Download Cloning and characterisation of a cysteine proteinase gene

International Journal for Parasitology 33 (2003) 445–454
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Cloning and characterisation of a cysteine proteinase gene expressed in
amastigotes of Leishmania (L.) amazonensisq
Fernanda Lasakosvitsch, Luciana Girotto Gentil, Márcia Regina Machado dos Santos,
José Franco da Silveira, Clara Lúcia Barbiéri*
Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu, 862, 6o andar,
04023-062 São Paulo, S.P., Brazil
Received 27 September 2002; received in revised form 24 December 2002; accepted 3 January 2003
Abstract
The present study describes the cloning and characterisation of a gene encoding a cysteine proteinase isoform, Llacys1, expressed in
amastigote forms of Leishmania (L.) amazonensis. Recombinant clones containing the Llacys1 gene were isolated from genomic DNA by
PCR amplification and screening of an amastigote cDNA library. Sequence analysis of the Llacys1 gene showed a high identity to sequence
of Leishmania (L.) pifanoi Lpcys1, Leishmania (L.) major cpa, Leishmania (L.) mexicana LCPa, and Leishmania (L.) chagasi Ldccys2. The
Llacys1 gene is present in a single copy per L. (L.) amazonensis haploid genome and was mapped on a chromosome of approximately 700 kb.
Two transcripts of the Llacys1 gene were identified, one of 2.4 kb transcribed in both forms of L. (L.) amazonensis, and another of 1.6 kb
weakly expressed in amastigotes. Related forms of Llacys1 gene exist in other species of Leishmania genus, including L. (L.) major, L. (L.)
mexicana, L. (L.) chagasi and Leishmania (V.) braziliensis. The Llacys1 expression in Escherichia coli was obtained when the nucleotide
sequence corresponding to the signal sequence was deleted, suggesting that this signal sequence was recognised by Escherichia coli and
cleaved, generating a truncated protein.
q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved
Keywords: Leishmania (L.) amazonensis; Amastigotes; Cysteine proteinase isoforms; Gene expression
1. Introduction
Protozoan parasites of the genus Leishmania present two
forms in their life cycle, promastigotes, which multiply in
the midgut of the sand fly vector, and amastigotes, the
obligate intracellular forms which live within phagolysosomes of macrophage from the vertebrate host. Species of
Leishmania cause a broad spectrum of diseases ranging
from cutaneous, mucocutaneous and visceral leishmaniasis
(Adler, 1964). The Leishmania (L.) mexicana complex
comprises species primarily associated with both the simple
and diffuse forms of cutaneous leishmaniasis characterised
by large, histocytoma-like lesions extremely rich in
parasites (Peters and Killick-Kendrick, 1987). Leishmania
q
Note: Nucleotide sequences reported in this paper are available in the
GenBank database under the accession numbers AF538038, AY141758 and
AY141759.
* Corresponding author. Tel.: þ55-11-5576-4532; fax: þ 55-11-55711095.
E-mail address: [email protected] (C.L. Barbiéri).
(L.) amazonensis is responsible for the high incidence of
human cutaneous leishmaniasis in the Amazon region,
Brazil.
Cysteine proteinases have been described in species
belonging to L. (L.) mexicana complex (North and Coombs,
1981; Coombs, 1982; Pupkins and Coombs, 1984; Pupkins
et al., 1986) and high enzyme activity has been detected in
the megasomes of amastigote form (Coombs, 1982; Pupkins
et al., 1986). Proteinases have been implicated with
virulence to vertebrate hosts (Mottram et al., 1996), and L.
(L.) mexicana mutants lacking one group of cysteine
proteinases (LCPb) exhibited reduced infectivity, compared
with wild-type parasites, to macrophages in vitro and in
BALB/c mice (Mottram et al., 1996). Furthermore,
evidence indicates that inhibitors of cysteine proteinases
prevent growth of amastigotes (Coombs et al., 1982;
Coombs and Baxter, 1984) and inhibitors of the LCPb
isoenzymes reduce the infectivity of Leishmania (Mottram
et al., 1996; Selzer et al., 1999), suggesting a chemotherapeutic value for these inhibitors (Coombs et al., 1982;
0020-7519/03/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved
doi:10.1016/S0020-7519(03)00010-9
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F. Lasakosvitsch et al. / International Journal for Parasitology 33 (2003) 445–454
Barret et al., 1999). Participation of Leishmania cysteine
proteinases in the escape of the parasite from the host’s
immune system has also been described (Alexander et al.,
1998) and their involvement in protective cellular responses
has been shown by experiments of immunisation of BALB/c
mice either with cysteine proteinase or DNA encoding
cysteine proteinases of Leishmania (L.) major (Rafati et al.,
2000, 2001b).
In L. (L.) amazonensis high activities of cysteine
proteinases with Mr of around 30 kDa were detected in
amastigote extracts (North and Coombs, 1981; Coombs,
1982; Pupkins and Coombs, 1984; Pupkins et al., 1986;
Alfieri et al., 1989). In a previous work we have shown that
one of these enzymes (p30) was implicated in lymphoproliferative responses of BALB/c mice mediated by CD4þ
Th1 and able to confer significant degree of protection
against homologous infection (Beyrodt et al., 1997).
Cysteine proteinase genes have not been characterised in
L. (L.) amazonensis. In the past few years, we attempted to
clone cysteine proteinase genes of L. (L.) amazonensis. In
this paper we describe the cloning, characterisation and
expression of a gene encoding a cysteine proteinase isoform
from L. (L.) amazonensis amastigotes.
2. Materials and methods
2.1. Parasites
Leishmania (L.) amazonensis (MHOM/BR/73/M2269),
L. (L.) mexicana (MNYC/BZ/62/M379), L. (L.) major
(MRHO/SU/59P),
Leishmania
(L.)
chagasi
(MHOM/BR/72/LD) and Leishmania (V.) braziliensis
(MHOM/BR/75/M2903) promastigotes were grown at
268C in 199 medium (Gibco-BRL) supplemented with 40
mM HEPES, 0.1 mM adenine, 2 mM L -glutamine, 5 mg/ml
hemin (in 50% triethanolamine), 100 U/ml penicillin, 100
mg/ml streptomycin, and 10% heat inactivated fetal bovine
serum (Gibco-BRL). The parasites were isolated from
exponential and stationary cultures (5 –6 days old) and
harvested at a density of 1 £ 109 cells for DNA extraction,
as described below. Leishmania (L.) amazonensis amastigotes were maintained by inoculation into footpads of
golden hamsters every 4 –6 weeks. Amastigote suspensions
were prepared by homogenisation of excised lesions,
disruption by four passages through 22-gauge needle, and
centrifugation at 250 £ g for 10 min; the resulting
supernatant was centrifuged at 1,400 £ g for 10 min, and
the pellet was resuspended in RPMI 1640. The suspension
was kept under agitation for 4 h at room temperature and
centrifuged at 250 £ g for 10 min. The final pellet contained
purified amastigotes which were essentially free of
contamination by other cells (Barbiéri et al., 1990).
2.2. Detection of proteinase activity in SDS-polyacrylamide
gels
Proteolytic activity of L. (L.) amazonensis amastigotes
and promastigotes was determined by zymography employing electrophoretic separation of parasite lysates under
unheated and non-reduced conditions resolved on 10%
acrylamide gels containing 0.1% copolymerised gelatin
(Gibco-BRL) by low-voltage (50 V) electrophoresis
(Robertson and Coombs, 1990). Proteinase activity was
detected after 1 h of incubation, under agitation, in 0.1 M
sodium acetate buffer, pH 5.0, containing 2.5% Triton X100, followed by 2 h of incubation in the acetate buffer in
the absence of Triton X-100 and Coomassie blue staining. In
some experiments proteinase inhibitor E-64 (trans-epoxisuccinil-L -leucinamide-(4-guanide-butane) or dithiothreitol (DTT) was added to all incubation solutions. Molecular
weights markers (Pharmacia LKB) were visible on the
background of stained gelatin when used in a 5-fold excess.
2.3. Nucleic acids isolation, Southern and Northern blot
analyses
Leishmania (L.) amazonensis genomic DNA was
extracted by incubation of 1 £ 109 promastigotes in lysis
buffer (50 mM Tris – HCl, pH 8.0, 62.5 mM EDTA, pH 9.0,
2.5 M LiCl and 4% Triton X-100) for 5 min at 378C. The
DNA was further purified by phenol-chloroform (1:1 V/V)
extraction and ethanol precipitation. The resulting pellet
was resuspended in 50 ml 10 mM Tris– HCl, pH 8.0, 1 mM
EDTA (TE) containing 0.6 mg/ml RNAse and incubated at
378C for 30 min. The DNA was precipitated with 2.5 V of
100% ethanol and 0.3 M sodium acetate and resuspended in
50 ml TE.
Leishmania (L.) amazonensis genomic DNA (2 mg) was
digested with restriction enzymes and subjected to electrophoresis in 0.8% agarose gel. Genomic DNA (2 mg) of L.
(L.) major, L. (L.) mexicana, L. (L.) chagasi and L. (V.)
braziliensis promastigotes, prepared as described above,
was used for digestion with Sma I and electrophoresis in
0.8% agarose gel. After electrophoresis the DNA was
transferred onto Hybond-N filter using standard blotting
procedures and fixed by UV crosslinker. The Llacys1 gene
was 32P-labelled using the random primer labelling method
(Feiberg and Volgelstein, 1983). Partial digestion of L. (L.)
amazonensis genomic DNA was carried out by incubation
of 2 mg DNA with restriction endonuclease Pvu II (0.1 U).
The Southern blot was performed as described above.
Total RNA was isolated from 1 £ 109 L. (L.) amazonensis exponential promastigotes and amastigotes using
TrizolR Reagent (Gibco-BRL) according to the manufacturer’s instructions. For Northern blot analysis 3 mg of RNA
were electrophoresed in formaldehyde agarose gels, transferred onto Hybond-N filter using 20 £ SSC (1 £
SSC ¼ 150 mM NaCl/15 mM Na-citrate) and hybridised
with labelled Llacys1 probe. Filters were also hybridised
F. Lasakosvitsch et al. / International Journal for Parasitology 33 (2003) 445–454
with tubulin probe and washed with SSC 2 £ containing
0.1% SDS at 428C and SSC 0.5 £ plus 0.1% SDS at 508C.
2.4. PCR amplification of Llacys1 gene from L. (L.)
amazonensis
Amplification of the encoding region of cysteine
proteinase gene was carried out with primers based on the
sequence of Lpcys1 gene from Leishmania (L.) pifanoi
(Traub-Cseko et al., 1993). The forward primers corresponding to the 50 end, nt 201 –216 (50 -GCA CGG ATC
CCC AAG ATG GCG CGC CGC AAC-30 ), 50 end nt 408 –
423 (50 -GCA CGG ATC CCC AAG ATG CAG ACA GCC
TAC-30 ) and the reverse primer corresponding to the 30 end,
nucleotide 1,248 –1,262 (50 -CCC GAA TTC GGC CGT
TGT CGT CGG-30 ) contained a Bam HI and Eco RI
restriction site sequences, respectively, for directional
cloning. For PCR reaction we used 200 ng of L. (L.)
amazonensis genomic DNA, 100 pmol of each primer, 2
mM dNTP mix and 3 mM MgCl2 in a volume of 50 ml. The
reaction was conducted for 60 cycles, with the following
thermal profile: 1 min at 948C; 30 s at 578C; 1 min at 728C.
PCR products were cloned in the Eco RI and Bam HI sites
of pUC18. DNA sequencing was carried out employing the
dideoxy-chain method in an ABI377 Applied Biosystems
Automatic Sequencer.
2.5. Screening of a cDNA library from L. (L.) amazonensis
amastigotes
A cDNA library from amastigotes of L. (L.) amazonensis
was constructed in l ZipLox and screened by use of the
Llacys1 gene as a probe. A total of 10,000 PFU was plated
and nylon filters were lifted. The filters were hybridised with
the Llacys1 gene previously labelled with [a-32P]dCTP at
428C overnight and washed with 2 £ SSC, 0.1% SDS, 0.1%
NaPi at 428C; 1 £ SSC at 508C and twice with 0.1 £ SSC at
508C. Plates presenting a strong radiolabelling were picked
and excised in vivo according to the ‘SuperScripte Lambda
System for cDNA Synthesis and l Cloning’ (Gibco BRL).
Sequence analysis was carried out employing the dideoxychain method in an ABI377 Applied Biosystems Automatic
Sequencer.
447
2.7. Pulsed field gel electrophoresis (PFGE)
Leishmania (L.) amazonensis promastigotes were grown
as previously described and 2.5 £ 109 cells were collected
by centrifugation. The pellet was washed with PBS,
resuspended in 1 ml of 1% low melting agarose, and
aliquots of 100 ml were distributed in glass capillaries. After
agarose solidification, the parasites were lysed and the
chromosomal bands separated by PFGE in a Gene
Navigator apparatus (Pharmacia) using a hexagonal electrode array, based on the clamped homogenous electric field
technique (Chu et al., 1986). PFGE was carried out in 1.2%
agarose gels in 0.5 £ TBE running buffer (45 mM Trisborate, 1 mM EDTA, pH 8.3) at 138C for 22 h. Separation
was performed with two phases of homogenous pulses with
interpolation at 200 V: phase 1, pulse time 60 s (run time 11
h); phase 2, 120 s (11 h). Gel was stained with 0.5 mg/ml
ethidium bromide, photographed and transferred onto nylon
filters.
2.8. Expression of recombinant Llacys1 in Escherichia coli
PCR products of Llacys1 gene amplified were cloned into
pGEX 3X vector previously digested with Bam HI and
Eco RI restriction enzymes in frame with glutathione-Stransferase (GST) (Smith and Johnson, 1988) and used to
transform E. coli DH5-a. Fusion proteins were obtained
from isopropylthio-b-galactoside-induced bacterial lysates
as described previously (Smith and Johnson, 1988).
2.9. SDS-PAGE and Western blotting
After growth, recombinant bacteria were pelleted at
4,000 £ g for 10 min, resuspended in sample buffer, and
subjected to SDS-12% PAGE. Western blotting was carried
out as described elsewhere (Towbin et al., 1979). After
electrophoresis, proteins from bacterial extracts were
transferred to nitrocellulose filter for 8 h at 200 mA. After
blocking with 0.5% powdered skim milk in PBS, the filter
was incubated with rabbit hyperimmune serum against L.
(L.) amazonensis amastigotes or monoclonal antibody
(mAb) anti-GST, washed with PBS-milk incubated with
peroxidase-conjugated secondary antibody, and developed
with diaminobenzidine and H2O2.
2.6. Reverse transcriptase-polymerase chain reaction (RTPCR) assay
3. Results
Analysis of Llacys1 gene transcription in amastigotes
and promastigotes from L. (L.) amazonensis was performed
using a reverse-transcription (RT)-PCR amplification kit
according to the manufacturer’s instructions (SuperScripte
Preamplification System For First Strand cDNA SynthesisGibco BRL). RNA from promastigotes and amastigotes was
reverse transcribed into single strand cDNA using oligo
(dT) as a primer. The second strand cDNA was synthesised
using specific primers for the Llacys1 gene.
3.1. Proteolytic activity of L. (L.) amazonensis
Extracts of multiplicative and stationary promastigotes
as well as amastigotes from L. (L.) amazonensis were
subjected to SDS-PAGE with gelatin-coupled gels. Amastigotes contained high proteolytic activities with apparent
Mr of around 30 kDa, whereas similar enzymes were absent
from multiplicative promastigote stage and stationary forms
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F. Lasakosvitsch et al. / International Journal for Parasitology 33 (2003) 445–454
(Fig. 1). The high proteolytic activity of L. (L.) amazonensis
amastigotes migrating around 30 kDa was completely
inhibited after incubation of gelatin-coupled gels in the
presence of E-64.
3.2. Cloning of a cysteine proteinase gene (Llacys1) from L.
(L.) amazonensis
By using genomic DNA of L. (L.) amazonensis
amastigotes and a pair of primers derived from evolutionary
conserved active sites cys25 and asp145 of Dictyostelium
discoideum cysteine proteinase (Eakin et al., 1990) a
fragment of 500 bp (Llacys23) was amplified by PCR.
This fragment was 98% identical at nucleotide level to the
cysteine proteinase gene Lpcys1 from L. (L.) pifanoi
(Traub-Cseko et al., 1993). Thus, as an aim to clone a
complete copy of the cysteine proteinase gene of L. (L.)
amazonensis, PCR amplification was performed using
genomic DNA of L. (L.) amazonensis and primers derived
from the ORF of Lpcys1 gene. A fragment of 1.08 kb,
named Llacys1, was amplified and cloned in pUC18 vector.
Nucleotide sequence analysis was carried out and showed
98% identity to Lpcys1 and LCPa cysteine proteinase genes
of L. (L.) pifanoi and L. (L.) mexicana, and 88% to cpa and
Ldccys2 of L. (L.) major and L. (L.) chagasi. Llacys1 has an
ORF encoding a protein with a predicted Mr of 40 kDa
which contains all the characteristics of cysteine proteinases
gene family. It displays the conserved cysteine and histidine
residues at positions 153 and 289, respectively, present in
the catalytic domain of cysteine proteinases. Glycine which
is involved in substrate binding in papain is also present at
position 151. In addition, other amino acid residue
important in catalysis, asparagine, is present at position
309. The peptide also includes an amino-terminal pre-region
containing the hydrophobic amino acids characteristic of the
signal sequence (Fig. 2) identified by the SignaIP Server
(http://www.cbs.dtu.dk/services/SignaIP). The pro-region is
cleaved between amino acid residues 128 and 129
generating a glycine. The putative motifs of MHC I and
MHC II were also identified by the BIMAS (http://bimas.
dcrt.nih.gov/) and Dnastar – Protean programs,
respectively.
In order to clone a complete copy of Llacys1 gene, a
cDNA library from L. (L.) amazonensis amastigotes was
constructed in lZipLox vector and screened with Llacys1
gene as a probe. Two clones were isolated and sequenced,
one of 1.6 kb (2A1) and another of 2.4 kb (3A4). They
showed an ORF encoding cysteine proteinase (ORF 1)
which is 100% identical to the ORF of Llacys1 gene,
however, they differ in the size of their 30 UTRs. It is
possible to observe two and nine stretches of polypyrimidines, respectively, in the 30 UTR of the clones 2A1 and 3A4
(Fig. 3). It is noteworthy that the clone 3A4 presents a
predicted C-terminal extension after the stop codon of its
ORF 1. Considering that the Ldccys2 gene from L. (L.)
chagasi also presents a C-terminal extension in its 30 UTR
(Omara-Opyene and Gedamu, 1997), besides the high
identity between this gene and Llacys1 (88%), the
nucleotide sequence of the Ldccys2 30 UTR was compared
with those from 3A4 and 2A1 clones (Fig. 3). The 30 UTR of
Ldccys2 gene presents two possible additional ORFs (ORF
II and III) which encode a cysteine proteinase precursor and
a putative RNA binding protein, respectively, whereas 3A4
clone contains only a second ORF in its 30 UTR (ORF 2)
which encodes a putative splicing factor (Fig. 3). We can
also observe that the presence of the first polypyrimidine
stretch in the 2A1 and 3A4 clones interrupts the ORF which
would encode a precursor of cysteine proteinase like in the
L. (L.) chagasi Ldccys2 gene.
3.3. Genomic organisation of Llacys1 gene
Fig. 1. Proteinase activity of L. (L.) amazonensis lysates. The parasite
extracts were subjected to SDS-PAGE on 10% acrylamide gel containing
0.1% gelatin under non-reducing conditions. After separation, gels were
incubated for 2 h at pH 5.0 in presence of 1 mM DTT (lanes 1, 3 and 5) or 1
mM E-64 (lanes 2, 4 and 6). 1, 2 – amastigotes; 3, 4 – exponential
promastigotes; 5, 6, – stationary promastigotes. The number at left
indicates the apparent molecular mass in kilodaltons (kDa).
Genomic DNA from L. (L.) amazonensis was digested to
completion with several restriction enzymes and hybridised
with Llacys1 gene, originating a single hybridisation band in
the majority of digests (Fig. 4A). Fig. 4C shows the
restriction map of the 10.5 kb genomic fragment containing
the Llacys1 gene.
In order to verify whether Llacys1 gene is arranged in a
tandem array manner, genomic DNA was partially digested
with Pvu II and analysed by Southern blotting using the
Llacys1 gene as a probe. The resulting hybridisation profile
(Fig. 4B) indicates that the Llacys1 gene has not a repetition
pattern, showing that it is not arranged in a tandem array.
Southern blot of chromosomes of L. (L.) amazonensis
promastigotes resolved by PFGE was probed with Llacys1.
The Llacys1 gene was located on a chromosomal band of
approximately 700 kb (Fig. 4E). Taken together, these
F. Lasakosvitsch et al. / International Journal for Parasitology 33 (2003) 445–454
449
Fig. 2. Comparison of the amino acid sequence of L. (L.) amazonensis cysteine proteinase (Llacys1) with those from different Leishmania species: Lpcys1,
LCPa, CPA and Ldccys2. Alignment of amino acid sequences was done by the Clustal W and Gene Doc programs. Light grey shaded depicts identical amino
acids. Conserved residues at the catalytic site of cysteine proteinase are also indicated in dark grey. The putative MHC I and MHC II motifs are underlined. The
arrow indicates the cleavage site of the mature protein and asterisks the first and the second methionines used to express the Llacys1 protein in E. coli with
pGEX expression vector.
results suggest that the Llacys1 gene is present in a single
copy per L. (L.) amazonensis haploid genome.
Aiming to study the distribution of Llacys1 gene among
different Leishmania species, Southern blots of genomic
DNA from L. (L.) mexicana, L. (L.) major, L. (L.) chagasi,
and L. (V.) braziliensis promastigotes cut with the restriction
endonuclease Sma I were probed with Llacys1. Fig. 4F
shows that Llacys1 probe hybridised to a single band,
ranging from 3.5 to 5.2 kb, in different Leishmania species.
Although a restriction length polymorphism could be
observed the Llacys1 gene is present in a single copy in
other Leishmania species.
weakly with the Llacys1 gene in amastigote form.
Transcription of Llacys1 gene in amastigotes and promastigotes from L. (L.) amazonensis was also demonstrated by
RT-PCR assay. Using primers to amplify the Llacys1 ORF a
fragment of approximately 1 kb was specifically detected in
amastigotes and promastigotes (Fig. 5B,C). These results
indicate that Llacys1 gene is transcribed in promastigote and
amastigote forms but the transcripts are accumulated in
amastigotes, suggesting that the steady state levels of
Llacys1 transcript are developmentally regulated.
3.4. Analysis of Llacys1 transcripts
The 1.08 kb fragment containing the ORF of cysteine
proteinase was cloned in pGEX 3X expression vector in
frame with the GST gene. Expression of Llacys1 gene in E.
coli resulted in a fusion protein of approximately 32 kDa
which strongly reacted with a mAb anti-GST (data not
shown). The size of this fusion protein (32 kDa) was
significantly lower than that expected (67.5 kDa). In order to
verify if either the N- or C-terminal portions of Llacys1 was
involved in the abnormal expression of the Llacys1 gene, the
Northern blot employing total RNA from L. (L.)
amazonensis exponential promastigotes and amastigotes
was hybridised with Llacys1 probe. Fig. 5A shows a
transcript of around 2.4 kb in both forms but much more
abundant in amastigotes which presented a strong hybridisation signal with the Llacys1 gene. In addition, it was also
observed a transcript around 1.6 kb which hybridised
3.5. Expression of L. (L.) amazonensis Llacys1 in E. coli
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F. Lasakosvitsch et al. / International Journal for Parasitology 33 (2003) 445–454
Fig. 3. Comparison of 30 UTR sequences of 2A1 and 3A4 cDNAs from L. (L.) amazonensis and Ldccys2 cDNA from L. (L.) chagasi. Schematic representation
of cDNAs 3A4 and Ldccys2 showing the predicted ORFs. Inset represents the alignment of the nucleotide sequences of the 30 UTRs from these clones. Light
grey shows nucleotide identity among the clones. Polypyrimidine tracts are marked in dark grey. Arrows indicate start of the ORFs II and III from L. (L.)
chagasi Ldccys2 gene and ORF 2 from L. (L.) amazonensis 3A4 clone. Asterisk indicates the stop codon of the first ORF from the clones.
50 and 30 regions encoding the N-and the C-terminal portions
were amplified, cloned in pGEX 3X and expressed in E.
coli. When the 30 region of the gene Llacys1 was cloned in
frame with the GST gene a fusion protein of the expected
size (53 kDa) was expressed by recombinant bacteria.
However, the construction carrying the 50 region of the
Llacys1 gene expressed a truncated form of N-terminal
domain of cysteine proteinase (32 kDa) (data not shown).
F. Lasakosvitsch et al. / International Journal for Parasitology 33 (2003) 445–454
451
Fig. 4. Genomic organisation of Llacys1 gene. (A) Southern blot of genomic DNA from L. (L.) amazonensis digested with several enzymes: (1) Bam HI; (2)
Eco RI; (3) HindIII; (4) Sma I; (5) Pst I; (6) Kpn I; (7) Sma I/Pvu II; (8) Pst I/Pvu II; (9) Kpn I/Sma I; (10) Kpn I/Pst I; (11) Kpn I/Pvu II; and (12)
Kpn I/Sma I/Pvu II and hybridised with Llacys1 probe. (B) Southern blot of partial digestion with Pvu II of genomic DNA from L. (L.) amazonensis hybridised
with Llacys1 probe. The time (in minutes) of digestion is indicated above the figure. (C) Restriction map of a 10.5 kb genomic fragment containing a copy of
Llacys1 gene. (D, E) Chromosomal localisation of the Llacys1 gene. (D) separation of L. (L.) amazonensis chromosomal bands by PFGE and staining with
ethidium bromide. (E) Hybridisation of the chromoblot with 32P-labelled DNA of L. (L.) amazonensis (1); and Llacys1 gene (2). Sizes of yeast chromosomes
are indicated at left in megabases (Mb). (F) Southern blot analysis of Llacys1 among different Leishmania species: genomic DNAs from L. (L.) major (1); L.
(L.) mexicana (2); L. (L.) chagasi (3); L. (V.) braziliensis (4); and L. (L.) amazonensis (5) were digested with restriction enzyme Sma I and hybridised with
Llacys1 gene.
Facing to these results, another strategy was used for
Llacys1 amplification. A fragment around 870 bp (denominated Llacys1 0 ) starting from the second methionine present
in the sequence was cloned in fusion with GST, originating
a protein with the expected size (62 kDa). This recombinant
protein was reactive with a mAb anti-GST as well as with a
hyperimmune serum against L. (L.) amazonensis amastigote
(Fig. 6).
4. Discussion
The present work focuses on the cloning and characterisation of a cysteine proteinase gene from L. (L.)
amazonensis. Although several cysteine proteinase genes
have been characterised in Leishmania species belonging to
the L. (L.) mexicana complex, like L. (L.) mexicana
(Robertson and Coombs, 1994; Souza et al., 1992; Mottram
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F. Lasakosvitsch et al. / International Journal for Parasitology 33 (2003) 445–454
Fig. 5. Transcription analysis of L. (L.) amazonensis Llacys1 gene. (A) Northern blot hybridisation. Total RNA from promastigotes (P) and amastigotes (A)
were run in formaldehyde gel, transferred to nitrocellulose membrane and hybridised with Llacys1 or tubulin probes. (B) RT-PCR assay: cDNA from
promastigotes (1, 2); and amastigotes (3, 4) were amplified using specific primers for the Llacys1 gene. Slots 2 and 4 contain the first strand cDNA synthesis
performed in absence of transcriptase reverse. (C) Southern blot of the same gel after transfer to nylon membrane and hybridisation with Llacys1.
et al., 1997) and L. (L.) pifanoi (Traub-Cseko et al., 1993),
they had not been described in L. (L.) amazonensis. Previous
data from the literature had shown high cysteine proteinase
activity in extracts of L. (L.) amazonensis amastigotes, in
contrast to promastigotes from exponential and stationary
cultures which exhibit very low proteolytic activity
(Pupkins et al., 1986). Our data corroborate these findings
showing several bands of activity around 30 kDa in
amastigotes of L. (L.) amazonensis but not in promastigotes
(Fig. 1). The cysteine proteinase gene of L. (L.) amazonensis
was isolated by PCR amplification from the genomic DNA
resulting in a fragment of 1.08 kb which was cloned in
pUC18 vector. This gene was denominated Llacys1 and
showed a high degree of identity with cysteine proteinase
genes from L. (L.) pifanoi (Lpcys1), L. (L.) major (cpa), L.
(L.) mexicana (LCPa) and L. (L.) chagasi (Ldccys2) (Fig. 2).
Fig. 6. Analysis of the expression of the recombinant protein Llacys1 0 in E.
coli by Western blot. Extracts from E. coli DH5-a transformed with pGEX
3X vector alone (lane 1) or pGEX 3X carrying the construct Llacys1 0 (lanes
2 and 3) were subjected to SDS-PAGE and transferred to nitrocellulose
membrane. 1 – pGEX vector expressing GST and incubation with MoAb
a-GST; 2 – Llacys1 0 expressing the recombinant protein and incubation
with MoAb a-GST; and 3 – recombinant protein and incubation with the
hyperimmune serum raised against L. (L.) amazonensis amastigotes.
Apparent molecular masses are indicated in kilodaltons (kDa).
The Llacys1 protein exhibits a pre-hydrophobic region
represented by residues 1– 30 and a pro-region which is
cleaved between amino acid residues 128 and 129
generating a glycine which is similar in Ldccys2 from L.
(L.) chagasi (Omara-Opyene and Gedamu, 1997).
Our results indicate that the Llacys1 gene is present in
one copy per haploid genome, and it is located in a 700 kb
chromosomal band. Southern blot analysis indicated the
presence of a single copy of related Llacys1 gene in L. (L.)
major, L. (L.) mexicana, L. (L.) chagasi and L. (V.)
braziliensis. Previous reports show that high cysteine
proteinase activity is characteristic of amastigote forms of
Leishmania species belonging to the L. (L.) mexicana
complex (Robertson and Coombs, 1990), and detection of
these enzymes in amastigotes of L. (L.) donovani and L. (L.)
major by use of gelatin-coupled SDS-PAGE has been
unsuccessful (Coombs, 1982; Pupkins et al., 1986).
However, recently the expression of two major cysteine
proteinases in L. (L.) major, cpa and cpb (Rafati et al.,
2001a; Sakanari et al., 1997; Robertson et al., 1996), has
been demonstrated, and two distinct cysteine proteinase
cDNAs from L. (L.) chagasi, a species belonging to the L.
(L.) donovani complex, were cloned and overexpressed in L.
(L.) chagasi promastigotes (Omara-Opyene and Gedamu,
1997). The high percentage of sequence identity observed
among several Leishmania cysteine proteinase genes and
Llacys1 (Fig. 2) is in agreement with the hybridisation
results observed (Fig. 4F).
The Llacys1 gene was used to screen a cDNA library
from L. (L.) amazonensis amastigotes constructed in l
ZipLox vector and clones 3A4 and 2A1 were sequenced
(Fig. 3). The comparison of the 30 UTR sequences of these
clones to the Ldccys2 C-terminal extension sequence
showed that they do not present an ORF encoding a
cysteine proteinase precursor as that found in the C-terminal
F. Lasakosvitsch et al. / International Journal for Parasitology 33 (2003) 445–454
extension of the Ldccys2 gene (ORF II). This can be
attributed to the presence of the first polypyrimidine tract
common to both cDNA clones but absent in the Ldccys2
sequence which could disrupt the corresponding ORF II and
III from 2A1 and 3A4 clones. On the other hand, another
ORF (ORF 2) is present downstream this region in clone
3A4.
In trypanosomatids the transcription is polycistronic and
the immature RNA is processed into monocistronic mRNA
by polyadenylation and addition of a capped 39 nucleotide
splice leader at 50 end. The polypyrimidine tracts play a role
in the binding of proteins which stabilise the mRNA (Hotz
et al., 1997). Clones 3A4 and 2A1 showed differences in
their 30 UTR due to the presence of variable number of
polypyrimidine tracts, two and nine stretches in clones 2A1
and 3A4, respectively. It could be possible that this
difference confers a reduced stability of the mRNA which
would impair the storage of the 1.6 kb transcript in the
parasite (see Fig. 5). In addition, trypanosomatids display
alternative RNA splicing which involves the insertion of the
poly(A) tail in 30 UTR at different positions. Our results
indicate that the 3A4 and 2A1 cDNAs were originated from
the same gene which undergoes an alternative splicing
generating transcripts from several sizes. In the 2A1 and
3A4 sequences we cannot observe the presence of splice
leader sequence, suggesting that these genes could not
represent a full-length cDNA of cysteine proteinase,
although the leader sequence has not been described in
cysteine proteinase genes from Leishmania.
The increase of Llacys1 mRNA steady-state level in the
amastigotes is consistent with the proteolytic activity
observed in this form (Fig. 1), as well as with previous
reports concerning the high activity of cysteine proteinase in
amastigotes from L. (L.) mexicana complex (North and
Coombs, 1981; Coombs, 1982; Pupkins and Coombs, 1984;
Pupkins et al., 1986). The transcriptional profile of the L.
(L.) amazonensis Llacys1 gene is similar to that of Lpcys1 of
L. (L.) pifanoi (Traub-Cseko et al., 1993), LCPa of L. (L.)
mexicana (Mottram et al., 1992) and Ldccys2 of L. (L.)
chagasi (Omara-Opyene and Gedamu, 1997). The mechanism responsible for the stage specific expression of the
Llacys1 gene is not clear and more studies are required to
clarify this point.
The Llacys1 gene was cloned in pGEX vector originating
a recombinant protein of 32 kDa instead of a protein of the
expected size of 67.5 kDa. Cysteine proteinases which are
targeted to an intracellular compartment or secreted exhibit
a hydrophobic amino terminal sequence comprising by 15 –
22 amino acid residues, termed signal sequence (Sajid and
McKerrow, 2002) which can be involved in folding,
transport and activity of the translated protein (Vernet
et al., 1995). Using programs as SignaIP Server we
identified a signal sequence in the predicted amino acid
sequence encoded by Llacys1 gene which would be
recognised by E. coli and cleaved in its periplasmic space
(Humphreys et al., 2000). The fusion protein of 32 kDa
453
resulting from the Llacys1 gene expression suggests that this
signal sequence was recognised and cleaved by E. coli
machinery. Thus, in order to test this possibility, the signal
sequence was removed by amplification of the Llacys1 gene
starting from the second methionine, originating a fragment
of around 870 bp (Llacys1 0 ) which was introduced in pGEX
resulting in a fusion protein of the expected size (62 kDa).
Previous results from our laboratory showed that the
recombinant protein of 43 kDa resulting from the cloning of
the Lacys23 fragment in pGEX expression vector elicits
lymphoproliferative responses in BALB/c mice previously
immunised with L. (L.) amazonensis. Moreover, secretion of
IFN-g in the supernatants of lymphocytes stimulated by p43
was also observed (Beyrodt, 1998, ‘molecular characterisation of a 30 kDa antigen from L. (L.) amazonensis
implicated in cellular immune responses in a murine
model’, PhD thesis). Furthermore, data from previous
work showed the implication of a cysteine proteinase of
30 kDa from L. (L.) amazonensis with lymphoproliferative
responses in BALB/c mice mediated by CD4þ Th1 and able
to confer significant degree of protection against homologous infection (Beyrodt et al., 1997). In agreement with
these results, several Class II and two Class I MHC motifs
were identified in the Llacys1 sequence (Fig. 2). Taken
together, these results suggest a possible involvement of
both, CD4þ and CD8þ lymphocytes in immune responses
induced by the protein encoded by the Llacys1 gene. The
immunogenicity of cysteine proteinases has been reported
and some data showed that the use of a recombinant L. (L.)
mexicana cysteine proteinase expressed in E. coli resulted in
the development of a potentially protective Th1 cell line
(Wolfram et al., 1995). More recent results showed that
immunisation of susceptible BALB/c with the cpa from L.
(L.) major protected the vaccinated animals against a lethal
challenge with L. (L.) major (Rafati et al., 2000), and
protection against L. (L.) major infection was also observed
in BALB/c mice following genetic vaccination with plasmid
DNA encoding L. (L.) major cpa and cpb (Rafati et al.,
2001b). The expression of a cysteine proteinase from L. (L.)
amazonensis in E. coli opens perspectives to use this
recombinant antigen in new strategies for immunisation in
experimental leishmaniasis.
Acknowledgements
We are grateful to Carolina Guilherme Prestes Beyrodt
for helpful discussion, Silvia B. Boscardin for suggestions,
Simone Katz and Renato A. Migliano Lopes for technical
assistance. This work was supported by the Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP) and
the Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico (CNPq) of Brasil.
454
F. Lasakosvitsch et al. / International Journal for Parasitology 33 (2003) 445–454
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