was isolated; and 5 µg of
total RNA from each time point was fractionated on
formaldehyde-containing 1% agarose gels. Shown is the hybridization
pattern of a TS probe containing the repeats and the percent
of intermediate forms detected.
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Treatment with actinomycin D, which blocks transcription in T. cruzi (52), was used to study the stability of amastin,
TS, and Tc85 mRNAs during intracellular
development of the parasite. Amastigote, intermediate, and
trypomastigote forms were treated for different incubation periods with
actinomycin D, and the amount of each mRNA species was studied by
Northern blot analysis. The concentration of actinomycin D used in the
experiments is known to block transcription in all stages of T. cruzi (5). The RNA loaded in each lane corresponded to total RNA
isolated from 1 × 108 extracellular
trypomastigotes, 3 × 107 amastigotes, or 3 × 107 intermediate forms. As shown in Fig.
5, amastin mRNA was quite stable in amastigotes with a half-life of 2-3 h, whereas
tubulin mRNA had a shorter half-life (data not shown).
Very little TS and Tc85 mRNAs were detected
in amastigotes, and therefore, their half-lives could not have been
estimated accurately. In contrast, TS mRNA was stable in
intermediate forms (half-life longer than 4 h) (Fig.
5b). In this case, tubulin mRNA decayed much
faster (half-life of ~1 h; data not shown).

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Fig. 5.
Amastin and TS mRNAs are stabilized in
amastigotes and intermediate forms. Intracellular amastigotes
(a) and intermediate forms (b) were collected
from L6E6 myoblasts infected with T. cruzi and incubated
without (control (C); ) or with ( ) 0.1 mg/ml
actinomycin D (Act. D) as detailed under "Experimental
Procedures." At the indicated times, total RNA was extracted,
fractionated on formaldehyde-containing 1% agarose gels, and
hybridized with the amastin and TS probes
containing the repeats. Shown are the autoradiographs and the
corresponding relative counts/min obtained by phosphor imaging.
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The mRNA stability of the amastin, TS, and
Tc85 genes was examined in trypomastigotes released from
infected cells. In the absence of actinomycin D, the level of mRNAs
coding for the TS gene decreased, whereas the
Tc85 mRNA levels were constant in recently released
trypomastigotes (Fig. 6). These
trypomastigotes were collected no more than 3 h after they were
released from the infected cell. When the parasites were incubated in
the presence of actinomycin D, Tc85 mRNA was the most
stable (half-life of ~3 h), followed by TS mRNA
(half-life of 1.5 h) and tubulin mRNA (half-life of
0.7 h) (Fig. 6). In trypomastigotes collected after 12 h, the
levels of TS, Tc85, and tubulin
mRNAs rapidly decayed (half-life of ~0.7 h; data not shown).

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Fig. 6.
Tc85 mRNA is stabilized in
trypomastigote forms. Trypomastigotes were collected from the
supernatant of infected cells and incubated with actinomycin D
(Act. D; 0.1 mg/ml) in Dulbecco's modified Eagle's medium
containing 10% fetal calf serum. Total RNA was extracted from the
drug-treated ( ) or control (C; ) parasites at the
indicated times; fractionated on formaldehyde-containing 1% agarose
gels; and hybridized with tubulin (a),
TS (b), and Tc85 (c)
probes. Each panel shows the respective autoradiographs and
quantitation by phosphor imaging.
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In higher eukaryotes, the abundance of some unstable mRNAs
(e.g. encoding histones, oncogene products, and cytokines)
can be increased dramatically by incubating cells in the presence of
protein synthesis inhibitors (53), and blocking protein synthesis can
reveal at what stage the control is exerted in the gene expression pathway. Thus, we studied the effect of cycloheximide, an inhibitor of
polypeptide chain elongation, on the levels of TS and
Tc85 mRNAs during intracellular development of T. cruzi. Different parasite stages were incubated in the presence of
250 ng/ml cycloheximide, which blocks protein synthesis in the T. cruzi stages studied (5, 43); after different periods of
treatment, the total RNA was extracted, and the amounts of
TS and Tc85 mRNAs were analyzed by Northern
blotting. In the amastigote forms, TS (but not
Tc85) transcripts increased with time in the presence of the
drug (Fig. 7, a and
b). Tc85 RNA was not detected in amastigotes even
after long exposures. In contrast, cycloheximide did not affect the level of TS mRNA, but increased the amount of
Tc85 transcripts by a factor of 3 in intermediate forms
after 2 h of treatment (Fig. 7, c and d). In
intermediate forms, TS RNA was stable. The addition of
cycloheximide to recently released trypomastigotes promoted an increase
in TS mRNA levels and only a small increase in
Tc85 mRNA levels (Fig. 7, e and
f). Thus, cycloheximide induces strong accumulation of
mRNA species mainly in the developmental stages preceding those in
which expression of a specific mRNA is maximal.

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Fig. 7.
A labile protein factor negatively controls
the onset of TS and Tc85 gene expression.
Intracellular amastigotes (a and b) and
intermediate forms (c and d) were collected from
infected L6E6 myoblasts. Trypomastigotes (e and
f) were collected from the supernatant of infected cells.
The parasites were incubated in the absence (control (C);
) or presence ( ) of cycloheximide (CH; 250 ng/ml). The
total RNA was extracted at the indicated times, fractionated on
formaldehyde-containing 1% agarose gels, and hybridized with
TS (a, c, and e) and
Tc85 (b, d, and f)
probes.
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 |
DISCUSSION |
We have shown that during the intracellular development of
T. cruzi, mRNAs coding for amastin, TS,
and Tc85 are sequentially expressed during intracellular
differentiation of amastigotes to trypomastigotes. The
amastin mRNA level is high in proliferating amastigotes;
TS mRNA is found mainly in the intermediate forms undergoing transformation from amastigotes to trypomastigotes; and
Tc85 mRNA is observed only in extracellular
trypomastigote forms. Fig. 8
schematically represents a model for the timing of the expression of
these three genes. It is noteworthy that TS gene expression
anticipates the expression of Tc85, suggesting that these
two proteins act at different times, despite the fact that both have
been implicated in cell invasion. It is important to note that the
amount of mRNA may not reflect directly the level of protein or
enzymatic activity. Nevertheless, TS might be mostly required at the
moment the parasite is released from the infected cell, whereas Tc85
may be important for the adhesion to new host cells, as suggested
before (25, 54). This hypothesis agrees with findings that recently
released parasites are poorly invasive, and invasion competence is
acquired after a certain time (55). Expression of some members of the
85-kDa glycoprotein gene family might be related to the acquisition of
this invasive capacity (26). In addition, the presence of TS in
recently released parasites could be important to promote parasite exit
from the infected cell, spreading from the local site of infection, and
access to the bloodstream.
The data we present indicate that the morphological transformations
observed during parasite development occur together with the
differential expression of the amastin, TS, and
Tc85 genes, and we propose that this process is influenced
by parasite-host cell interactions. The results shown in Fig. 4 suggest
that the number of T. cruzi divisions in the host cell does
not control expression of these genes since intermediate forms and
TS mRNA appear earlier in infection when the cells are
infected with a higher number of parasites. We found that the larger
the number of infecting parasites, the earlier we could detect
intermediate forms and the accumulation of TS mRNA. The
nature of the signals that induce the transformation and accumulation
of TS mRNA is unknown. They might be related to the
limitation of nutrients, as shown for the differentiation of
epimastigotes to metacyclic forms (56). Alternatively, products derived
from the host cell might act directly or indirectly as signals for
differentiation. For example, peptides generated by the catabolism of
hemoglobin are able to induce activation of the epimastigote adenylate
kinase via a Gi protein pathway (23). Furthermore, an
increase in cAMP seems to promote differentiation into infective
metacyclic forms, whereas activation of protein kinase C seems to be
important for T. cruzi proliferation (18, 20).
We have provided evidence supporting the notion that differential
expression of the TS and Tc85 genes is mostly
controlled at the post-transcriptional level, as shown for several
other genes differentially expressed in trypanosomes. The transcription assays using permeable cells suggest that transcription is constitutive for all these genes, and that the TS gene is transcribed as
a polycistronic RNA. In addition, inhibition of transcription by actinomycin D revealed that amastin, TS, and
Tc85 mRNAs are stable in the developmental stages where
they are known to accumulate. Specifically, amastin mRNA is
stable in amastigotes; TS mRNA is stable in intermediate
forms; and Tc85 mRNA is stable in recently ecloded
trypomastigotes. On the other hand, we found that at least in
trypomastigotes, TS mRNA decays faster than tubulin
mRNA, indicating that at this stage, TS mRNA is
unstable. The data presented derive from trypomastigotes recently
released from infected cells. However, when trypomastigotes age, the
amount of RNA decreases due to a general decrease in
transcription.2 Thus, decay of TS and
Tc85 mRNAs is much faster in trypomastigotes that are
collected 12 h after eclosion (data not shown), even in absence of
actinomycin D, in agreement with the fact that the level of these
mRNAs is substantially decreased in aged trypomastigotes. The
highly invasive forms that arise after this aging period are known to
have much lower levels of cellular RNA than at other parasite stages.
The mechanism that promotes TS and Tc85 mRNA
stabilization could occur by the removal of labile factors. Protein
synthesis inhibition by cycloheximide treatment shows that the level of TS mRNA increases in amastigotes and that
Tc85 mRNA increases in intermediate stages. In both
cases, the increase is observed immediately before the stage at which
stabilization occurs. The addition of cycloheximide at the stages where
mRNA levels normally start to decrease also results in some
accumulation. Just after the period in which Tc85 mRNA
stabilization occurs, a small increase in mRNA accumulation was
observed (see Fig. 7f), but after longer incubation, the
levels of mRNA rapidly decreased, suggesting that other factors may
be present to promote degradation and removal of the stable mRNAs.
At the stages that show maximal RNA accumulation, the stabilization
could be additionally supported by formation of stable polysomes. The
effect of cycloheximide in this case could be a consequence of
inhibition of translation, without release of the mRNA from the
polysomes, as found in other trypanosomes. Procyclin-encoding mRNA
accumulates in bloodstream forms of Trypanosoma brucei after
treatment with cycloheximide (57). In Leishmania, protein
synthesis inhibition induces accumulation of the mRNA encoding the
major surface glycoprotein (gp63), which has been shown to be
negatively regulated by a labile, sequence-specific protein that
targets this RNA for rapid degradation (58). Recently, it has been
shown that inhibition of proteasome function prevents transformation in
T. cruzi (59), arguing that stabilization of labile proteins
can also influence differentiation.
RNA stability seems to be mediated by the interaction of molecules with
regulatory elements present in the 3'-untranslated region (3'-UTR) of
mRNAs. These elements have been identified in T. brucei
procyclin mRNA as 16-mer stem-loop and 26-mer polypyrimidine tract
sequences in the 3'-UTR, which, upon interaction with certain proteins,
promote stabilization and translation of the target RNA, respectively
(12, 13). In T. cruzi, the stability of mRNA coding for
reporter genes is also modulated by the 3'-UTR of some members of the
85-kDa glycoprotein family (6). 3'-UTRs from members of the gp85 gene
family (Tt34c1, SA85.1, and TSA-1), which
accumulate in trypomastigotes, exhibit an inhibitory effect on the
expression of reporter genes in epimastigotes, metacyclic trypomastigotes, and amastigotes, but not in trypomastigotes. This
probably represents a common mechanism to down-regulate gp85. The
3'-UTR of amastin mRNA, which is constitutively transcribed, also
decreases the steady-state levels of reporter mRNAs 6-14-fold in
epimastigotes as compared with epimastigote-specific 3'-UTR sequences.
This inhibitory effect is not observed in amastigotes in which
amastin mRNA accumulates (5).
We cannot exclude the possibility that a mechanism besides mRNA
stability controls the expression of TS and Tc85. In fact, control at
the translational level has been found for Tc85 (6). The
rates of transcription initiation, elongation,
trans-splicing, polyadenylation, and RNA transport could
also participate in the control. For example, binding of splicing
factors to unprocessed RNAs could inhibit RNA export from the nucleus,
leading to rapid degradation, as shown in other eukaryotes. An increase
in transcription initiation and elongation rate, although not detected
using the lysolecithin-permeable cell assay, could also modulate expression.
In summary, these results suggest that stage-specific proteins
expressed in the intracellular forms of T. cruzi are
post-transcriptionally regulated by factors involving stabilization of
mRNAs. The differential expression of the TS and
Tc85 genes also provides a convenient system to study the
signaling mechanism occurring in T. cruzi differentiation.
Expression of these genes can be measured after addition of molecules
that interfere with signaling mechanisms known to induce
differentiation through an increase in cAMP levels (17, 18, 20, 21,
60-63), induction of calcium transients (62, 63), or inhibition of
protein kinases.
 |
ACKNOWLEDGEMENTS |
We thank Elisabetta Ullu and Christian
Tschudi (Yale University) for very helpful suggestion and discussions
and Elisabetta Ullu for laboratory use. We thank Norma W. Andrews (Yale
University) and Victor Nussenzweig (New York University Medical Center)
for supplying some reagents and some of the parasites used in this work
and Lys Guilbride for reading the manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from the
Fundação de Amparo à Pesquisa do Estado de São
Paulo (Thematic Projects 94/3496-4 (to S. S.) and 95/4562-3 (to W. C.
and M. J. M. A.)), the Conselho Nacional de Desenvolvimento
Científico e Tecnológico, and the Ministério da
Ciência e Tecnologia (Banco Inter-Americano de Desenvolvimento-Financiadora de Estudos e Projetos) of Brasil.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Fellows of the Fundação de Amparo à Pesquisa do
Estado de São Paulo.
Performed part of this work (with Elisabetta Ullu) during the
National Science Foundation-Conselho Nacional de Desenvolvimento Cientifico e Tecnológico International Collaborative Program. To
whom correspondence should be addressed. Fax: 55-11-5715877; E-mail:
sergio{at}ecb.epm.br.
2
Pereira-Chioccola, et al.,
manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
TS, trans-sialidase;
3'-UTR, 3'-untranslated
region.
 |
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