Type I signal peptidase from Leishmania is a target of the immune response in human cutaneous and visceral leishmaniasis

Molecular & Biochemical Parasitology 135 (2004) 13–20

Type I signal peptidase from Leishmania is a target of the immune response in human cutaneous and visceral leishmaniasis夽 Sima Rafati a,∗ , Ali-Hatef Salmanian b , Tahere Taheri a , Slavica Masina c , Cedric Schaff c , Yasaman Taslimi a , Nicolas Fasel c a

Department of Immunology, Pasteur Institute of Iran, P.O. Box 11365-6699, Tehran, Iran b National Research Center for Genetic Engineering and Biotechnology, Tehran, Iran c Department of Biochemistry, University of Lausanne, Lausanne, Switzerland Received 15 August 2003; accepted 22 December 2003

Abstract The gene encoding type I signal peptidase (Lmjsp) has been cloned from Leishmania major. Lmjsp encodes a protein of 180 amino residues with a predicted molecular mass of 20.5 kDa. Comparison of the protein sequence with those of known type I signal peptidases indicates homology in five conserved domains A–E which are known to be important, or essential, for catalytic activity. Southern blot hybridisation analysis indicates that there is a single copy of the Lmjsp gene. A recombinant SPase protein and a synthetic peptide of the L. major signal peptidase were used to examine the presence of specific antibodies in sera from either recovered or active individuals of both cutaneous and visceral leishmaniasis. This evaluation demonstrated that sera from cutaneous and visceral forms of leishmaniasis are highly reactive to both the recombinant and synthetic signal peptidase antigens. Therefore, the Leishmania signal peptidase, albeit localised intracellularly, is a significant target of the Leishmania specific immune response and highlights its potential use for serodiagnosis of cutaneous and visceral leishmaniasis. © 2004 Elsevier B.V. All rights reserved. Keywords: Leishmania major; Cutaneous leishmaniasis; Visceral leishmaniasis; Signal peptide; Signal peptidase; Human immunoreactivities

1. Introduction Leishmania parasites belonging to the order Kinetoplastida are the causative agents of a large spectrum of diseases in humans, varying from localised cutaneous infection to visceral dissemination, which is often fatal if not treated [1]. Leishmania parasites exist in two basic developmental forms. Upon feeding, an infected sandfly inoculates the flagellated extracellular promastigote, which enters the mammalian host macrophages and differentiates intracellularly into a non-flagellated amastigote that can survive and divide in the macrophage phagolysosome. Definitive diagnosis of cutaneous leishmaniasis (CL) depends on detecting either the amastigotes in clinical specimens, or the promastigotes in culture from biopsies. For visceral leishmaniasis (VL, 夽 Note: Nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under the accession number: AY129954. ∗ Corresponding author. Tel.: +98-21-6953311-20 Ext. 2112; fax: +98-21-8742314-5. E-mail address: s [email protected] (S. Rafati).

0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2003.12.011

kala-azar), definitive diagnosis often requires the detection of amastigotes in patient specimens obtained via invasive methods such as splenic puncture. The use of DNA probes, nested PCR and immunoblot analysis techniques are possible, but require additional reagents, equipment and standardisation. Alternatively, proteins involved in the biological function of the parasite may be used in diagnosis if they are able to be recognised by the human immune system. Secretory proteins contain a signal sequence at their amino-terminal which directs them toward the secretory pathway of cells. The signal sequence is removed by a signal peptidase (SPase) either during, or shortly after translocation across the membrane of the rough endoplasmic reticulum [2,3]. The type I SPase functions to cleave away the signal peptide from the translocated pre-protein, thereby releasing secreted proteins from the membrane and allowing them to locate to their final destination in the periplasm, outer membrane, or extracellular milieu [4]. In 1980, the first prokaryotic SPase was isolated from Escherichia coli and a year later the gene encoding this SPase was cloned and sequenced [4]. It has been demonstrated that SPase is an essential enzyme in both prokaryotes and eukaryotes [5].

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In the absence of SPase, protein precursors are translocated, however, they are found to accumulate on the outer surface of the endoplasmic reticulum inner membrane and cause eventual death [6]. For this reason, it has been suggested that the bacterial type I SPase could be an attractive target for the design of novel antimicrobial compounds. Initial comparisons of the substrate specificities in bacterial and eukaryotic SPase revealed that they are possibly related enzymes that belong to the serine protease family [7]. The mechanism of action of SPase is not completely defined but significant progress has been made with isolation and characterisation of the proteins and genes that encode them [8]. Here we describe the isolation and characterisation of the type I SPase from Leishmania major. This work demonstrates that L. major contains a homologous type I SPase to eukaryotes and prokaryotes, with the presence of five conserved domains (A–E) [8]. In the present study, we have examined the specificity of the human humoral immune response to this type I SPase. For this purpose, sera from individuals with active and recovered cases of CL and VL, in parallel to sera from normal individuals were evaluated for the presence of specific antibodies against a recombinant and a synthetic peptide form of the L. major SPase.

2. Materials and methods 2.1. Parasites The following strains of L. major MHRO/IR/75/ER (IR75), MRHO/SU/59/P (LV39), MHOM/IL/80/FRIEDLIN (Friedlin) and L. infantum MCAN/ES/98/LLM-877 were used. Promastigotes of both species were grown at 26 ◦ C in RPMI 1640 medium (Sigma) supplemented with 10% heat inactivated fetal calf serum (Gibco, BRL), and 0.1 mg ml−1 gentamicine (Sigma). Stationary phase promastigotes were harvested at a density of 2 × 108 parasites per millilitre. 2.2. Preparation of Leishmania parasite genomic DNA Genomic DNA was isolated from 2 × 108 promastigotes in lysis buffer (10 mM Tris–HCl, pH 8.0; 1 mM EDTA, pH 8.0 and 100 mM NaCl) containing 10% sodium N-lauraylsarcosinate. The samples were incubated at 37 ◦ C for 20 min. RNase A (6 ␮g ml−1 , Roche) was added and further incubated for 15 min at 37 ◦ C. Subsequently, proteinase K (20 mg ml−1 , Roche) was added and incubated for 30 min at 37 ◦ C. The DNA was then purified by a phenol–chloroform extraction and ethanol precipitation [9]. 2.3. PCR amplification of Lmjsp To isolate the Lmjsp gene, two primers were designed based on the comparison between consensus sequences of several eukaryotic and prokaryotic SPases. For the forward primer, we took advantage of five conserved amino acids

(GSMEP) found at the N-terminal of domain B. The reverse primer was designed based on five conserved amino acids (LTKGD) found in domain E of the SPase in eukaryotes. Considering codon preferences for L. major, two primers were designed corresponding to the 5 spl (5 GC GGA TCC GGC AGC ATG GAG CCC 3 ) and 3 sp2 (5 GC AAG CTT GTT GTC ACC CTT GGT CAA 3 ), containing BamHI and HindIII restriction sites, underlined, respectively, for directional cloning. L. major genomic DNA (100 ng) was used in the standard PCR mix using PrimZyme DNA polymerase (Biometra) in a 50 ␮l reaction with 500 pg of each primer in the presence of 3 mM Mg2+ . The amplification program was: 95 ◦ C, 1 min; 62 ◦ C, 2 min and 72 ◦ C for 3 min, for a total of 30 cycles. To obtain 5 upstream and 3 downstream sequences of the Lmjsp gene, total RNA was isolated from L. major (IR75) promastigotes using TRIZOL (Gibco BRL). The isolated RNA was reverse transcribed to cDNA by an oligo dT (5 TCC GGA TCC TTT TTT TTT TTT TTT TTT 3 ). The 5 flanking sequence was amplified by RT-PCR using the L. major splice leader sequence (5 TCA GGA TCC AAC GCT ATA TAA GTT ATC AG 3 ) as the forward primer, incorporating a BamHI site, underlined, and sp2 as a reverse primer. The 3 downstream sequence was amplified by using the sp1 as forward primer and oligo dT as a reverse primer. The following program was used for 5 UTR amplification: 95 ◦ C, 1 min; 60 ◦ C, 1.5 min and 72 ◦ C, 10 min for a total of 35 cycles. The program for 3 end amplification was the same as for 5 upstream sequences except for the annealing temperature which decreased to 48 ◦ C. 2.4. Subcloning and sequencing of the Lmjsp gene Both PCR and RT-PCR products were digested with BamHI and HindIII, gel purified (QIAGEN agarose gel purification kit) and subcloned into the pGEM-II vector (Promega). Plasmid DNA was purified by the alkaline lysis method [10]. Restriction enzyme digestion and sequencing (dideoxy chain termination method, sequence kit, Pharmacia), verified the presence of positive clones. Percent identity between sequences was analysed using ALIGN software available under the Expasy Molecular Biology Server (http://www.expasy.org/tools/align). Multiple sequence alignment was performed using ClustalW (http://bioweb.pasteur.fr/seqanal/interfaces/clustalw-simple. html). 2.5. Southern blot analysis Genomic DNA from three different strains of L. major (LV39, IR75 and Friedlin) was isolated and digested with PstI, SalI, NruI and HincII restriction enzymes (Roche). Digested DNA (10 ␮g per lane) was separated on a 0.8% agarose gel and then transferred to a Genescreen Plus membrane (NEN Research Product) according to standard techniques [10]. The Lmjsp DNA was labeled with 32 P-dCTP,

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using the random priming method [10], and used as a probe. The membrane was hybridized at 65 ◦ C for 16 h in a solution containing 0.5 M NaPO4 , pH 7.2, 5 mM EDTA, 7% SDS, 0.25% skim milk powder, and 5 × 106 cpm ml−1 labeled probe. The membrane was washed twice at room temperature in 0.16 M NaPO4 , 1% SDS for 10 min, and once at 65 ◦ C for 1 h in the same solution. The membrane was exposed on BIOMAX MR film (Kodak). 2.6. Expression and purification of recombinant SPase An Lmjsp insert of 378 bp commencing with an ATG in domain B of the mature sequence, was subcloned into the pQE-30 vector (QIAGEN) using BamHI and HindIII restrcition enzymes and renamed pQE-SP. pQE-SP enables the expression of a fusion protein with a 6× His-tag at the N-terminus. The E. coli M15 pREP4 strain was transformed with pQE-SP and grown at 37 ◦ C with agitation in LB broth supplemented with 100 ␮g ml−1 ampicillin and 25 ␮g ml−1 kanamycin. For the production of rSP, the culture was grown to an optical density of 0.8 at 600 nm, and protein expression was induced with 1 mM IPTG overnight at 37 ◦ C. The rSP was purified using a Ni-NTA resin (QIAGEN) and concentrated as previously described [11]. 2.7. Peptide synthesis The Sy-SP peptide corresponding to amino acids 55–135 of the full length mature sequence with a calculated molecular weight of 9.6 kDa, was chemically synthesised (Dictagene, http://www.dictagene.ch) by solid-phase Fmoc chemistry as described [12] and purified to homogeneity under GLP conditions. Amino acid analysis, analytical HPLC and mass spectrometry were used to determine the purity of the final product (greater than 90%). 2.8. Production of polyclonal antiserum Rabbit polyclonal antiserum was raised against the rSP antigen. The recombinant protein (60 ␮g) was mixed with Poloxamer 407 (8%, Sigma) then injected twice at 3-week intervals. Antisera were collected 2 weeks after the second injection. For the synthetic SP peptide, rabbits were immunized with 200 ␮g of peptide three times, according to standard operating procedures (Eurogentec S.A, Belgium). 2.9. NP40 lysis and immunoblotting Parasites were collected from in vitro cultures by centrifugation (10 min, 1500 × g, 4 ◦ C), washed in PBS and lysed in 140 mM NaCl, 1.5 mM MgCl2 , 10 mM Tris–HCl (pH 8.6) and 0.5% NP40. Following lysis, the parasites were pelleted at 4000 × g for 3 min at 4 ◦ C. The pellet was used as the NP40 insoluble fraction and the supernatant was taken as the soluble fraction. Quantification of the amount of protein was performed by BCA® assay (Pierce) according to

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manufacturer’s instructions. For immunoblotting, samples were separated on a 12.5% SDS–PAGE in SDS loading buffer. The proteins were then transferred to nitrocellulose (Schleicher and Schuell) in 3 g l−1 Tris, 14.4 g l−1 glycine and 20% methanol during 1 h at 100 V (Biorad). Membranes were blocked with 5% dried milk powder and 0.05% Tween 20 in TBS (10 mM Tris–HCl, 150 mM NaCl, pH 7.4). Rabbit anti-SP peptide anti-serum in blocking solution (1:1000 in 1% dried milk) was added to the membranes and incubated overnight at 4 ◦ C. The membranes were washed four times in 0.05% Tween 20 in TBS and then incubated with horse radish peroxidase-conjugated anti-rabbit antibody (Sigma) in blocking solution (1% dried milk) for 1 h at room temperature, washed and developed by chemiluminescence (Amersham). 2.10. Human sera Sera were collected from human clinical cases of CL and VL. These included, in group A, sera from active patients. For VL, group A sera was taken from 12 individuals ranging from 1 to 3 years old that had been diagnosed by the detection of Giemsa-stained amastigotes in bone marrow aspirates and clinical evaluation (fever, splenomegaly), together with an indirect immunofluoresence assay with whole parasites as antigen. Biopsy samples from bone marrow were cultured on NNN slants overlaid with RPMI (Sigma) containing 20% FCS. CL active cases consisted of six patients, 18 to 21 years old, with CL skin lesions. These individuals had been diagnosed by detection of Leishmania parasites following microscopic examination of histopathological sections taken from skin lesion biopsy samples, or by culture in NNN. Group B included sera from recovered individuals. VL samples were from Meshkinshahr in the North West of Iran and consisted of five individuals ranging from 5 to 15 years old. They were diagnosed with VL based on similar criteria to active cases and had received standard intravenous antimony treatments. The average recovery period was 4.5±1.5 years. The recovered cases of CL consisted of 11 individuals with an average recovery of 2.6 ± 1.06 years, with duration of clinical skin lesion being 5.8 ± 1.86 months. Group C consisted of three sera from normal individuals of a non-endemic part of Iran with no previous history of CL or VL. 2.11. ELISA Microtiter plates (maxisoipt, Nunc) were coated overnight with either freeze/thawed (F/T) parasite antigen (L. major or L. infantum, 10 ␮g ml−1 ), rSP or Sy-SP antigens (l0 ␮g ml−1 ) diluted in PBS. Following four washes with PBS containing 0.05% Tween 20 (PBS-Tween), plates were blocked with 200 ␮l of 1% BSA for 2 h. After washing, 100 ␮l of sera, diluted 1:50 in 1% BSA/PBS-Tween, was added to the wells and incubated for 2 h. Plates were washed four times and 100 ␮l of horseradish peroxidase-conjugated goat anti-human IgG (Sigma) di-

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luted 1:1000 in 1% BSA/PBS-Tween was added to each well. The binding of conjugate was visualised with 100 ␮l of O-phenylenediamine (Sigma) in citrate phosphate buffer (citric acid 0.1 M, Na2 PO4 0.2 M, pH 5.5). The reaction was stopped with 4 M H2 SO4 and the absorbance was read using a spectrophotometer at 492 nm. The cut-off point was taken as three standard deviations (S.D.) above the mean absorbance for normal sera. 2.12. Statistical analysis The differences in the level of antibody production was determined by student’s t-test. Differences were considered significant when p < 0.01.

ues for the L. major type II (17%), E. coli (16%) and Plasmodium falciparum (13%) SPs. It should be noted that the P. falciparum sequence is not yet 100% complete. 3.2. Gene copy number of Lmjsp Genomic DNA from different strains of L. major (LV39, IR75, and Friedlin) were isolated and analyzed by Southern blotting using various restriction enzymes to estimate copy number (Fig. 3). Only one band was detected with the restriction enzymes PstI (21 kb), SalI (13 kb), NruI (17 kb) and HincII (8 kb). This data suggests that the Lmjsp gene may be present only at one copy per haploid genome although we cannot formally exclude that the obtained fragments are present in tandem arrays.

3. Results 3.1. Isolation of the signal peptidase encoding gene To isolate the type I SPase gene from genomic DNA of L. major, PCR was performed using the two primers sp1 and sp2. Following sequence confirmation, full length cDNA (732 bp) was isolated via RT-PCR from L. major promastigotes (accession number AY129954). Lmjsp contains an open reading frame of 543 bp with an ATG codon located 189 bp downstream of the splice leader sequence. Translation of the full length nucleotide sequence of Lmjsp (type I SPase) is shown in Fig. 1. The five domains (A–E) conserved within other SPs are highlighted. The multiple sequence alignment (Fig. 2) depicts the conserved residues found amongst various SPs. Upon comparison of the L. major type I SP with the other species listed, 38% and 36% conserved identity, respectively, was obtained with the Saccharomyces cerevisiae (S. cerevisiae) and Canis familiaris (C. familiaris) SPs, with significantly lower val-

3.3. Production of a recombinant signal peptidase and its detection in parasite lysate The Protean DNA STAR program was used to estimate the antigenic index of Lmjsp. Accordingly, the Lmjsp coding region (378 bp) commencing from the ATG in domain B was introduced in frame into the bacterial expression vector pQE-30. A commercial anti His-tag antisera was used in immunoblots to determine whether the rSP protein had been expressed (data not shown). A strongly reactive band of the expected size (16 kDa) was observed in the crude induced bacterial lysate of pQE-SP. The E. coli expressed rSP was purified by Ni-NTA column and analyzed by SDS–PAGE. The gel was stained with coomassie blue and a protein band at 16 kDa was visualised (Fig. 4A). The signal peptidase was detected in Leishmania parasite insoluble and soluble fractions (Fig. 4B) as evidenced by immunoblotting using serum raised against the SP peptide. A band at 16 kDa was detected for the rSP (Fig. 4B, lane 1) and a band at around 21 kDa was detected in the parasite fractions (lane 2 insoluble, lane 3 soluble) which is close to the predicted molecular weight (20.5 kDa) of the full length SP. The insoluble fraction contains some lower molecular weight bands at around 16 kDa which may represent a truncated form of the SP. 3.4. Immunoreactivities of sera from cutaneous and visceral leishmaniasis cases

Fig. 1. Amino acid sequence of L. major type I signal peptidase (accession number AAN08877). The five conserved domains (A–E) are boxed.

Human sera from CL and VL cases was analysed by standard ELISA for humoral response against parasite, F/T, rSP or Sy-SP antigens. The response was considered positive when optical density values were at least three standard deviations above the mean of normal sera from non-infected control donors. Both recovered and active cases of CL showed a strong response to parasite F/T antigen, as would be expected (Fig. 5A). The same sera recognised the rSP and Sy-SP antigens at similar intensities, albeit to a lesser degree than F/T antigen (Fig. 5B and C; Table 1).

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Fig. 2. Multiple sequence alignment of signal peptidases. Conserved residues are shaded in black and grey. Accession numbers are: L. major I (AAN08877), L. major II (A1160655), P. falciparum (CAD52351), S. cerevisiae (CAA30533), C. familiaris (P13679) and E. coli (AAA24064). The ClustalW program was used to perform the alignment.

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Fig. 3. Southern blot analysis of the Lmjsp gene. Genomic DNA (10 ␮g per lane) from three strains of L. major (LV39 (L), IR75 (I), and Friedlin (F)) were digested with the PstI, SalI, NruI and HincII restriction enzymes, electrophoresed on a 0.8% agarose gel, transfered to a nylon membrane and hybridized with a SPase gene probe. Molecular weight markers for 21 and 5.1 kb are shown.

Fig. 4. Purification of rSP and detection of signal peptidase in L. major. (A) rSP was purified from E. coli lysate by a single step affinity chromatography procedure using Ni-NTA resin. Lane 1: crude lysate of E. coli transformed with the pQE-SP prior to induction with 1 mM IPTG; lane 2: crude lysate following induction for 3 h; lane 3: purified rSP (l6 kDa). The 15% SDS–PAGE was stained with Coomassie blue. (B) Recombinant signal peptidase, 0.5 ␮g (lane 1), L. major NP40 insoluble fraction, 20 ␮g (lane 2) and L. major NP40 soluble fraction, 20 ␮g (lane 3), were electrophoresed on SDS–PAGE and detected by immunoblotting using anti-rabbit SP peptide antiserum. Molecular weight markers for 21.5 and 14.4 kDa are shown.

Table 1 Total IgG repsonse of F/T, rSP and Sy-SP antigens to sera from active and recovered cases of human cutaneous and visceral leishmaniasis F/T Normal (n: 3) Active CL (n: 6) Recovered CL (n: 11) Active VL (n: 12) Recovered VL (n: 5)

0.35 1.84 1.79 2.29 1.64

rSP ± ± ± ± ±

0.01 0.41 0.20 0.20 0.39

0.24 0.66 0.79 0.38 0.55

Sy SP ± ± ± ± ±

0.01 0.17 0.19 0.10 0.09

0.19 0.76 0.55 0.49 0.51

± ± ± ± ±

0.01 0.26 0.15 0.2 0.07

Average values (measured at 490 nm) and standard deviations for each group are indicated; n: number of samples.

Fig. 5. Total IgG antibody response of active and recovered cutaneous leishmaniasis individuals against L. major F/T, rSP and Sy-SP antigens. The cut-off values were obtained as three standard deviations above the mean absorbance of normal sera. Reactivity toward F/T, rSP and Sy-SP antigens are shown in panels A, B and C, respectively. All antigens (10 ␮g/ml) were screened by sera from active CL (n = 6), recovered CL (n = 11) and normal individuals (n = 3).

A similar pattern of humoral antibody response was seen with sera from individuals from recovered or active VL. Sera from the active form recognised F/T antigen at a higher intensity than that of recovered cases. With regard to rSP and Sy-SP antigens, it was sera from the recovered VL cases

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4. Discussion

type I and type to that observed Leishmania. Southern blot copy gene, as is

Signal peptidase type I has been characterised in many organisms [5,8,13,14]. In this study, the SPase encoding gene of L. major (Lmjsp) was isolated and characterised. Nucleotide sequence analysis of Lmjsp showed similarity with other eukaryotic and prokaryotic SPases. At the amino acid level, L. major SPase shares a high number of conserved identities with the SPases of S. cerevisiase [15] and C. familaris [16]. These homologies are predominantly found in five domains denoted as A–E in Fig. 1, and are present in all known type I SPases. Domain A at the N-terminus consists of the hydrophobic residues Ile-Val-Val-Val-Leu, presumably residing in the membrane-spanning regions of SPs [17]. Domain B contains a conserved Ser-Met sequence that is likely positioned near the membrane surface and may be involved in catalysis [17]. Domain C contains a conserved RGD motif. This motif is present in many adhesion proteins, such as fibronectin, laminin and vitronectin [18]. It is also seen in proteins for which its function in cell adhesion is less evident [19]. Furthermore, it has been demonstrated that not all RGD-containing proteins mediate cell attachment [20], likely due to the RGD sequence not always being available at the surface of the protein or due to its presentation in a context that is not compatible with integrin binding [21]. Among less developed organisms, the RGD motif was described in unicellular parasites such as Leishmania, several bacteria and viruses. In these organisms it appears to be involved in pathogenicity and interaction with host cells [22]. The fourth domain, domain D contains a His-Arg sequence which is strictly conserved in the mitochondrial SPase enzyme [7]. In bacterial SPs, a Lys is found in place of the His [7]. Domain E has a conserved tripeptide Gly-Asp-Asn followed with a conserved Asp and a conserved Arg. The SP sequences contain no conserved Cys residues [7]. The enzymatic activity of SPase is shown to be resistant to normal classical peptidase inhibitors of serine, cysteine, aspartate or metallo-proteases. The conserved Ser in domain B is essential for catalytic activity as was demonstrated for bacterial SPases [23,24]. This shared unusual feature of SPases being resistant to general peptide inhibitors, indicates the critical role of such enzymes in the processing of many membrane and secreted proteins to their mature products [25,26]. It should be noted that there are three different types of bacterial SPases. In addition to the type I SPase, there is the type II SPase that cleaves the signal peptides from lipid modified proteins and a type III SPase specialised in the cleavage of the prepillin proteins [8]. Unlike the type I SPase, both type II and III SPases are not thought to be essential for bacterial viability [27]. There is quite a degree of sequence diversity between the

Fig. 6. Total IgG antibody response of active and recovered visceral leishmaniasis individuals against L. infantum F/T, rSP and SY-SP antigens. The cut-off values were obtained as three standard deviations above the mean absorbance of normal sera. Reactivity toward F/T, rSP and Sy-SP antigens are shown in panels A, B and C, respectively. All antigens (10 ␮g/ml) were screened by sera from active VL (n = 12), recovered VL (n = 5) and normal individuals (n = 3).

which showed a marginally higher antigen specific humoral response.

II bacterial SPases [27] which is similar between the type I and type II SPases of analysis indicated that Lmjsp is a single the case for many bacterial SPs [17], this

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may be further proved via gene knockout studies. The SP protein was shown to be present in L. major soluble and insoluble extracts, at the expected size, following immunoblot analysis with an anti-SP antiserum. A smaller molecular weight band was additionally present in the insoluble extract which may represent a form of SP not completely processed from the membrane. Computer analysis indicated that the most antigenic part of the protein SPase commences from domain B. Therefore, we decided to express the gene fragment of Lmjsp spanning domains B–E. Both the CL and VL forms of leishmaniasis are major health problems, therefore, we evaluated the humoral responses of CL and VL cases against recombinant and synthetic forms of L. major SPase. We report here that the sera of both forms of leishmaniasis in active and recovered conditions recognise rSP and Sy-SP with similar intensity, thereby indicating that the SPase, albeit localised intracellularly, is antigenic and recognised by both CL and VL sera. The precise role of anti-SPase antibodies during active and healed leishmaniasis is unknown and requires further investigation. Presently, we are attempting to evaluate the protective immunity raised by SPase in the BALB/c mouse model using different concentrations and combinations of antigens and immunization schedules Fig. 6.

Acknowledgements The authors wish to thank Shiva Eslami, Fatemeh Doustdari and Myriam Corthesy for their technical assistance. This investigation received financial support from the UNDP/World Bank/WHO Special program for Research and Training in Tropical Disease (TDR), ID no. 970556, A10115 to S.R. and from the FNRS (grant nos. 3100-047342 and 3100-059450.00) to N.F.

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