The expression of HSP83 genes in Leishmania infantum is affected by temperature and by stage-differentiation and is regulated at the levels of mRNA stability and translation
© Larreta et al; licensee BioMed Central Ltd. 2004
Received: 25 March 2004
Accepted: 03 June 2004
Published: 03 June 2004
Exposure of Leishmania promastigotes to the temperature of their mammalian hosts results in the induction of a typical heat shock response. It has been suggested that heat shock proteins play an important role in parasite survival and differentiation.
Here we report the studies on the expression of the heat shock protein 83 (HSP83) genes of Leishmania infantum. Confirming previous observations for other Leishmania species, we found that the L. infantum HSP83 transcripts also show a temperature-dependent accumulation that is controlled by a post-transcriptional mechanism involving sequences located in the 3'-untranslated region (3'-UTR). However, contrary to that described for L. amazonensis, the accumulation of the HSP83 transcripts in L. infantum is dependent on active protein synthesis. The translation of HSP83 transcripts is enhanced during heat shock and, as first described in L. amazonensis, we show that the 3'-UTR of the L. infantum HSP83 gene is essential for this translational control. Measurement of the steady-state levels of HSP83 transcripts along the promastigote-to-amastigote differentiation evidenced a specific profile of HSP83 RNAs: after an initial accumulation of HSP83 transcripts observed short after (2 h) incubation in the differentiation conditions, the amount of HSP83 RNA decreased to a steady-state level lower than in undifferentiated promastigotes. We show that this transient accumulation is linked to the presence of the 3'-UTR and flanking regions. Again, an 8-fold increase in translation of the HSP83 transcripts is observed short after the initiation of the axenic differentiation, but it is not sustained after 9 h.
This transient expression of HSP83 genes could be relevant for the differentiation of Leishmania, and the underlying regulatory mechanism may be part of the developmental program of this parasite.
Leishmania parasites are dimorphic protozoa that pass through markedly different environments during their life cycle. The flagellated promastigote develops in the alimentary tract of the sandfly vector and is inoculated into a mammalian host during a blood meal. In the mammal, the parasite is phagocytosed by macrophages and switches to the amastigote form. After intracellular multiplication and cellular lysis, amastigotes enter other macrophages, spreading the infection. The molecular basis of the promastigote-to-amastigote transformation remains poorly understood although it is well established that several environmental factors such as pH and temperature trigger this cytodifferentiation process . Indeed, a temperature upshift in vitro from 25° to 37°C, combined with acidification of the growth medium, is a sufficient stimulus to induce a promastigote-to-amastigote differentiation in several Leishmania spp. [2, 3].
Early studies showed that exposure of Leishmania to temperatures typical of the mammalian host induces a stress response and synthesis of heat shock proteins (HSP), suggesting that HSP could be important players in the differentiation process . Thereafter, genes coding for HSP have been cloned and extensively studied, mainly those coding for HSP70 and HSP83 [reviewed in ]. Given the conserved and universal nature of the heat shock response, heat shock genes provide a suitable model for studying the unusual features of gene expression in trypanosomes. In these early-branched eukaryotes, the RNA polymerase II transcription is not regulated and gene expression is exclusively controlled at the post-transcriptional level [5, 6].
HSP70 and HSP83 genes are transcribed constitutively and their transcription is not induced by heat shock [7–9]. Instead, the HSP70 and HSP83 are synthesized at elevated rates during heat shock, indicating that the stress response in Leishmania is regulated exclusively at a post-transcriptional level . Nevertheless, the induced synthesis of HSP70 and HSP83 does not increase significantly the steady-state levels of these proteins. This is probably due to the striking abundance of these proteins in unstressed Leishmania promastigotes: HSP70 and HSP83 make up to 2.1% and 2.8%, respectively, of the total protein . The mechanisms regulating the HSP83 expression during heat shock in L. amazonensis has been examined in detail by Shapira and co-workers [7, 10, 11]. Transcripts for HSP83 accumulate upon temperature elevation by a post-transcriptional mechanism that induces variations in mRNA turn-over: the HSP83 transcript was rapidly degraded at normal temperatures, whereas heat shock leads to its stabilization. The fast decay of HSP83 mRNA at lower temperatures depended on active protein synthesis . By transfection experiments with plasmid constructs, they found that the regulated decay of HSP83 mRNA is controlled by sequence or conformational elements present in both upstream and downstream untranslated regions (UTRs) . Recently, they identified by deletion analysis a regulatory element located between positions 201 and 472 in the 3'-UTR of the HSP83 gene as responsible for the preferential translation of the HSP83 mRNA during heat shock .
In this work, we have carried out an analysis of the mechanisms of HSP83 gene expression in another Leishmania species, L. infantum. This species causes visceral leishmaniasis, a severe, often fatal, disease common in the Mediterranean basin and in Latin America . We found that the L. infantum HSP83 mRNA accumulation and its enhanced translation during heat shock is driven by sequences located in the 3'-UTR, even though the translational efficiency is conditioned by the nature of the 5'-UTR. Noticeably, we found that the HSP83 mRNA abundance and the HSP83 translation rate show fluctuations along the promastigote-to-amastigote differentiation in L. infantum, reinforcing the postulated role for this protein in morphological differentiation of Leishmania .
Results and Discussion
The HSP83 mRNAs of L. infantum are stabilized in response to a temperature increase
The 3'-UTR of the L. infantum HSP83 gene confers stability to a reporter transcript in response to a temperature increase
Most of the regulatory elements that control gene expression in Leishmania and other kinetoplastids have been located at the UTRs of the mRNAs, more frequently at the 3'-UTRs [17, 21–24]. The boundaries of the 5'- and 3'-UTR of the HSP83 mRNA were defined by an RT-PCR approach (see Materials and methods for details). The trans- splicing acceptor site was located 290 nucleotides upstream from the beginning of the HSP83 ORF, and the polyadenylation site of the 3'-UTR was determined to be at nucleotide 896 from the stop codon (GenBank™/EBI Data Bank accession number X87770).
Sequences within the 3'-UTR of HSP83 are required for enhanced translation at elevated temperatures
First, we determined the relative content of both cat transcripts and CAT protein in the promastigote cultures transfected with pXCATIRs, pXIRPstCATIR, pX5HisCATIR, and pX5HisCAT3His (Fig. 5B and 5C, respectively). As expected, the level of cat transcripts increased after heat treatment in promastigotes transfected with constructs containing the HSP83 3'-UTR (pXCATIRs, pXIRPstCATIR and pX5HisCATIR), but increase was not observed in promastigotes transfected with the construct pX5HisCAT3His, containing the H2A4 3'-UTR. Remarkably, it was observed that pXCATIRs-transfected promastigotes, accumulating more cat transcripts than pXIRPstCATIR- or pX5HisCATIR-transfected promastigotes, expressed a 2- to 3-fold lower amount of CAT protein than those transfected with either pXIRPstCATIR or pX5HisCATIR (Fig. 5B). Therefore, these results indicate that cat transcripts derived from pXCATIRs construct are poorly translated relative to cat transcripts expressed by the other constructs. It is conceivable that the different translational efficiencies are linked to differences in the 5'-UTRs.
The HSP83 mRNA abundance transiently varied during the axenic promastigote-to-amastigote differentiation
The involvement of the 3'UTR of the L. infantum HSP83 gene in the regulation of HSP83 RNA abundance during differentiation was analyzed using pXCATIRs-transfected parasites. As shown in Figure 8C, the relative abundance of cat transcripts varied in a similar manner to that of the HSP83 transcripts during the differentiation process. This suggests that the HSP83 3'-UTR contains the regulatory sequences responsible for the transient accumulation of the transcript.
A transient, preferential translation of HSP83 transcripts occurs during the promastigote-to-amastigote differentiation
In order to determine the involvement of regulatory cis-elements within the HSP83 UTRs, we analyzed de novo synthesis of proteins in an L. infantum cell line transfected with construct pXIRPstCATIR (Fig. 9B). A 2-fold increase of de novo CAT synthesis was observed in this cell line after 3 h of differentiation. Similar increases were determined in cell lines transfected either with pXCATIRs or with pX5HisCATIR, but not in parasites transfected with pX5HisCAT3His (data not shown), indicating that this preferential translation that occurs after 3 h of differentiation is linked to the presence of the HSP83 3'-UTR. However, it must be noticed that the increase of CAT synthesis was found to be lower than the increase determined for the endogenous HSP83.
The parallelism between accumulation of HSP83 transcripts (Fig. 8A) and de novo HSP83 synthesis (Fig. 9A) during the promastigote-to-amastigote differentiation suggest that both processes are linked in the same molecular mechanism.
The lack of transcription initiation control in kinetoplastid parasites reveals the importance of specific post-transcriptional processes in regulation of gene expression [5, 6]. The heat shock genes have been classically studied as excellent models to study gene regulation in a broad range of organisms, including Leishmania . The key advantage of this system is that the expression of HSP genes can be induced experimentally in promastigote cultures by raising the temperature. Results shown in this work revealed that L. infantum HSP83 gene expression is regulated post-transcriptionally by molecular mechanisms operating at two levels: i) accumulation of transcripts at 37°C; and ii) enhanced translation of HSP83 during heat shock. The stabilization of HSP83 transcripts in L. infantum during heat shock depended on active protein synthesis, suggesting that a temperature-activated protein factor is involved. This result is in contrast with a previous report in L. amazonensis in which, even though it was found that HSP83 transcripts accumulated upon temperature, the stabilization of the transcripts at elevated temperatures seemed the result of a heat-shock dependent, blocking effect on a specific mRNA degradation apparatus that is very active at 26°C . Therefore, some differences in the stabilization mechanism of the HSP83 mRNA may exist for these two Leishmania species. Remarkably, the regulation of the HSP83 RNA stabilization was found to be similar to that described for the regulation of L. infantum HSP70 gene expression , suggesting that a common mechanism could be responsible for the regulation of both heat-shock genes in L. infantum. Another observation made in this study is that the temperature-dependent accumulation of L. infantum HSP83 transcripts is regulated by sequences located at the 3'-UTR. This data is in agreement with the studies in L. amazonensis that show that HSP83 mRNA stability is conferred by a sequence element in the 3'-UTR . There is a growing number of Leishmania genes whose mRNA levels are stage-specific regulated by cis-elements within the 3'-UTRs [21, 22, 24, 28].
The second level of regulation of HSP83 gene expression is through a translational control. As shown in this work and in other reports [8, 11], translation of heat shock transcripts in Leishmania increases dramatically upon temperature elevation. Similar to the process of HSP83 mRNA stabilization during heat shock, sequences located in the 3'-UTR are needed to confer preferential translation to HSP83 transcripts. Also, the 5'-UTR of HSP83 mRNAs has an effect on the translational efficiency of these transcripts, even though it can be replaced with a 5'-UTR from a different gene. Thus, we have found that the presence of the L. infantum HSP83 or H2A 5'-UTR in the cat transcripts significantly increases the translation rate of the CAT protein (Fig. 6). In conclusion, our data and those of Zilka et al.  support a model in which the enhanced translation at elevated temperatures of Leishmania HSP83 transcripts is driven by sequences located exclusively in the 3'-UTR. There are several examples of mRNAs that are regulated at the translation level via elements in the 3'-UTR. It has been demonstrated for different organisms ranging from viruses to humans that the interaction of RNA-binding proteins with 5'- and 3'-UTRs of the mRNAs leads to an "RNA circularization" that increases translational efficiency; this structure allows a 3'-UTR-mediated translational control [reviewed in  and ].
The observation that the steady-state level of HSP83 transcripts varies during the axenic promastigote-to-amastigote differentiation is especially interesting. A rapid increase in the HSP83 RNA level was observed after 2 h of differentiation, but after 9 h the amount of HSP83 transcripts decreased to the initial level. After 24 h of differentiation, the HSP83 RNA level decreased further to levels lower than those present in promastigote cultures. This variation in the HSP83 RNA levels seems to be a consequence of incubating the parasites in differentiation growth conditions (37°C and pH 5.5), since promastigotes heat-shocked at 37°C along a 24-h period showed a continuously increased in the amount of HSP83 transcripts. The variations in HSP83 mRNA levels in the early steps of the differentiation process were accompanied by a drastic fluctuation in the de novo synthesis of HSP83 (Fig. 9). Thus, an 8-fold increase in the synthesis of HSP83 was observed after 3 h of differentiation relative to that observed in the promastigote forms. However, the rate of synthesis dropped to the initial level at 9 h of differentiation and was 4-fold lower after 24 h of differentiation than in promastigotes. In addition, it was demonstrated that the transient accumulation of HSP83 transcripts and the fluctuations in the de novo HSP83 synthesis during the promastigote-to-amastigote differentiation are driven also by the HSP83 3'-UTR. The parallelism in the regulation of Leishmania HSP83, both in heat shock and in differentiation, reinforce the general believe that temperature is a factor for differentiation in Leishmania .
An interesting question raised by the present data is whether the fluctuations in HSP83 gene expression observed during the axenic differentiation were reflecting a physiological reality or were only an experimentally induced artifact. Following experimental procedures of differentiation, in which environmental conditions that mimic the in vivo process are used [2, 3], we differentiate L. infantum promastigotes into parasite forms that are morphologically similar to amastigotes. In addition, these axenic amastigotes express A2 transcripts, a product of the A2 gene family that is detectable only in fully differentiated amastigote forms [22, 26]. Assuming that the amastigote-like forms obtained in this work represent truly L. infantum amastigotes, it is likely that the variations in HSP83 RNA and protein levels are relevant for the differentiation process of the parasite. In an outstanding work, Wiesgigl and Clos  showed that inactivation of L. donovani HSP83 (named HSP90 in that work) by geldanamycin or radicicol induces the differentiation from the promastigote to the amastigote stage. Thus, the decrease both in HSP83 RNA and in preferential translation of HSP83 transcripts observed after 9 h of differentiation could be a regulated cellular event that triggers the promastigote-to-amastigote differentiation. This finding supports the hypothesis that indicates that HSP83 homeostasis controls stage differentiation in Leishmania . Increasing experimental evidence suggests that heat shock proteins HSP70 and HSP90 (=HSP83) interact with multiple key components of signaling pathways that regulate growth and development [31, 32]. In particular, HSP90 is a molecular chaperone associated with the folding of signal-transducing proteins, such as steroid hormone receptors and protein kinases [33, 34]. Pharmacologically impairment of the HSP90 has demonstrated that this protein is involved in several developmental pathways [35, 36], leading to the suggestion that HSP90 is an evolutionary capacitor . A revealing example of how fluctuations in HSP90 levels can affect a signal transduction pathway is found in the interaction of HSP90 with Ras/Raf-1 [ and references therein], a signaling pathway involved in cell proliferation and differentiation. The current model for HSP90/Raf-1 interaction suggests that HSP90 is essential for maturation of Raf-1, but it needs to be released for activation of Raf-1. Thus, naturally occurring variations in the expression levels of HSP90 may serve as a triggering factor for Raf-1 activation. In the case of Leishmania, taking into account the HSP90/Raf-1 interaction model and the HSP83 inhibition experiments in L. donovani , we can hypothesize that the variation observed in the levels of newly synthesized HSP83 during the promastigote-to-amastigote differentiation is a triggering factor of the Leishmania developmental program. Thus, a simplified picture on the involvement of HSP83 in the differentiation process can be depicted as follows. Initially, the heat stress, experienced by the promastigotes after transmission to the mammalian host, induces the synthesis of HSP83, which is needed for stabilizing partially denatured or newly synthesized regulatory proteins. Then, the inhibition of the HSP83 synthesis by an amastigote-specific mechanism leads to a decrease in chaperone capacity of the cell and, consequently, to the liberation of cellular regulators in an active conformation that triggers the differentiation program. Future studies based on this framework will help to elucidate the molecular role played by HSP83 (and other HSPs) in the Leishmania differentiation.
Parasites and treatments
L. infantum promastigotes (MCAN/ES/96/BCN150) were cultured at 26°C in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco), adding penicillin G (100 U ml-1) and streptomycin (0.1 mg ml-1). Logarithmic phase cultures (5–9 × 106 promastigotes ml-1) were used in all the experiments described. For heat shock experiments, L. infantum promastigotes were incubated at different temperatures (37, 39 or 41°C) for different periods of time. For axenic differentiation of L. infantum promastigotes, late logarithmic phase cultures (1–2 × 107 cells ml-1) were transferred to RPMI 1640 medium (Gibco) titrated to pH 5.5 by addition of 20 mM succinate (Sigma); the medium was supplemented with 25% FCS, penicillin G (100 U ml-1) and streptomycin (0.1 mg ml-1). Finally, parasites were incubated at 37°C (5% CO2). Under these conditions, promastigotes differentiated to amastigote-like forms within 96 h.
Plasmid DNAs used in transfections were obtained with the Plasmid Maxy Kit (Qiagen, Hilden, Germany). Transfection of L. infantum promastigotes was essentially performed as described previously . Briefly, logarithmic phase promastigotes of L. infantum were harvested by centrifugation (1000 × g, 10 min) and resuspended at a density of 108 ml-1 in ice-cold EPB buffer (21 mM Hepes, pH 7.5, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, and 6 mM glucose). Afterwards, 0.4 ml of cell suspension (4 × 107 promastigotes) were put into a 0.2-cm electroporation cuvette and chilled on ice for 10 min. Thirty μg of plasmid DNA were added and the cells were pulsed two times (500 μF, 2.25 kV cm-1) using a Bio-Rad Gene Pulser with a capacitance extender module. Afterwards, samples were transferred to 10 ml of RPMI medium supplemented with 20% FCS and incubated for 24 h at 26°C. After incubation in medium lacking antibiotics, transfected cell lines were selected in the presence of 20 μg ml-1 of geneticin (G418; Roche Diagnostics, Mannheim, Germany). Stable cultures were obtained after 10–15 days of incubation at 26°C.
Northern blot analysis and nuclear run-on assays
Total RNA was isolated from L. infantum promastigotes using the Total Quick RNA Cells and Tissues kit (Talent, Trieste, Italy). Northern analysis of RNA samples was carried out by separation of RNA in 1% agarose-formaldehyde gels and transferred to nylon membranes using the Transfer Power Lid system (Hoefer, San Francisco, USA). DNA probes were labeled by nick-translation according to standard techniques . Hybridization was performed as reported earlier . For reuse, blots were treated with 0.1% SDS for 30 minutes at 95°C to remove the previously hybridized probes.
Nuclear run-on assays were performed as described  using freshly isolated nuclei of exponential-phase promastigotes. Nuclei were isolated from promastigotes grown at 26°C or additionally incubated at 37°C for 10, 30, and 60 min.
Mapping of RNA ends by RT-PCR
To determine the sites of poly(A)+ and spliced leader addition in the HSP83 transcripts, cDNA was generated by reverse transcription of total RNA using a (dT)18 primer that included the Eco RI restriction sequence (5'-CGGAATTCTT TTTTTTTTTT TTTTTT-3'). Reverse transcription steps were performed with MuLV reverse transcriptase (Perkin-Elmer Corp.) following the manufacturer's protocol (1 μg of total RNA per reaction). To determine the 3' boundary of the HSP83 transcript, a cDNA aliquot was PCR amplified using the following primers: forward, 5'-ATGTCCAAGA AGACGATGGC-3'; reverse, the (dT)18-attached primer (see above). To map the 5' end, the cDNA was PCR-amplified using the following primers: forward, 5'-GCTATATAAG TATCAGTTTC TGTAC-3' (derived from the Leishmania spliced leader sequence); reverse, 5'-TGCAGGTAAT ACTAATGGTT-3' (L. infantum HSP83 specific primer). PCR products were cloned into pSTBlue-1 vector (Novagen, Madison, WI) and sequenced to determine the HSP83 gene boundaries. The clone containing the 3' end of the L. infantum HSP83 gene was further digested with Pst I and religated in order to eliminate coding sequences, this clone was named pE2 and the Pst I-Sal I insert was used as an HSP83 3'-UTR specific probe.
The genomic clone gLiB1.2 , containing the 5'-end of the L. infantum HSP83 gene cluster, was digested with Sal I, and the 3.8-kb restriction fragment was cloned in pUC18 to generate clone 10S. This clone was used as template to PCR-amplify different regions of the L. infantum HSP83 gene. The relevant regions of each construct were confirmed by DNA sequencing.
In order to obtain plasmids pXCATIRs and pXCATIRas, clone 10S was digested with Bgl II and Bam HI and the 2.0-kb fragment was cloned, downstream from the cat ORF, into the Bgl II site of pXcat plasmid . This genomic fragment comprises the 3'UTR, IR and 5'UTR sequences located between the first and the second HSP83 genes of the L. infantum gene cluster. Both orientations were selected: sense (pXCATIRs) and antisense (pXCATIRas). It should be noted that pXcat is a pX63NEO derivative , in which the cat ORF was inserted in the Bam HI site of pX63NEO .
The following constructs were first generated in pBluescript KS (-) prior to subcloning into pX63NEO . In these constructs, cat ORF was PCR-amplified from plasmid pC4cat  using the following primers: forward, 5'-GCGATATC AT GGAGAAAAAA ATCACTGG-3', and reverse, 5'-CCCAAGC TT A CGCCCCGCCC TGCCACT-3' (the sequence coding for the cat termination codon is indicated in italics). The PCR product was double digested with Eco RV+Hin dIII and cloned into pBluescript to generate plasmid pBlCAT. The 3'UTR region of HSP83 gene was amplified using the primers: forward, 5'-CCAAGCTT AG GTGGACTGAG CCGGTA-3', and reverse, 5'-GCGTCGACG GATCC GCGGG TTATTGCTTT GCATT-3'. The PCR product was double digested with Hin dIII+Sal I and cloned into pBlCAT to generate plasmid pBlCAT3H. Finally, the cat-HSP83 3'-UTR insert was excised from pBlCAT3H as a Bam HI fragment and subcloned into the corresponding site of pX63NEO to generate the construct pXCAT3H.
The region 3'UTR+IR+5'UTR located between coding regions of genes 1 and 2 of the HSP83 gene cluster was amplified with different pairs of primers in order to orientate the cloning. Thus, when this region was to be placed upstream the cat ORF, the following primers were used: forward, 5'-CGGGATCC CG CGCACTGCTC TTTACATT-3', and reverse, 5'-TGCCTGCAG C GTCTCCGTCA TGGTTGCAG-3'. After digesting the PCR product with Bam HI+Pst I, the 1407-pb band was cloned into pBlCAT to generate pBl-IRCAT. On the other hand, when this region was to be placed downstream the cat ORF, the following primers were used: forward, 5'-CCAAGCTT AG GTGGACTGAG CCGGTA-3', and reverse, 5'-GCGTCGACGG ATCC GCTCTC CGTCATGGTT GCAG-3'. The PCR product was digested with Hin dIII+Sal I and cloned into pBl-IRCAT; after digestion with Bam HI, the chimeric gene was cloned into pX63NEO to generate plasmid pXIRPstCATIR.
Control constructs were generated using regulatory regions derived from the L. infantum H2A gene cluster . The 5'UTR along with upstream sequences were PCR-amplified using as a template the clone pS4KHE; this clone contains the H2A4 gene (GenBank™, EMBL and DDBJ accession number AJ419627) and was obtained by insertion of an Eco RI+Hin dIII genomic fragment, derived from L. infantum locus 2, into pBluescriptII. For amplification, the following primers were used: 5'-GTAAAACGAC GGCCAGT-3' (M13/pUC Sequencing Primer (-20), New England Biolabs, Beverly, MA), and 5'-GCGATATC CA TGGCTGCGAT GGGTAGGT-3' (derived from L. infantum H2A4 gene). The 590-bp PCR-product was digested with Bam HI+Eco RV and cloned into pBlCAT to generate clone pBl5HisCAT. Afterwards, the 3'UTR+IR+5'UTR fragment derived from HSP83 gene cluster (see above) was cloned downstream cat gene to generate clone pBl5HisCATIR. Finally, the chimeric gene was excised by Bam HI digestion and inserted into pX63NEO to yield the construct pX5HisCATIR.
The 3'UTR and downstream sequences derived from L. infantum H2A4 gene was PCR-amplified using the following primers: forward, 5'-CCATCGAT GT CCTCCGGCCT GACAGCGC-3'; and reverse, 5'-GCGTCGACGG ATCC CATGGT TGCGGAAAGG AGAC-3'. The 639-bp PCR-product was double digested with Cla I+Sal I and cloned into pBl5HisCAT to generate clone pBl5HisCAT3His. Finally, the insert was excised by Bam HI digestion and cloned into pX63NEO to generate the construct pX5HisCAT3His. Further details on the sequence of the plasmids can be obtained from the authors upon request.
After appropriate treatment, 3 × 107 parasites were collected and resuspended in 100 μl of DMEM without methionine (Dulbecco's Modified Eagle Medium, Met-; Gibco, Paisley, UK) supplemented with 10% of FCS. Parasites were pre-incubated at different temperatures or pHs for 30 min and then labeled with 100 μCi of [35S]methionine/cysteine protein labeling mix (Redivue Pro-mix L-[35S], >1000 Ci mmol-1; Amersham, Aylesbury, UK) and incubated 2 h further in the same conditions. After labeling, cells were harvested, washed twice with phosphate-buffered saline, and lysed in SDS-polyacrylamide gel sample buffer. Incorporation of 35S was measured using the Liquid Scintillation Counter 1209 RacKbeta (Wallac, Turku, Finland). Samples of proteins corresponding to a similar number of cells were separated over 10% SDS-polyacrylamide gels.
For immunoprecipitation of CAT, parasites (6 × 107 cells) were harvested by centrifugation, washed with PBS and treated with 100 μl of lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and a protease inhibitor cocktail (1 mM PMSF, 8 μg ml-1 of leupeptin, 4 μg ml-1 of pepstatin and 4 μg ml-1 of aprotinin). The mixture was incubated at 4°C for 30 min with gentle shaking, and sonicated for 10 min. The lysates were microcentrifuged at 4°C for 15 min. Seventy-five μl of soluble proteins were incubated with 60 μg of rabbit anti-CAT serum (Sigma-Aldrich, St. Louis, MO) for 12 h at 4°C with gentle shaking. Fifteen μl of Protein A-agarose slurry (Sigma-Aldrich) were equilibrated in 50 μl of lysis buffer and added to the Leishmania extract-CAT antiserum mixture. After incubation on an orbital rotator for 1 h at 4°C, the beads were collected by centrifugation and washed three times in 0.4 ml of buffer A (10 mM Tris-HCl (pH 8.0), 30 mM NaCl, and 2% Triton X-100), two times in 0.4 ml of buffer B (10 mM Tris-HCl (pH 8.0), 50 mM NaCl, and 0.1% Triton X-100) and one time in 0.4 ml of buffer C (10 mM Tris-HCl (pH 8.0) and 0.05% Triton X-100). Finally, the beads were resuspended in 60 μl of Laemmli's buffer. Immunoprecipitated proteins were resolved by SDS-PAGE on acrylamide gels.
For immunoprecipitation of Leishmania HSP83, the following modifications were introduced: (a) the lysis buffer contained 50 mM Tris-HCl (pH 8), 10 mM EDTA (pH 8), 1% SDS, and the above indicated protease inhibitors; (b) after lysis, 75 μl of soluble proteins were mixed with 675 μl of ChIP buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl, and the protease inhibitor cocktail) and 20 μl of anti-HSP83 rabbit serum .
Coomassie blue-stained gels and autoradiographs were scanned with the GS-710 Calibrated Imaging Densitometer (Quantity One version 4.3.1; BIO-RAD). Measurements were performed under conditions in which a linear correlation existed between the amount of proteins or RNA and the intensities of the bands.
List of abbreviations
heat shock protein
open reading frame
This work was supported by Spanish Ministerio de Ciencia y Tecnología Grant BMC2002-04107-C02-01. Also, an institutional grant from Fundación Ramón Areces is acknowledged.
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