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International Journal for Parasitology 33 (2003) 445–454 www.parasitology-online.com 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)00010-9 446 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 448 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 450 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 452 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. 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