Parasite strains and culture
For our analyses we used the Lo8 strain of L. donovani, a gift from D. Zilberstein. Promastigotes were routinely cultivated at 25°C in supplemented M199 medium . Cell density was monitored using a Schaerfe Systems CASY Cell Counter. In vitro promastigote to amastigote differentiation was performed as described .
PCR amplification of CPN10 DNA
Four primers were delineated from an amino acid sequence comparison of known GroES and CPN10 proteins. The extreme bias in Leishmania towards G and C residues in the wobble positions of codons was used to create primers with minimal degeneracy:
primer CPN10.1: CCGCTGTTCG ACCGCGTGCT GG
primer CPN10.2: GGCGGCATCR TGCTGCC
primer CPN10.3: CGGCAGCAYG ATGCCGCC
primer CPN10.4: CAGCACCTTG TCGCCCACCT TCAC
100 ng of L. donovani genomic DNA was mixed with 40 ng of oligonucleotide primer pairs, 25 nmoles of each dNTP, 10 × reaction buffer, and 1 unit of Taq DNA polymerase (Beckman) in a 50 μl reaction. The reaction mix was incubated for 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C. The cycle was repeated 35 times. 10% of the reaction was analysed on a 1% agarose gel. Negative controls included each primer by itself and omission of template DNA. Bands absent from the negative controls were recovered by preparative agarose gel electrophoresis and by using glass beads adsorption (PureGen Kit, Biozyme). PCR products were subcloned using the TA cloning kit (Invitrogen) and subjected to sequence analysis..
Reverse transcriptase (RT-) PCR
Total RNA from promastigotes stage parasites strain Lo8 was prepared using a Ribolyser Kit (Hybaid). cDNA synthesis was carried out using the First Strand synthesis kit (Pharmacia) with the enclosed (dT)18 primer and 5 μg of total RNA. The cDNA was then amplified enzymatically using the primers CPN10.1 and CPN10.4 The amplification was carried out as described above.
We used a cosmid library of L. donovani previously described in . The library was screened by hybridisation with digoxigenin-labeled DNA probes derived from the subcloned amplification products. Hybridisation was performed at 65°C in HYB 9 solution (Biozyme) for 16 h. Washes were performed at decreasing SSC concentrations (6 × to 0.2 × SSC, 0.5 % SDS) at 65°C. Filters were developed using anti-digoxigenin FAB/AP conjugate (Boehringer Mannheim) followed by colorimetric staining with NBT/BCIP. Positive cosmid clones were picked, rescreened, and verified in an amplification reaction using CPN10-specific primers.
Subcloning and sequence analysis
Cosmid DNA from positive clones was subjected to restriction endonucleased digest and Southern Blot to determine suitable restriction endonucleases. Cosmid DNA was then digested at a preparative scale and subcloned into an appropriately cleaved pBluescript KS+ vector (Stratagene) or pJC45 vector . Plasmid subclones were then subjected to bidirectional primer walking sequence analysis, starting from the known sequences of the PCR amplificates. Sequencing was performed on an Applied Biosystems Model 370 sequencer using the DyeTerminator Kit.
Contig alignment, in silico translation, and sequence alignment were performed using the MacMolly Tetris and the MacVector software packages. Phylogenetic analyses were performed using the clustalW algorithm included with MacVector at default settings.
Recombinant Expression of CPN10
The putative open reading frame of CPN10 was amplified using specific primers that modify the start codon sequence into a NdeI site and the stop codon sequence into an EcoRI site. The amplification products were cleaved with NdeI and EcoRI and ligated into the expression vector pJC45 , between the NdeI site and the EcoRI site. The expression plasmids were transformed into the bacterial strain BL21 (DE3) [pAPlacIQ], a gift from Olivier Payet, CNRS Toulouse. Bacteria were grown in CircleGrow medium (BIO 101) supplemented with 50 μg/ml Ampicillin and 10 μg/ml Kanamycin to OD600 = 0.5. IPTG was added to 1 mM and incubation was continued for 1 h. The purification of His-tagged proteins from bacterial lysates by metal chelate chromatography has been described [12, 14].
To express CPN10::GFP chimera, the CPN10 coding sequence, minus the stop codon, was enzymatically amplified to create BamHI and HindIII sites at the 5' and 3' ends, respectively. Digested with BamHI and HindIII, the amplification products were ligated between the BamHI and HindIII sites of the yeast plasmid p426/PQ25 . By this procedure, CPN10 and GFP coding sequences were fused in frame. The chimeric CPN10::GFP coding sequence was then excised with BamHI and XhoI and ligated into pIRmcs3- . The plasmid pIRCPN10::GFP and the parent plasmid pIRmcs3- were linearised using SwaI and transfected into L. donovani by electroporation. Recombinant parasites were placed under ClonNAT (Werner Bioreagents) selection (100 μg/ml) and analysed by fluorescence microscopy.
Immunisation and antibody preparation
The immunisation of laying hens and the preparation of antibodies from egg yolk has been described .
SDS-PAGE and Western Transfer were performed as described [14, 19]. Briefly, membranes were treated with blocking buffer (4% skim milk powder and 0.1% Tween 20 in DPBS), with antibodies at appropriate dilutions in blocking buffer, and with secondary antibody/alkaline phosphatase conjugate (Dianova) in blocking buffer. Blots were stained with nitrobluetetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate.
1 × 108 promastigotes were harvested by centrifugation and lysed in 500 μl solubilising buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, and 2.5 μg × ml-1 each of aprotinin, leupeptin, pepstatin, and antipain). Lysates were centrifuged at 13000 × g, 4°C. Soluble proteins were incubated with 20 μl anti-CPN10 antibody for 1 h at 4°C. 350 μl of protein A-sepharose slurry were preincubated with anti-chicken IgG from rabbit and added. The slurry was further incubated at 4°C for 2 h. The immunoabsorbent was centrifuged and washed three times in washing buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0,2% NP-40, 2 mM EDTA), twice in washing buffer B (as above, except NaCl is 500 mM) and once in washing buffer C (10 mM Tris-HCl pH 7.5). For analysis on SDS-PAGE 250 μl of SDS sample buffer was added and the samples were heated for 5 minutes at 95°C. After centrifugation, 50 μl of the supernatant was loaded onto a 12% PA gel.
Immune electron microscopy
Immune electron microscopy was performed essentially as described . LR-White embedded microsections were treated with chicken antibody at 1:500 (anti-CPN10) or 1:2000 (anti-CPN60.2) in PBS (0.1 % Tween 20, 1% BSA). Anti-chicken IgG (rabbit) and Protein-A immunogold particles (10 nm) were used for detection. For detection of CPN10::GFP chimera, anti-GFP monoclonal antibody and 5 nm anti-mouse immunogold conjugate (Sigma) were used.
L. donovani promastigotes expressing CPN10:GFP chimera were grown to 2 × 107 cells ml-1. 1 ml of parasite culture was subjected to centrifugation for 10 min at 1500 × g. The supernatant was discarded and the cell pellet was resuspended in 200 μl of Dulbecco's PBS. 2 μl was applied to a multiwell microscope slide and subjected to fluorescence microscopy at 63-fold magnification. Samples were analysed in bright field and in the FITC channel using a Hitachi Model monochrome CCD camera. Images were taken and merged using the Improvision Open Lab® software package and exported in TIFF format. Cropping and juxtapositioning were done using Adobe Photoshop® software.
Pulsed Field Gel Electrophoresis
Leishmania cells were harvested by centrifugation and washed twice in PBS. Following centrifugation, parasites were resuspended in PBS and mixed with an equal volume of prewarmed 1,5 InCert agarose (FMC BioProducts, Rockland). This mixture was aliquoted in block formers (2 × 107 parasites per block, Pharmacia). To lyse parasites, the agarose blocks were incubated in 2 mg/ml proteinase K (1% Laurylsarkosyl, 0,5 m EDTA pH 9.0) at 37°C for 48 h, and stored in 0.5 EDTA at 4°C.
PFGE was performed at 13°C in 0,25 × TBE buffer under the following conditions: intervall in sec: 100-10 log; switching angle: -110 lin; voltage: 200-150 log (Rotaphor, Biometra).
Saccharomyces cerevisiae strain YPH80 chromosomes (New England Biolabs) were used as size standards.
After staining with ethidium bromide (1 μg ml-1) and strand breakage by a 5 min exposure to UV radiation (254 nm), the gel was blotted onto a positively charged nylon membrane (Qiagen) by alkaline transfer . The membrane was hybridised with a digoxigenin-labeled CPN10 probe. Blots were stained as described above.
Primary experimental data were digitalised either on a flatbed scanner (Microtek Scanmaker 8700) or on a 35 mm film scanner (NIKON LS-4000). Phosphor imager (Molecular Dynamics) data were imported directly as TIFF images. Images were cropped and optimised for colour saturation using Adobe Photoshop® software. No filters or other image altering functions were employed on the images or parts thereof. Images were combined with vector graphics and text using ClarisDraw® software, version 1.0d.