Leishmania donovani Heat Shock Protein 100

We report the cloning and molecular analysis of the Leishmania donovani clpB gene. The protein-coding region is highly conserved compared with its L. major homologue, while 5′- and 3′-flanking DNA sequences display considerable divergence. The encoded mRNA has an unusually long 5′-leader sequence typical for RNAs, which are translated preferentially under heat stress. The gene product, a 100-kDa heat shock protein, Hsp100, becomes abundant only during sustained heat stress, but not under common chemical stresses. Hsp100 associates into trimeric complexes and is found mostly in a cytoplasmic, possibly membrane-associated, localization as determined by immune electron microscopy. Hsp100 shows immediate early expression kinetics during axenic amastigote development. In its absence, expression of at least one amastigote stage-specific protein family is impaired.

Protozoa of the genus Leishmania parasitize the tissue macrophages of their mammalian hosts, where they survive and proliferate inside the phagosome. This intracellular proliferation is the key for their survival inside the mammalian host.
Leishmania parasites encounter heat stress as a regular feature of their digenic life cycle during the transmission of the promastigote stage from poikilothermic sandfly vectors into mammalia. The elevated temperatures they encounter within a mammalian host serve as a key trigger for the development from the promastigote into the intracellular stage, the amastigote (1). In some Leishmania species, elevated temperatures alone can induce stage development (2,3). However, in Leishmania donovani, a heat shock alone is insufficient and must be complemented by a shift of pH into the acidic range similar to the conditions inside the phagosomes of mammalian macrophages to induce cellular differentiation toward the amastigote (1).
A temperature upshift similar to the one encountered during host invasion can indeed induce heat shock protein synthesis, and it has been proposed that such elevated Hsp synthesis may protect the parasite against the adverse effects of elevated temperature (4,5). Quantitative analyses have shown, however, that the transient induction of Hsp synthesis does not result in significant changes of most of the Hsp's steady state concentrations. Moreover, the major Hsps such as Hsp70 and Hsp83 (Hsp90) are highly abundant in Leishmania promastigotes under all culture conditions and thus not likely to play amastigote stage-specific roles during infection (6).
A newly identified heat shock protein, Hsp100, member of the ClpB family of heat shock proteins (7), is, in contrast, barely detectable in unstressed promastigotes of several Leishmania species, but its concentration increases severalfold during a heat stress (8). It is also highly abundant in Leishmania major amastigotes isolated from infected lymph node tissue. Replacement of the L. major clpB alleles that encode Hsp100 dramatically reduces the virulence in L. major (9).
We are interested in the L. donovani clpB gene and its product, Hsp100, mainly because expression of Hsp100 is more tightly regulated in L. donovani than in L. major (8) and because L. donovani promastigotes readily differentiate into axenic amastigotes in vitro, which allows to study the role of Hsp100 in this process. In this article, we report the cloning and sequence analysis of the L. donovani clpB gene. We show the expression kinetics and the intracellular organization of Hsp100, and demonstrate its impact on amastigote differentiation.

EXPERIMENTAL PROCEDURES
Parasite Culture and Strains-Promastigotes of L. donovani were cultivated in M199 medium supplemented with 25% fetal calf serum and 20 g/ml gentamycin. For cultivation under acidic conditions, the medium was buffered with 40 mM MES 1 buffer (pH 5.0) and pH was adjusted to 5. For animal passage, 4 ϫ 10 8 Lo8 strain promastigotes were injected intraperitoneally into BALB/c mice. After 8 weeks of incubation, the animals were sacrificed, and parasites were grown as promastigotes from spleen tissue and stored frozen in liquid nitrogen.
For in vitro amastigote development, frozen promastigotes fresh from passage were brought back into liquid culture and subjected within 2 weeks to the stage differentiation regimen; cells were first heat-shocked for 24 h at 37°C, harvested by mild centrifugation, and resuspended in fresh medium at pH 5.5. Cultivation was continued at 37°C for 4 days, and stage development was monitored by microscopy and by SDS-PAGE and immunoblot using antibodies against the amastigote-specific A2 proteins (10,11).
DNA Library Constructs-The FIX genomic DNA library of L. donovani strain DD8 was prepared essentially as described for the equivalent L. major DNA library (8).
A cosmid DNA library of L. donovani strain Lo8 was prepared by cleavage of the shuttle cosmid vector pcosTL (12) with SmaI and BamHI and ligation with size-selected Sau3AI partial digest products of L. donovani strain Lo8 genomic DNA. After packaging using the Gigapack Gold II kit (Stratagene), the complexity of the library was tested, 2 and the library was amplified and stored in liquid nitrogen.
Library Screens and Sequence Analysis-clpB-positive clones were isolated from both phage and cosmid libraries by screening with digoxigenin-labeled L. major clpB DNA probes.
Two FIX clones were isolated from the phage library, which showed identical clpB-positive band patterns in Southern blot analysis after restriction digests with several restriction endonucleases. The clpB locus was reconstituted from two BamHI subfragments derived from one clone (clone 3). Both BamHI subfragments were subcloned into pBluescript (Stratagene). The plasmids that carried the two BamHI subfragments were subjected separately to a Nested Deletion reaction (Pharmacia Biotech Inc.). The deletion mutants were size-selected and used as templates for automated sequence analysis on an Applied Biosystems model 130A system using a Dye Terminator kit (Applied Biosystems). The use of specifically synthesized oligonucleotide primers in automated sequencing served to bridge the gaps between partial sequences obtained from individual deletion products.
We also isolated several clones from the cosmid DNA library. One of these cosmids, cos100.4A, was also used as template in the sequence analysis. By using specific primers, we analyzed the 450 bp located immediately upstream of the 5Ј BamHI subfragment.
Electrotransfection-The cosmid cos100.4A was purified from the E. coli host strain using a Qiagen Maxiprep kit. Plasmid DNA was extracted by alkaline lysis and purified over twice by density gradient centrifugation (13). Linear DNA for homologous recombination was excised from pL.m.clpB-NEO or pL.m.clpB-HYG (9) by a single XbaI digest. Singlestranded overhangs were removed by nuclease S-1 treatment.
Electrotransfection of Leishmania promastigotes was carried out as described (14,15). Briefly, cells were harvested during late log phase of growth, washed twice in ice-cold PBS and once in prechilled electroporation buffer, and suspended at a density of 1 ϫ 10 8 cells/ml in electroporation buffer. Chilled DNA was mixed with 0.4 ml of the cell suspension, which was immediately used for electroporation using a Bio-Rad Gene Pulser apparatus.
Electrotransfection of linear DNA was carried out at 3.000 V/cm and 25 microfarads in a 4-mm electroporation cuvette whereas circular DNA was transfected at 3.750 V/cm and 25 microfarads. Mock transfection of L. donovani wild type was performed in identical fashion, however without plasmid DNA, to obtain positive control strains for phenotypical analyses.After electroporation, cells were kept on ice for 10 min before being transferred to 10 ml of drug-free medium. The appropriate drugs for selection of transfectants were added after 24 h.
RT-PCR-Recombinant strain Lo8 (cos 100.4A) was cultivated at 37°C for 24 h and harvested by centrifugation. Total RNA was prepared following standard procedures (16). For mapping of the poly(A) site, first strand cDNA synthesis was carried out using the First Strand cDNA synthesis kit (Pharmacia) with the enclosed (dT) 18 primer and 5 g of total RNA. The cDNA was then amplified enzymatically using the primers Ld100 -52 (GCACGGTGAAAGTGACTCTC) and NOT-18 (GG-AAGAATTCGCGGCCGCAG), which anneal immediately upstream of the Hsp100 stop codon and to the 5Ј half of the (dT) 18 primer, which bears sites for NotI and EcoRI. Amplification was carried out with 5 units of Taq DNA polymerase (Beckman) in the first strand synthesis mix at 95°C (1 min), 52°C (1-2 min), and 72°C (1-2 min) with 40 cycles. The product was digested with NarI and EcoRI and ligated into pBluescript cut with EcoRI and ClaI. After transformation plasmid minipreps were carried out and restriction analysis was performed using enzymes expected to cut within the 3Ј-untranslated region of the clpB gene. Candidate plasmids were sequenced. The sequences were aligned to the clpB DNA sequence.
RT-PCR of the 5Ј leader sequence was carried out in an analogous way. cDNA was prepared from 5 g of RNA from the Lo8 (cos100.4A) strain using the First Strand synthesis kit but with 100 ng of primer Ld100 -56 (GGGAATTCTAACGCTATATAAGTATCAG). After cDNA synthesis, 40 pmol of primers Ld100.71 (TGGATACGGGAGAGTTCA-AG), directed against the 5Ј end of the noncoding strand of the ClpB open reading frame, and SL-20 (GGGAATTCTAACGCTATATAAGTA-TCAG), directed against the spliced leader sequence, and 5 units of Taq DNA polymerase were added, and amplification was carried out at 95°C (1 min), 50°C (1 min), and 72°C (1 min) with 40 cycles. Negative controls that lacked either of the primers or were carried out with RNA from wild type Lo8 strain were included. The products were analyzed by electrophoresis in 2.5% Small DNA Agarose (Biozym). A 420-nucleotide band absent from the controls was purified from the gel using the Gene Clean II Kit (BIO 101). The gel-purified PCR product was then ligated into plasmid pOCUS-T (Novagen) overnight in a refrigerator at 4 -8°C using T4 ligase (MBI Fermentas). The subcloned PCR product was then sequenced using primers directed against the T3 and T7 promoters of the vector. The sequences were aligned with the clpB gene sequence.
Quantification of Hsps in the immunoblot was carried out as described (6). Briefly, recombinant Hsps carrying a 20-amino acid aminoterminal extension were mixed at defined concentrations with L. donovani lysates, and the mixtures were analyzed by SDS-PAGE and immunoblot. Band intensities for natural and recombinant protein were compared densitometrically for each lane, and those recombinant protein concentrations were determined that matched the staining intensity of the natural protein. The amount of each Hsp per cell was calculated from the number of cells used in the analysis, and the number of molecules per cell was deduced using Avogadro's number.
Sucrose Gradient Centrifugation-A linear sucrose gradient (5-20%) was overlayered with sample and spun at 20°C and 200,000 ϫ g for 18 h in a Beckman SW-41 rotor. The gradient was fractionated from the bottom using a peristaltic pump. Fractions were analyzed by SDS-PAGE and silver staining or by immunoblot analysis.
Gel Filtration-A Sephacryl S-400 column (10 mm ϫ 400 mm) was packed at 15 ml h Ϫ1 . The column was equilibrated with running buffer (25 mM Tris/HCl, pH 6.8, 75 mM KCl). V o was determined as 12 ml using blue dextran. V t was 28 ml. The column was calibrated with the HMW calibration kit (Pharmacia) at a flow rate of 4.5 ml h Ϫ1 . Crude cytoplasmic extracts from L. donovani (cos100.4A) were applied and eluted at 4.5 ml h Ϫ1 . Fractions (0.25 ml) were collected and analyzed by immunoblot with anti-Hsp100 antibodies. Relative signal intensities were quantitated by using NIH image software on an Apple Power PC computer with a UMAX Vista-S8 flatbed scanner.
Protein Cross-linking-Cross-linking was performed with dithiobis(succinimidyl propionate) (DSP, Pierce). Promastigotes or amastigotes (2 ϫ 10 7 ) were washed once in PBS. The cell pellet was resuspended in 1 ml of PBS. DSP was added to 1 mM, and the suspension was incubated at room temperature for 30 min. 100 mM Tris-HCl, pH 7.5, was then added to stop the reaction; the mixture was incubated for another 30 min on ice. Cells were washed twice with PBS and lysed in SDS loading buffer. Cross-linking was reversed by boiling the sample with 50 mM DTT in SDS sample buffer. Cross-linking products were analyzed by 5% SDS-PAGE and immunoblot.
Immune Electron Microscopy-L. donovani promastigotes were incubated for 1 h at 37°C, harvested by gentle centrifugation, washed twice, and fixated for 24 h at 37°C in 200 mM sodium cacodylate buffer with 4% paraformaldehyde. Fixated cells were dehydrated in ethanol and embedded in LR-White. Ultrathin sections were prepared on an Ultracut E (Reichert) and placed on 200-mesh Ni grids.
Anti-Hsp100 antibodies (1:500) or preimmune antibodies (1:500) were incubated with the grids for 1 h at 37°C and then overnight at 4°C. The sections were then treated for 1 h each with rabbit anti-chicken antibody (1:300, Jackson Immunolab) and with protein A-gold (10 nm, 1:100, Biocell). Staining of subcellular structures was performed in 1% UO 2 in water and Pb-citrate (Reynolds' procedure). Electron micrographs were taken on a Philips CM-10 transmission electron microscope.
Imaging-Halftone images were digitalized on a flatbed scanner. Digital images were cropped and juxtaposed using Adobe Photoshop™ software, version 3.0.5, on an Apple Power PC™ computer. Line drawings were generated and combined with halftone images using Claris Draw™ software, version D 1.5.

RESULTS
Cloning and Sequence Analysis of the L. donovani clpB Gene-We isolated two bacteriophage clones from a FIX genomic DNA library of L. donovani strain MHOM/IN/80/DD8 using the L. major ClpB gene (8) as hybridization probe. We subcloned two BamHI subfragments, which were positive for hybridization into pBluescript and analyzed the sequence of both inserts. One BamHI subfragment of 3.6 kb represents 2. We also isolated clones from the L. donovani strain MHOM/ IN/??/Lo8 cosmid library. One cosmid, cos100.4A, was included in the sequence analysis to analyze sequences beyond the 5Ј-BamHI site. We thus obtained 7.7 kb of sequence spanning the entire clpB locus with 2.6 kb of coding sequence, 2.5 kb of 5Ј-flanking DNA, and 2.6 kb of 3Ј-flanking DNA.
The sequence of the L. donovani ClpB gene (GenBank accession no. Z94053) was compared with its L. major homologue (Ref. 8, GenBank accession no. Z35058) (data not shown). The sequence of the Hsp100-coding region is well conserved between both species with 96% sequence identity. The flanking regions are less conserved with sequence identities of 87% both for the 5Ј-and 3Ј-flanking DNA. The sequence mismatches result mostly from short insertions or deletions, while long stretches are almost perfectly conserved (data not shown).
We attempted to map the 5Ј end of the L. donovani Hsp100 mRNA by primer extension analysis and by RT-PCR but did not succeed, probably due to the very low abundance of Hsp100 mRNA (8). We therefore transfected L. donovani Lo8 strain with the cosmid cos100.4A, which contains the clpB gene locus in a cosmid shuttle vector and selected for recombinant parasites in G418-supplemented medium. The recombinant strain produces 5-10-fold more Hsp100 compared with wild type L. donovani as estimated by SDS-PAGE and Western blot (see Fig. 2B). We isolated RNA from this strain after incubation at 37°C for 24 h and performed RT-PCR using primers directed against the spliced leader and against clpB gene sequences immediately upstream of the coding region. We observed a PCR product of 420 nucleotides, which was absent from the controls and from a reaction with wild type L. donovani RNA (data not shown). The RT-PCR product was subcloned into pBluescript, and DNA from several bacterial clones was sequenced. The sequence of the RT-PCR products map the trans-splicing donor site to an AG consensus at position Ϫ379 (Fig. 1A).
We next performed RT-PCR to map the polyadenylation site again using RNA from the Hsp100-overproducing strain and primers against the poly(A) tail and a sequence immediately upstream of the stop codon. We amplified a 725-bp DNA, which again was subcloned and subjected to sequence analysis. The products map the polyadenylation site to a position 691 nucleotides downstream of the stop codon (Fig. 1B).
Hsp100 Synthesis Is Inducible by Heat Only-We measured Hsp100 synthesis in heat-shocked promastigotes of L. donovani by metabolic pulse-labeling with [ 35 S]Met and immune precipitation ( Fig. 2A). Within 30 min after the onset of a heat stress, Hsp100 synthesis increases 8-fold as determined by PhosphorImager analysis (data not shown). The synthesis rate decreases only slightly after 2 h and remains well above the noninduced rates for at least 30 h. Unlike induction of Hsp70 and Hsp83 synthesis (6), we find that induction of Hsp100 synthesis is not transient but persists for the duration of the temperature stress.
In prokaryotes and in eukaryotes, the heat shock response is inducible not only by heat but also by a variety of physiological or chemical stresses, i.e. low pH, ethanol, heavy metal ions, and arsenite (17). The response to these stresses is usually mediated by the activation of a heat shock transcription factor, which, upon activation, binds to the promoters of heat shock genes and activates transcription (17,18). Since transcription of heat shock genes is not inducible in Leishmania (6,19), we tested whether chemical stresses affect Hsp100 expression. We determined the LD 50 for several chemicals and exposed L. donovani promastigotes to 50% of the respective LD 50 for 24 h. We also subjected promastigotes to acidic pH 5.5 and to 37°C for the same period. The promastigotes were harvested and subjected to SDS-PAGE and immunoblot analysis with anti-Hsp100 antibodies. The result, shown in Fig. 2B (lanes 1-7), suggests that Hsp100 levels are inducible only by heat stress. The small induction observed with arsenite (lane 7) is not always reproducible and probably not significant. It appears from this that the lack of transcription control in Leishmania forestalls chemical induction of the heat shock response.
We also analyzed the Hsp100 concentration in the recombinant strain Lo 8 (cos100.4A) before and after heat stress (Fig. 2B,  lanes 8 and 9). The protein is overexpressed from the episome in unstressed cells. Nevertheless, a heat stress still induces Hsp100 synthesis. We conclude from this that Hsp100 does not participate in a feedback regulation of its own synthesis.
The heat-inducible synthesis rates observed correspond to a significantly increased intracellular concentration. We quantitated Hsp100 in L. donovani promastigotes from 25°C culture and after 24 h of 37°C using a calibrated immunoblot analysis (6). The result is shown in Fig. 3A and displayed graphically in Fig. 3B. We observe a 6 -7-fold increase of Hsp100 during the first 24 h of heat stress. Longer incubation at 37°C further increases Hsp100 numbers to approximately 2.5 ϫ 10 5 molecules/cell after 36 h (data not shown).
Leishmania Hsp100 Forms Trimeric Complexes-Some Hsp100 family members have been shown to form multimeric complexes in solution (20,21). When we performed a density gradient sedimentation analysis with extracts from the strain L. donovani (cos100.4A), we found Hsp100 to co-sediment with the catalase standard, indicating a native size of approximately 230,000 or 11.3 Svedberg units (Fig. 4A). Gel filtration analysis of Hsp100 on a Sephacryl S-400 resin yielded a Stoke's radius of 65 Å (Fig. 4B), corresponding to an apparent size of approximately 410,000. By applying the algorithms described by Siegel and Monty (22), we calculated the native size of Hsp100 to 302,400 (Table I). Given a calculated monomeric molecular weight of 97,100, this native size assessment indicates a trimeric organization of Hsp100 in L. donovani. The frictional ratio (f/f 0 ϭ 1.47) indicates a deviation of the Hsp100 complex from a pure spherical shape. This may account for the observed discrepancies of apparent size in gel filtration and sedimentation analysis.
We also performed protein cross-linking experiments to further investigate the oligomeric state of Leishmania Hsp100. Promastigotes of strain Lo8 and of the recombinant strain Lo8 (cos100.4A) were subjected to DSP cross-linking in vivo and analyzed by SDS-PAGE and immunoblot (Fig. 5). Both wild   2 and 4), albeit with different intensities due to the overexpression of Hsp100 in Lo8 (cos100.4A). DSP cross-links two bands, one with a molecular weight of 200,000 and one with a molecular weight of 300,000. These bands disappear when the cross-linking is reversed under reducing conditions (lanes 6 and 8) and are not detectable in the absence of the cross-linking agent (lanes 1 and 3). The same pattern of bands of 200,000 and 300,000 was observed when we cross-linked live axenic amastigotes of strain Lo8 (cos100.4A) (lane 12). We do not observe any bands that may indicate cross-linked complexes larger than 300,000. We also performed immune precipitation studies with anti-Hsp100 antibodies (data not shown). We could not identify any protein species that was co-precipitated in stoichiometric quantities. Therefore, Hsp100 appears to form homotrimeric complexes in solution.
Hsp100 Is Clustered inside the Cytoplasm-As a first step toward unraveling the function of Hsp100, we attempted to determine its subcellular localization. Light microscopic analyses with indirect gold staining and silver enhancement had indicated a predominantly cytoplasmic localization for the L. major Hsp100. This was further supported by the results of biochemical fractionation studies with L. donovani (8). To study Hsp100 localization at a microstructural level, we performed immune electron microsopy with heat-stressed Lo8 strain promastigotes. The results are shown in Fig. 6. Little background staining is observed with the preimmune antibody preparation (Fig. 6, a and b). With the anti-Hsp100 antibodies, we observe gold particles mostly close to the plasma membrane (Fig. 6, c and d) with only some staining in the nucleus. The Hsp100 immune complexes are found mainly in clusters (Fig. 6, e and f), which may be a manifestation of the observed association of Hsp100 into oligomeric complexes.
Hsp100 Synthesis Is Induced Early during Amastigote Development-L. donovani promastigotes when exposed sequentially to heat stress and to acidic stress spontaneously develop into axenic amastigote-like forms, which are indistinguishable from true, tissue-derived amastigotes (1,10,11,23,24). This allowed us to monitor Hsp100 levels during amastigote development; we triggered L. donovani strain Lo8 promastigotes to develop into amastigotes by first raising the culture temperature to 37°C for 1 day and then lowering the pH to 5.5 at 37°C. At day 3 of this treatment, amastigote-specific marker proteins, the A2 proteins (10, 11), become detectable (Fig. 7, lower  panel, day 3) and reach maximal levels at day 5. Hsp100 concentration increases earlier, immediately after the temperature shift, and continues to increase for another day before reaching a plateau (Fig. 7, upper panel). Two other heat shock proteins, Hsp70 and Hsp83, also increase, but only by approximately 50% during the amastigote development. It is clear from this that Hsp100 is expressed in amastigotes of L. donovani induced by the elevated temperature, which is a key trigger for the stage differentiation. Additionally, Hsp100 synthesis occurs very early during amastigote development and is not inducible by other common physiological stresses.
b Predicted M r was calculated from the amino acid composition of Hsp100, under the assumption that Hsp100 bears no post-translational modifications.
c Frictional ratio was calculated using the equation described in Ref. This view was supported by results obtained with ⌬clpB gene replacement strains, which do not express Hsp100. The construction of these mutants by homologous recombination, a careful analysis of the mutants' genotypes, and the full phenotype of the mutants will be published elsewhere. 3 Briefly, we have replaced both clpB alleles with the genes encoding hygromycin phosphotransferase and neomycin phosphotransferase following established procedures (25). As tested by restriction analysis and Southern blot, the selection marker genes were faithfully and exclusively inserted into the L. donovani clpB gene locus. 3 The L. donovani ⌬clpB mutant strains display no apparent phenotype as promastigotes in standard in vitro culture either at 25°C or at 37°C. 3 Neither is the morphological development toward amastigote-like stages impaired, as assessed by scanning electron microscopic analysis of wild type and mutant axenic amastigote cultures (Fig. 8A).
We tested three L. donovani ⌬clpB mutant strains and a mock-transfected wild type L. donovani strain for their amastigote stage-specific expression of the A2 gene product. As shown in an immunoblot analysis in Fig. 8B (upper panel), Hsp100 appears in wild type L. donovani during the first day of the stage development, while none of the ⌬clpB mutant strains show any Hsp100 expression in the course of the experiment. Whereas, in the wild type, A2 proteins become prominent on day 4 and remain at high levels on day 5, their synthesis is largely impaired in all three ⌬clpB gene replacement mutants (Fig. 8B, lower panel). This results in an overall Ͼ80% reduction of A2 protein levels in the mutant amastigotes, as quantitated by densitometrical scanning of the immunoblot. Therefore, the lack of Hsp100 significantly affects the expression of at least one amastigote stage-specific marker protein family. DISCUSSION We have isolated and characterized the gene, clpB, which encodes the L. donovani Hsp100. The gene shows a high degree of sequence conservation when compared with its L. major homologue. We could map the 5Ј-splice donor site to a position 379 bp upstream of the open reading frame and the polyadenylation site to 694 nucleotides downstream of the stop codon. The long 5Ј leader RNA could be involved in the regulation of Hsp100 synthesis. Such long leader sequences have already been reported for other heat shock mRNAs in higher eu-karyotes and have been implicated in the regulation of heat shock RNA translation (26 -28). Heat shock gene expression in Leishmania is regulated post-transcriptionally, possibly on the level of translation (6,19,29), and it should be interesting to test the potential of the Hsp100 5Ј leader RNA to mediate post-transcriptional regulation of reporter gene expression.
Hsp100 synthesis fails to be induced by several chemical stresses, a feature that was also reported for heat-inducible translation of Hsp70 mRNA in Drosophila (27). In higher eukaryotes, but also in yeast, chemical induction of the stress response is mediated by the activation of heat shock transcrip-3 S. Krobitsch and J. Clos, manuscript in preparation.

FIG. 7.
Expression of Hsp100 during axenic amastigote development. Samples of a L. donovani promastigote culture (day 0) and of an amastigote development culture (days 1-6) were taken daily and collected by centrifugation. Equal amounts of protein was analyzed by 8% SDS-PAGE and immunoblot, using either a mix of antibodies directed against Leishmania Hsp70, Hsp83, and Hsp100 (upper panel) or a monoclonal antibody against the amastigote-specific A2 gene product (10,11). The lanes with the blotted marker proteins were cut off the filters and stained separately for 30 s with Coomassie Brilliant Blue. The sizes of selected marker proteins are indicated on the left. tion factors and induction of RNA synthesis from heat shock genes (17,18). The lack of transcription regulation in Leishmania may thus prevent a response to chemical stresses.
Hsp100 Is an Amastigote-specific Heat Shock Protein-Hsp100 is expressed under conditions of heat stress, both in the promastigote and in the amastigote stage of L donovani. In the dynamic natural situation, however, the heat shock encountered by promastigotes occurs during inoculation into a mammalian host and therefore must be seen as a prelude to amastigote stage differentiation. In this regard, the distinction between heat-shocked promastigotes and axenic amastigotes is artificial. Rather, Hsp100 is a heat shock protein that is synthesized early during amastigote development and probably all through the mammalian stage of the life cycle. In fact, Hsp100 synthesis was observed in true, tissue-derived amastigotes of L. major (9).
In the promastigote stage, Hsp100 is barely expressed, which suggests the protein is not required in this stage. Indeed, we found that disruption of the clpB genes both in L. major (9) and in L. donovani 3 yielded perfectly viable promastigotes with wild type-like proliferation rates. Therefore, both the expression and the role of Hsp100 are most likely restricted to the mammalian stage of Leishmania parasites.
Association of Hsp100 into Membrane-associated Oligomers-By applying sedimentation analysis, gel filtration analysis, protein-protein cross-linking, and immune precipitation, we have shown Leishmania Hsp100 to be organized as homotrimer in solution. This is reflected in the immune electron microscopic studies, which showed the immunogold complexes in clustered arrays indicating association of Hsp100 into oligomers. Therefore, Hsp100, like the Clp protease subunits in E. coli or the yeast Hsp104 (20,21,30), forms homooligomers. Whether the ability to associate into trimers is inherent in the Hsp100 polypeptide is the subject of an ongoing study using recombinantly expressed protein.
Hsp100-immunogold complexes were found distributed mostly close to the plasma membrane but also within the nucleus. This supports our earlier report, where we found Hsp100 predominantly in the cytoplasmic fraction of L. donovani with traces in the nuclear fraction (8). These trace amounts of Hsp100 were therefore not due to contaminations of the nuclear fraction. The close proximity of the Hsp100 immunogold complexes to the plasma membrane is an unexpected result and may hint at a role in membrane trafficking of biomolecules, a role not previously ascribed to ClpB family proteins. One must note, however, that the biochemical fractionation studies have shown Hsp100 to be in the soluble cytoplasma fraction (8,31) and, therefore, Hsp100 cannot be tightly bound to or integrated into the cell membrane. This result may be relevant to our efforts to unravel the function of Hsp100 in Leishmania.
A Role during Amastigote Development?-The fact that Hsp100 is expressed early during the amastigote development and its important role in the mammalian stage of L. major (9) suggest a role for this protein during the stage development. The ability of L. donovani to develop into axenic amastigotes in vitro is a valuable asset in the study of Hsp100 function. Our finding that lack of Hsp100 impairs expression of a family of amastigote-specific proteins, the A2 gene family products (10), is highly interesting in this regard. All A2 family members are affected by the lack of Hsp100. The expression of the A2 proteins occurs downstream of the Hsp100 expression (Fig. 7) and is regulated by a mechanism that involves modulated mRNA stability (11). Moreover, it was shown recently that a lack of A2 expression in L. donovani, induced by expression of antisense RNA, significantly reduced the virulence in infected animal hosts (32). This makes the effect of the clpB gene replacement on the expression of the A2 gene family all the more significant.
Further analysis of other biochemical markers of amastigote development in the ⌬clpB mutant strains has been initiated and will show whether repression of such markers is universal or selective. In the former case, Hsp100 is pivotal in amastigote development; in the latter case, its role would be more restricted. First results indicate a more selective impact of Hsp100 in the mammalian stage of L. donovani. 4