Leishmania parasites were recovered from a naturally infected dog from the Madrid area, Spain. They were characterised as L. infantum zymodeme 1 (MON-1) by isoenzyme analysis 23 and transformed into promastigotes in vitro before being stored under liquid nitrogen as JPC (MCAN/ES/98/LLM-724). Five independent clones (JPCM1 to JPCM5) were derived from this stock by colony cloning and tested for infectivity in hamsters. Two of the cell lines were recovered from the spleen 15 weeks after infection confirming that they had conserved their infection potential. Clone M5 (JPCM5; MCAN/ES/98/LLM-877) was selected for all further studies.

JPCM5 grew well as promastigotes in vitro in many of the media used to grow Leishmania, including RPMI, SDM79 and NNN. A temporary increase of serum concentration from 10 to 20% (v/v) promoted the parasite's growth following adaptation to a different medium, cloning, transformation from, or to, amastigotes and transfection experiments. In HOMEM medium with 10% (v/v) serum, the parasites had a doubling time of 16.0 ± 2.7 h. After 4 to 5 days in culture, when stationary phase had been reached, most promastigotes had a metacyclic morphology, indicating that metacyclogenesis had occurred. Stationary phase promastigotes of JPCM5, grown in HOMEM medium, were transformed in vitro into amastigote-like forms using conditions previously described for other L. infantum lines 24. The transformation of the entire culture, according to morphological parameters, required 3 to 4 days at 37°C in acidic medium (pH 5.5 for SDM or pH 6.5 for MAA). This was accompanied by changes in protein profile as determined by SDS-PAGE (data not shown). The axenic amastigotes could be maintained, with the help of regular sub-passages, or transformed back into promastigotes by switching the culture conditions to 28°C in HOMEM medium with 10% (v/v) FCS. It generally required four days for transformation fully back to promastigotes.

L. infantum JPCM5 were infective to human and dog macrophages (see below) and hamsters. JPCM5 parasites could be recovered from spleen, liver and bone marrow of infected hamsters less than two months post infection. Long-term in vitro culture of some Leishmania species has been reported to result in loss of virulence 25. To test whether this applied to JPCM5, promastigotes were sub-passaged in vitro for 8, 17 and 25 weeks (corresponding to about 10, 20 and 30 sub-passages, respectively) before being inoculated into hamsters. In each case, amastigotes were recovered from the spleen after less than three months indicating that the promastigotes had not lost their virulence and were still able to establish an infection. These in vitro and in vivo results indicated that JPCM5 is suitable for most cell-culture studies.

The complete CPA gene of L. infantum JPCM5 (LiCPA), including the ORF and the 5' and 3' flanks, was amplified by PCR using primers derived from the L. mexicana CPA 21 and sequenced. The LiCPA ORF sequence predicted a protein of 354 amino acids corresponding to a size of 38.9 kDa, an isoelectric point of 7.6 and possessing a 10 amino acid C-terminal extension similar to the L. mexicana CPA 21. Comparison of the deduced amino acid sequence with the translated CPA sequences from several Leishmania species is shown in Figure 1. LiCPA shares 97.7% amino acid identity with the published L. chagasi CPA (LcCPA) 26, 87.3% with L. major CPA (LmjCPA) and 80.3% with L. mexicana CPA (LmxCPA) protein sequences, respectively. There are three potential N-glycosylation sites in LiCPA, two in the mature domain (²⁰⁸NGT²¹⁰ and ¹⁷⁰NHS¹⁷²) and one at the beginning of the C-terminal extension (³⁴⁵NTS³⁴⁷); the last two being conserved in all sequences except in L. mexicana and L. braziliensis. However, amino-acid residues important for catalysis, ¹⁵³C, ²⁸⁹H and ³⁰⁹N, as well as the predicted glutamine of the oxyanion hole ¹⁴⁷Q, are conserved in all sequences 21. LiCPA is identical in size to CPA from L. major, L. mexicana and L. donovani, but differs from LbCPA, which has a 123 amino acid C-terminal extension. A C-terminal extension is characteristic of the cathepsin L-like CPB cysteine peptidases of Leishmania and other trypanosomatids 2027, but has not been observed before in any CPA protein.

Southern blot analysis showed that LiCPA is single-copy (Figure 2, panels A and B) and Northern blotting showed that LiCPA is expressed at similar levels in log phase and stationary phase promastigotes and also amastigotes (Figure 2C). A LiCPA deletion construct containing the SAT gene flanked by about 800 bp of LiCPA 5' and 3' flank regions was generated and used to transfect JPCM5 parasites. The parasites were selected in 96-well plates with HOMEM medium supplemented with the antibiotic nourseothricin. In these conditions, one clone was obtained seven weeks post-transfection. Southern blot analysis (Figure 3B) and PCR analyses (Figure 4A) showed correct integration of the SAT construct into the LiCPA locus. Moreover, the wild type alleles could not be amplified with CPA gene specific primers (Figure 4A), and CPA mRNA could not be detected (Figure 4C), confirming that this nourseothricin-resistant clone (named ΔLicpaC1) is a null mutant resulting from loss of heterozygocity.

A second transfection was performed on JPCM5 promastigotes with a second deletion construct derived from the previous one by replacement of the SAT marker by the BLE gene. The transfection and selection steps were performed as previously, except that 10 μg/ml of phleomycin was used in place of nourseothricin. Four clones grew onto agar plates after two weeks. PCR (Figure 4A) and Southern blot (not shown) analysis demonstrated that these four clones were ΔLicpa heterozygote mutants having correctly integrated the BLE construct into one LiCPA allele. One of these (clone 3047) was selected and re-transfected with a third CPA knockout construct, pGL813, carrying the HYG gene. A total of 16 clones were obtained from four 96-well plates after two weeks of selection in the presence of both phleomycin and hygromycin. Southern analysis of one of these clones, called ΔLicpaC2, confirmed that it was indeed a hygromycin- and bleomycin-resistant ΔLicpa null mutant (Figure 3C) and RT-PCR analysis of cDNA showed the absence of CPA mRNA (Figure 4C). The LiCPA gene was re-introduced into the CPA locus in both ΔLicpa mutants. The re-integration construct, pGL793, contained the LiCPA ORF surrounded by the native 5' flank with 3' sequence derived from the DHFR locus, and the blasticidin-resistance gene enclosed between the 5' and 3' DHFR flank sequences. Both ΔLicpa mutants were transfected with the re-integration cassette and selection achieved in the presence of 10 μg/ml of blasticidin. Several clones were isolated from each transfection (the ones characterised in this study were named ΔLicpaC1::CPA andΔLicpaC2::CPA) and the correct replacement of previously integrated deletion cassettes was confirmed by PCR (Figure 4A) and Southern blot (Figure 4B). LiCPA re-expression in ΔLicpaC1 and ΔLicpaC2 was demonstrated by RT-PCR (Figure 4C).

The ΔLicpaC1 and ΔLicpaC2 mutants were grown as promastigotes in HOMEM medium. In these conditions, they exhibited no apparent growth reduction compared with wild type JPCM5. They were also both able to transform into axenic amastigotes. In order to assess whether the mutants lacking CPA were virulent, their infectivity toward human macrophages was investigated. In comparison with JPCM5, the ΔLicpaC1 and ΔLicpaC2 mutants had a reduced ability to infect human macrophages (Figure 5A) and a reduced number of amastigotes/infected macrophage (Figure 5B). The infectivity was restored to wild type levels by re-expression of CPA in the ΔLicpaC2 mutant, but ΔLicpaC1::CPA had a virulence phenotype midway between the ΔLicpaC1 mutant and wild type parasites. Some cell lines were also tested for their ability to infect macrophages of dogs (Figure 5C and 5D). No statistically significant differences (ANOVA; p ≤ 0.05) were observed between wild type, ΔLicpaC1 orΔLicpaC1::CPA with respect to percentage of infected macrophages or the number of amastigotes per infected macrophage.

The virulence of ΔLicpaC1 to hamsters was found to be significantly less than for the wild type JPCM5. In a hamster infection model (Figure 6) parasites were not detected in the liver and only very low levels were detected in the spleen, with the number of parasites decreasing between 1 and 6 months post-infection. The overall parasite burden for these clones was between 10²- and 10⁴-fold lower compared with JPCM5 at 1 and 6 months post-infection, respectively. The virulence of ΔLicpaC1::CPA was also compared in vivo to JPCM5. Low numbers of parasites were found in the spleen of ΔLicpaC1::CPA infected hamsters 3 months post-infection, whereas no parasites were detected in spleen or liver of hamsters at other time points, indicating the virulence was not restored in the re-expresser clones.

L. infantum JPC (MCAN/ES/98/LLM-724) was isolated in the WHO Collaborating Centre for Leishmaniasis, ISCIII, Madrid, Spain from the spleen of a naturally-infected dog residing in the area in 1998. The parasites were maintained in Novy-MacNeal-Nicolle (NNN) medium (66% (v/v) bacto-agar, 33% (v/v) defibrinated rabbit blood) at 28°C. The resulting promastigotes were grown in RPMI medium before cloning by limiting dilution in agar blood plates. Five clones were isolated, JPCM1–JPCM5, but all further analyses were carried out with JPCM5 (MCAN/ES/98/LLM-877).

JPC and JPCM5 promastigotes were grown in modified Eagle's medium (designated HOMEM medium, RPMI, SDM79 or NNN media with 10% (v/v) heat-inactivated foetal calf serum (FCS) at 28°C. Changes between media were accompanied by temporary elevation of FCS concentration to 20%(v/v). Cultures were sub-passaged to 0.5–1 × 10⁶ cells/ml in fresh medium every 3–4 days when the culture had reached late logarithmic phase. The exponential curve regression, the specific growth rate and the doubling time were calculated using Microsoft Excel software from four independent experiments.

Axenic cultures of amastigote-like forms were performed in MAA medium following the protocol previously published with minor modifications 24. Briefly, promastigotes were grown for 4 to 5 days in HOMEM medium supplemented with 10% (v/v) FCS and 24.5 mM hemin. Then 1–5 × 10⁷ parasites were pelleted, washed once in PBS, re-pelleted and re-suspended in 5 ml of MAA2 medium (modified medium 199 with Hank's salts (Gibco BRL), 0.5% soybean trypto-casein (Promega), 20% (v/v) FCS, 4.8 mM L-glutamine, 24.5 mM hemin, 4 mM NaHCO₃ and 25 mM HEPES pH 6.5). The parasites were then incubated at 37°C in the presence of 5% CO₂ for 2 to 3 days before the medium was first changed. Thereafter the medium was changed every 4 to 5 days.

Transformation from amastigotes to promastigotes was performed by transferring 10⁶–10⁷ amastigotes, which were washed once in PBS, into 10 ml of HOMEM medium supplemented with 20% (v/v) FCS. After incubation at 28°C under air until more than 90% of the parasites were flagellated (generally after 4 to 5 days), the medium was changed to HOMEM supplemented with 10% (v/v) FCS.

Transfections were performed as previously reported 46 using 20 μg of linear DNA. 24 h after the transfections, mutant parasites were selected using one or more antibiotics: 20 μg/ml nourseothricin (Hans Knoll Institute, Germany), 50 μg/ml hygromycin B (Roche, Germany), 10 μg/ml phleomycin (Cayla, France) and/or 20 μg/ml blasticidin (Cayla, France). Clones were either obtained on 0.7% agar-HOMEM plates or in liquid medium, in presence of the appropriate antibiotics, and propagated in HOMEM medium supplemented with the same antibiotics.

The U937 human monocyte line, which is non-adherent, was differentiated to macrophages in RPMI with 5% (v/v) FCS supplemented with 0.1 ng/ml phorbol 12-myristate 13-acetate (PMA) at 37°C in presence of 5% CO₂. Differentiation had occurred by 72 h when the cell line was adherent. The cells were washed with RPMI to remove non-adherent cells before adding the JPCM5 parasites at a ratio of 1:20 (macrophages:Leishmania). The infected cultures were incubated at 37°C in the presence of 5% CO₂. After 3 hours the cultures were washed to remove free Leishmania. Samples of infected macrophages were fixed and stained with Giemsa stain at 24, 48, 72 and 96 h post-infection. For each time point, at least 100 macrophages were observed and the number of amastigotes per infected cell was counted. The results were expressed in terms of the % infected macrophages and the number of amastigotes/infected macrophage.

Infections of the canine monocyte-macrophage cell-line DH82 were performed essentially as described by Kiderlen and Kaye 47. Briefly, adherent and non-adherent DH82 cells were collected in polypropylene round-bottom tubes and stationary phase promastigotes were added at an effector-to-target ratio of 1:8. The cell suspension was incubated for 2 hours at 37°C with gentle agitation. Remaining extra cellular parasites were washed off by differential centrifugation (220 × g, 8 min, 4°C, four repeats); the resulting cell suspension was seeded into 4-chamber LabTek tissue culture slides (Nunc, Denmark) and incubated for 120 hours. Slides were stained with Giemsa solution (Merck, Germany). For each time point at least 400 macrophages were observed and the number of amastigotes per infected cell was counted; results were expressed as the % infected macrophages and the number of amastigotes/infected macrophage.

Infections of Golden Syrian hamsters were initiated by intraperitoneal (i.p.) inoculation with 10⁷ stationary phase promastigotes in PBS. The hamsters' weight was monitored every week. The hamsters were sacrificed at 3, 6 and 9 months post-infection and the spleens were weighed and sliced into pieces to collect amastigote parasites. These were transformed in vitro at 28°C in HOMEM medium supplemented with 20% (v/v) FCS to establish promastigote cultures.

Hamsters used for the study of parasite virulence were inoculated i.p. with 10⁸ stationary phase promastigotes and sacrificed at 1, 3 and 6 months post-infection. Spleen and liver samples were used for parasite quantification by culture microtitration 48.

Genomic DNA was prepared from large scale promastigote cultures and extracted using phenol as previously published 21. PCR was carried out using 50 ng of genomic DNA as template, 100 ng (about 30 pmoles) of each primer, 5% DMSO and 2.5 U Taq polymerase (Perkin-Elmer). Conditions were 1 cycle of 94°C for 5 min then 25 cycles of 94°C for 1 min, 60°C for 2 min and 72°C for 2 min followed by 1 cycle of 72°C for 5 min.

JPCM5 CPA flanking regions were amplified from L. infantum DNA with primers OL670–OL671 and OL672–OL673 originally designed to amplify L. mexicana sequences and integrated in place of the L. mexicana flank sequences in each side of the DHFR-SAT cassette of the Lmxcpb-sat deletion construct 22 to generate pGL545. The LiCPA BLE (pGL726) and HYG (pGL813) deletion constructs were derived from pGL545 by replacing the SpeI/BamHI fragment containing the SAT resistance gene by the appropriate antibiotic resistance gene extracted from pGL53 and pGL345 (p Lmxcpb-ble and p Lmxcpb-hyg constructs), respectively. A 1.55 kb DNA fragment corresponding to the LiCPA 5' flank and ORF was amplified with primers OL1005 and OL1006 in order to generate SacII and PstI sites for sub-cloning. The LmxCPC 5' and 3' flank sequences from pGL545 49 were then replaced by this PCR product and the LiCPA 3' flank, amplified as described above, to generate the LiCPA re-expression construct pGL793.

For Southern blot analysis, 5 μg L. infantum genomic DNA was digested to completion with the appropriate restriction enzymes before being separated and transferred onto Nylon membrane as described previously 22. For wild-type L. infantum DNA analysis, the membrane was treated and probed with a non-radioactively labelled PCR product corresponding to the LiCPA ORF as described above. ΔLicpa mutant blots were probed with radioactively labelled PCR products corresponding to the LiCPA ORF 5' or 3' flanks, essentially as published 50 except that the membranes, after high stringency washes, were exposed on Phosphorimager plates. The plates were scanned at high resolution on a Typhoon 8600 apparatus (Molecular Dynamics).

Total RNA was extracted from 10⁷ to 10⁸ parasites using Trizol (Invitrogen, UK) or RNAEasy kit (Qiagen, Germany) according to the manufacturer's instructions. The amount of total RNA extracted was quantified by spectrophotometry and 5 μg were loaded on 0.8% agarose gel prepared in DEPC-treated 0.5× TBE buffer. After separation, gels were washed once in DEPC-water and transferred onto membrane using to the same procedure as for Southern blot. Hybridization with radioactively labelled probes took place overnight at 65°C. Stringent washes were performed in 0.2× SSC, 0.1% SDS and the membranes were exposed onto Phosphorimager plates. The plates were scanned at high resolution after two days exposure.

cDNA was prepared from 5 μg of total RNA using the AMV RT module from the GeneRacer kit (Invitrogen, UK). A first RT-PCR was performed on 1 μl of cDNA mixture with 2 units of Thermozyme enzyme (Invitrogen, UK) and 100 ng of first-round primers in 50 μl reaction volume. The resulting PCR mixture was diluted 30 times in water and 1 μl of it was used as template for a nested reaction performed in a 20 μl reaction volume with 1 unit of TaqDNA polymerase (ABGene, UK) and 100 ng of nested primers. The first-round primers used were the Splice Leader primer (OL618)/OL137 for the 5' amplification, and OL67/GeneRacer Oligo dT primer for the 3' amplification. The primers for the nested PCR were OL618/OL136 for the 5' amplification and OL1194/GeneRace 3' Nested primer for the 3' amplification. OL1984 and OL1985 were used to PCR amplify LinJ36.2050.

Oligonucleotides used in this study.

OL67 5'-CAGAACATGCAGACAGCC-3'

OL136 5'-GAGCGGAACCGTAGCACA-3'

OL137 5'-GACAGCGTCCGCAGTGGTG-3'

OL618 5'-AAGTATCAGTTTCTGTACTTTATG-3'

OL670 5'-ATATAAGCTTCTACTGCACCAGGTACTG-3'

OL671 5'-ACGTGTCGACGAGAAGGACGTGACGGGG-3'

OL672 5'-GGTGCCCGGGGCTGTGCACAAACACGACC-3'

OL673 5'-GTCGAGATCTCACTGTCCATGGCACGCCTG-3'

OL1006 5'-CTGCAGCTAGGCCGCTGTCGTCGG-3'

OL1194 5'-GACAGCGTCCGCAGTGGTG-3'

OL1984 5'-GCACCCGGGATGGCGGCAACTCATCTTAC-3'

OL1985 5'-GCAGGATCCTCACTTCGGCAAACCGTTCTTTC-3'