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. 2004 Jul;9(3):294–303. doi: 10.1379/CSC-15R1.1

The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite Leishmania

Christian Brochu 1, Anass Haimeur 1,*, Marc Ouellette 1,1
PMCID: PMC1065288  PMID: 15544167

Abstract

Antimony-containing drugs are still the drugs of choice in the treatment of infections caused by the parasite Leishmania. Resistance to antimony is now common in some parts of the world, and several mechanisms of resistance have been described. By transfecting cosmid banks and selecting with potassium antimonyl tartrate (SbIII), we have isolated a cosmid associated with resistance. This cosmid contains 2 copies of the heat shock protein 70 (HSP70) and 1 copy of the heat shock cognate protein 70 (HSC70). Several data linked HSP70 to antimony response and resistance. First, several Leishmania species, both as promastigotes and amastigotes, increased the expression of their HSP70 proteins when grown in the presence of 1 or 2 times the Effect Concentration 50% of SbIII. In several mutants selected for resistance to either SbIII or to the related metal arsenite, the HSP70 proteins were found to be overexpressed. This increase was also observed in revertant cells grown for several passages in the absence of SbIII, suggesting that this increased production of HSP70 is stable. Transfection of HSP70 or HSC70 in Leishmania cells does not confer resistance directly, though these transfectants were better able to tolerate a shock with SbIII. Our results are consistent with HSP70 and HSC70 being a first line of defense against SbIII until more specific and efficient resistance mechanisms take over.

INTRODUCTION

The protozoan parasite Leishmania is responsible for several pathologies ranging from cutaneous lesions to visceral infections that can be fatal if untreated (Herwaldt 1999). The incidence of leishmaniasis increases, and its control is further exacerbated by the high incidence of resistance to pentavalent antimony (SbV)–containing drugs, which are still the mainstay in the treatment of Leishmania infections in most parts of the world (Murray 2001; Guerin et al 2002; Berman 2003). Miltefosine is now used in treating leishmaniasis in parts of India, but resistance to miltefosine, at least in vitro, can readily occur (Perez-Victoria et al 2003), suggesting that SbV is likely to remain an important drug in the control of Leishmania infections. The mode of action of SbV is poorly understood, but it is believed that SbV is a prodrug that needs to be reduced to trivalent antimony (SbIII), which would be the toxic species against Leishmania. It is not known yet whether most of the reduction occurs in the parasite or in the host cell (Sereno et al 1998; Shaked-Mishan et al 2001). Analysis of mutants selected for resistance to SbIII and to its related metal arsenite (AsIII) led to the development of a model for metal resistance. In this model, SbV and SbIII appear to enter Leishmania cells by means of different transporters (Brochu et al 2003), and mutations in one of these could lead to resistance. As part of the detoxification process, the metal would be conjugated to trypanothione, a spermidine-glutathione conjugate specific to kinetoplastidae (Fairlamb and Cerami 1992), which is increased in resistant cells by virtue of the amplification or overexpression (or both) of genes coding for enzymes involved in glutathione or spermidine biosynthesis (Grondin et al 1997; Haimeur et al 1999; Guimond et al 2003). The Sb-trypanothione conjugate is then either sequestered inside a vacuole by the ATP-binding cassette transporter PGPA (Légaré et al 2001) or extruded from the cell by a thiol-X pump (Dey et al 1996).

Although the above model has received considerable support from in vitro work, it is possible that other resistance mechanisms operate in field isolates. In fact, it was suggested in a recent report that amplified deoxyribonucleic acid (DNA) derived from chromosome 9 might be involved in clinical SbV resistance (Singh et al 2003). Other mechanisms of resistance have also been described in antimony-resistant mutants (Haimeur and Ouellette 1998; Shaked-Mishan et al 2001). One powerful strategy to isolate novel resistance genes in Leishmania is functional cloning. This technique was useful to isolate transporters involved in resistance to nucleoside (Vasudevan et al 1998), folate (Kündig et al 1999), and phospholipid analogs (Perez-Victoria et al 2003) or soluble proteins mediating resistance to inhibitors of nucleoside and ergosterol metabolism (Cotrim et al 1999). This strategy was used here in an attempt to isolate novel genes mediating resistance to SbIII. Characterization of 1 cosmid, named X6, revealed the presence of genes coding for the homolog of the heat shock protein HSP70 and heat shock cognate protein HSC70.

Leishmania have a dimorphic life cycle and are found as promastigotes in the sandfly vector (25°C) and amastigotes in the mammalian macrophage (37°C). The Leishmania life cycle can be mimicked in vitro, and temperature is indeed an important trigger of differentiation (Zilberstein and Shapira 1994), and some HSP homologs were suggested to be important for Leishmania differentiation (Wiesgigl and Clos 2001). The HSP70s of Leishmania are part of a multigene family, with varying numbers in different species (Quijada et al 1997; Zurita et al 2003)(www.genedb.org). In contrast to most eukaryotes, the regulation of HSPs in Leishmania was suggested to occur exclusively at the post-transcriptional level (Hunter et al 1984). This was first demonstrated for the HSP83 gene (Argaman et al 1994), for several Leishmania HSP genes (Brandau et al 1995), and in details for HSP70 (Quijada et al 1997). Recently, gene transfection experiments have shown that HSP70 can bestow a heat-inducible increase in resistance to peroxide (Miller et al 2000). The role of HSPs in Leishmania in response to antimony is unexplored, with only 1 report showing that HSPs of varying molecular weight were increased in the presence of AsIII (Lawrence and Robert-Gero 1985). The isolation of a cosmid coding for HSP70 has allowed us to analyze the association between antimony resistance and HSP70 in Leishmania in more detail.

MATERIALS AND METHODS

Reagents

Potassium antimonyl tartrate (SbIII) was obtained from Aldrich. Sodium m-arsenite (AsIII) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were obtained from Sigma.

Cell lines and cultures

The Leishmania tarentolae cell lines TarII wild-type, As50.1, As20.3rev (selected for resistance to AsIII), and Sb400.4 (selected for resistance to SbIII) have been described previously (Ouellette et al 1991; Dey et al 1994; Haimeur et al 2000), as have L infantum strains MHOM/MA/67/ITMAP-263 and Sb4000.3 (selected for resistance to SbIII) (Brochu et al 2003). L tarentolae and L infantum strains were grown in SDM-79 (Brun and Schonenberger 1979) at 25°C as promastigotes and in MAA/20 for L infantum (Sereno et al 1998) at 37°C as axenic amastigotes.

Viability test

The MTT micromethod was used to estimate the viability of cells after heat or metal treatments (Sereno et al 1998). Briefly, cells in their exponential growth phase were grown in SDM-79 for 12 hours with 2 times the EC50 of SbIII (0.1 μM for L tarentolae and 225 μM for L infantum) or for 2 hours at 42°C. A 100-μL aliquot was transferred to a 96-well flat-bottom microtray, where 10 μL of MTT (7.5 mg/mL) was added to each well for 4 hours. The reaction was stopped by the addition of 100 μL of 50% isopropanol–10% sodium dodecyl sulfate. The plates were incubated for 30 minutes under agitation before observing the optical density at 570 nm. Statistical significance was calculated by the Fisher's protected least significant difference test using StatView SE + Graphic software v1.03.

DNA manipulations

The pSP72α-NEO-α vector (Papadopoulou et al 1994) transfected in the wild-type strains of L tarentolae or L infantum was used as a control in viability tests. Polymerase chain reaction fragments, using the cosmid X6 as template for the L tarentolae HSC70 (5′-GACGACCACTGCCGCAGAGATG-3′ and 5′-GTCCTTGCTCAGCCGGCCCTTG-3′), and the L tarentolae HSP70 (5′-CATCGATGAGCGTCACAGAG-3′ and 5′-ATGCACCGAGTTGCACAGTC-3′) were cloned in the expression vector pSP72α-NEO-α and were transfected in L tarentolae or L infantum. Pulse field gel electrophoresis of Leishmania chromosomes was done as described previously (Tamar and Papadopoulou 2001) using a Bio-Rad CHEF DR III apparatus.

Functional cloning

The genomic cosmid library, made from a partial Sau3AI digest of the DNA of the mutant As20.4, was described previously (Kündig et al 1999). L tarentolae promastigotes were transfected by electroporation (Papadopoulou et al 1992) using 20 μg of the cosmid library, and transfectants were grown in 5 mL of SDM-79 medium. After 24 hours of growth, an equal volume of fresh medium was added with hygromycin B (final concentration 300 μg/mL) and incubated for an additional 24 hours. The cells were transferred to a 15-mL conical tube, centrifuged at 2500 rpm at 4°C for 10 minutes, and resuspended in fresh SDM-79 medium at a density of 2.5 × 107 cells/mL. Aliquots (200 μL) were plated carefully on hygromycin B– containing plates, which were then sealed with parafilm and incubated at 29°C. Colonies usually appeared between 5 to 10 days of incubation. Colonies were scraped off the plates with a glass spreader in 1 mL of medium and resuspended in SDM-79 medium at a concentration of 5 × 106 cells/mL. Cells were grown for 24 hours in the presence of 1 μM of hygromycin B, and 5 × 106 cells were spread on plates containing SbIII (0.5 to 2.5 μM) and hygromycin (300 μM) and incubated at 29°C. Colonies appearing after 2 to 3 weeks of incubation were then picked and grown individually in 5 mL of SDM-79 medium with hygromycin at 300 μM. Cosmids were isolated as previously described (Kündig et al 1999).

DNA sequencing

DNA sequencing was carried out on an Applied Biosystem 377 automated sequencer. Analysis of the sequences was performed using the GCG software package (Genetics Computer Group, 1994) and PAUP* 4.0 (Swofford 2003) The nucleotide sequences reported here will appear in the GenBank sequence database under the accession numbers AY423867 for L tarentolae HSP70 and AY423868 for L tarentolae HSC70. The following sequences, with their accession number in parentheses, were used for the phylogenetic analysis: L donovani HSP70 (X52314), L infantum HSP70 (X85798), L braziliensis HSP70 (AF291716), Trypanosoma brucei HSP70 (P11145), T brucei HSC70 (P20030), Human HSP70 (P08107) and Escherichia coli DNAK (P04475). The L major HSP70 (LmjF28.2780) and L major HSC70 (LmjF28.2820) sequences are available at www.genedb.org.

Immunoblotting

Cells were grown to log-phase then harvested and lyzed by sonication on ice 3 times, (30 seconds of sonication, followed by a 30-second recovery) and diluted in sodium dodecyl sulfate (SDS) containing Laemmli buffer. Equal amounts of total proteins were loaded on a 10% SDS– polyacrylamide gel electrophoresis gel followed by immunoblotting using anti-HSP70 and anti–α-tubulin antibodies (Harlow and Lane 1988). The reaction was detected by using the enhanced chemiluminescence method (Amersham). The mouse monoclonal antibody anti–heat shock protein 70 Mab (5A5) (Alexis Biochemical) reacts with HSP70 members across a broad range of species, including those from Leishmania. It recognizes an epitope located between amino acids 122–264 of human HSP70, a very conserved region shown to be involved in adenosine triphosphate (ATP) binding. The mouse monoclonal antibody anti–α-tubulin (bovine) 236–10 501 (Molecular Probes) was also used as a control. The intensity of the reactions was determined by densitometry analysis, using an alphaImager 2000 (Alpha Innotech) with the software AlphaEase version 3.3b and compared with the signal intensity of α-tubulin.

RESULTS

Isolation of an HSP70-containing cosmid by functional cloning in Leishmania

To isolate SbIII resistance genes, we transfected in wild-type L tarentolae cells a cosmid bank of genomic DNA derived from As20.4, an AsIII-selected mutant highly cross-resistant to SbIII (Dey et al 1994). Transfectants were selected on SbIII-containing plates, and a few colonies were grown on plates with 2.5 μM SbIII. Five different cosmids were isolated from several transfectants, whereas the same cosmids were isolated from different colonies. Only 2 of these cosmids, termed “XS” and “X6”, were still associated with resistance on retransfection (A. Haimeur and M. Ouellette, personal communication). The gene on cosmid XS is currently under investigation, and the retransfection of cosmid X6, isolated from 1 of these colonies, in wild-type L tarentolae conferred a 25-fold increase in resistance to AsIII (10 μM) and more than a 600-fold resistance to SbIII (60 μM) (Table 1). The link between X6 and resistance to SbIII was not straightforward, however, because all further attempts to isolate the episomal cosmid from secondary transfectants failed. Instead, it was found that the cosmid had integrated in the genome of these highly resistant selected cells. We found that in the initial transfectant, the cosmid X6 was both existing as an extrachromosomal circle and integrated in the genome (not shown). The transfection of X6 repeatedly gave SbIII-resistant colonies on selective plates, and despite its integration in the genome, we reasoned that a gene present on this cosmid might contribute to the metal resistance phenotype. Cosmid X6 was partially mapped and sequenced and found to contain 2 copies of the HSP70 gene. The evidences for integration of the cosmid (or part of it) in the chromosome encoding HSP70 are provided in Figure 1. One large molecular weight L tarentolae chromosome hybridized to the HYG probe in transfectant X6, whereas no bands hybridized in the wild-type cells (Fig 1A, lanes 1 and 2). Extrachromosomal circles, as shown in cells transfected with the control cosmid cLHyg, do not migrate similarly and usually give rise to smears (Fig 1A, lane 3) representing different forms of circular DNA (White et al 1988; Grondin et al 1993). A similar CHEF blot run at the same time was hybridized to an HSP70 probe. One high–molecular weight chromosome hybridized to the HSP70 probe in the wild-type cell (Fig 1B, lane 2), whereas the same band and 1 slightly higher hybridized in transfectant X6 (Fig 1B, lane 1). It is this higher band that also hybridized to the HYG probe. To further prove that the transfected cosmid (or part of it) integrated in the L tarentolae chromosome harboring the HSP70 locus, a similar blot was hybridized to the Leishmania-activated protein kinase C receptor gene (LACK) that is, according to the L major genome, ∼8 kb upstream of the HSP70 genes on L major chromosome 28 (www.genedb.org). The same hybridization profile was seen with the LACK probe as with the HSP70 probe (Fig 1 B,C), showing that the cosmid had indeed integrated in the HSP70-coding chromosome. The integration of cLHyg increases the size of the chromosome 28 version of L tarentolae. This could be the result of the integration of the whole cosmid, hence increasing the copy number of HSP70. This does not seem to be the case, however, because the copy number of HSP70, as determined by Southern blot analysis, is not drastically changed in the X6 transfectant compared with wild-type cells, although some minor rearrangements did occur, as suggested by the presence of additional hybridizing bands (Fig 1D). The HYG gene and plasmid sequences were clearly integrated in the Leishmania genome (Fig 1 E,F). Multiple bands hybridized with these markers (Fig 1 E,F, lanes 4 and 6), suggesting that the rearrangement probably selected during drug selection is complex, but contained mostly plasmid-derived sequences.

Table 1.

 Susceptibility to metals in Leishmania

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Fig 1.

Fig 1.

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 Chromosomal integration of the cosmid X6 and its characterization in antimony-resistant transfectant. Leishmania cells were isolated and their chromosomes resolved by pulse field gels. Deoxyribonucleic acid (DNA) was transferred and hybridized to an HYG (probe a) (A), HSP70 (probe b) (B), and LACK (C) probes. LACK and HSP70 are linked on chromosome 28 of Leishmania major (www.genedb.org). 1, L tarentolae cells retransfected with X6 and selected for resistance to SbIII; 2, L tarentolae wild-type cells; and 3, L tarentolae cells transfected with the cosmid cLHYG (Ryan et al 1993). The smear in lane 3 is indicative of the episomal nature of the transfected DNA. The integration event was further studied by Southern blot analysis of total genomic DNA hybridized to HSP70 (D), HYG (E), and vector sequences (probe b) (F). The DNA was derived from wild-type cells (odd numbers) or the X6 retransfectant (even numbers) and digested with XbaI, 1, 2; NotI, 3, 4; BglII, 5, 6. (G) A partial map of cosmid X6 was determined using a selection of restriction enzymes. The equivalent region in the L major genome (taken from www.genedb.org) is found below the map of the cosmid. Arrows without gene names indicate hypothetical proteins. The location of the probes (a,b,c) is indicated below the map. Only the HSPs were sequenced in cosmid X6. B, BglII; H, HindIII; N, NotI, X, XbaI

The cosmid X6 was further characterized, and 8.5 kb downstream of HSP70, a copy of the more distantly related HSC70 was found (Fig 1G). The HSP70 gene is 1971 base pairs and encodes a putative protein of 657 amino acids for a predicted molecular weight of 71.6 kDa. The protein shares more than 96% identity with the HSP70 of L major, L donovani, and L infantum and is also very close to HSP70 of T brucei (87.8%) and humans (73.2%) (Fig 2). The other gene, HSC70, is 1992 base pairs and encodes a putative protein of 664 amino acids for a predicted molecular weight of 72.4 kDa. According to the phylogenetic tree (Fig 2A), L tarentolae HSC70 is distinct from the HSP70 proteins. It is highly related (89.4% identity) to a new copy of a HSP70-related gene on chromosome 28 of L major and to the T brucei HSC70 (Fig 2). The L tarentolae HSC70 shares 58% identity with the T brucei HSC70 but less than 50% identity with the different HSP70s of Leishmania sp. The N-terminal domain, including the ATP-binding domain, is very conserved between the L tarentolae HSC70 and the other HSP70 proteins, with most differences located in the C-terminal 10 kDa of the protein.

Fig 2.

Fig 2.

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 Phylogenetic analysis and sequence conservation of the Leishmania HSP70 protein. (A) Phylogenetic analysis was performed using the GCG software package and PAUP*4.0 and the amino acid sequences of various members of the HSP70 family. (B) Pairwise identities (% identity) among different proteins. L tarentolae HSP70 (LTHSP70), L tarentolae HSC70 (LTHSC70), L major HSP70 (LMHSP70), L major HSC70 (LMHSC70), L donovani HSP70 (LDHSP70), L infantum HSP70 (LIHSP70), L braziliensis HSP70 (LBHSP70), Trypanosoma brucei HSP70 (TBHSP70), T brucei HSC70 (TBHSC70), human HSP70 (HHSP70), and Escherichia coli DNAK (DNAK)

Induction of HSP70 by SbIII in Leishmania

To look further into the role of HSP70 and antimony resistance, we tested whether antimony could induce the expression of the HSP70 proteins. To monitor HSP70 expression, we used a commercial monoclonal antibody (Mab 5A5) that reacts with HSP70 members of a broad range of species because it recognizes a highly conserved epitope located in the ATP-binding domain of HSP70. This antibody can react equally well to HSP70 and HSC70 of L tarentolae (Fig 3A), both having the conserved ATP-binding domains. We also observed that the HSP70-HSC70 proteins were overproduced in the X6 transfectant with its integrated cosmid (Fig 3B). We showed that the L tarentolae HSP70 (and or HSC70) was induced by several fold (>4) when cells were incubated for 12 or 24 hours with either 1 or 2 times the EC50 of SbIII (Fig 3C). Similarly, the HSP70s of L infantum promastigotes were also induced dramatically (>4-fold) on shock with the EC50 of SbIII (Fig 3D). The induction of HSP70s was more rapid in L infantum promastigotes compared with L tarentolae because we could not observe induction of HSP70s in the latter species after a 2-hour shock (results not shown). L infantum can be grown as axenic amastigotes, and although the basal level of HSP70s is higher in these cells that are grown at 37°C, a shock with SbIII can also increase (∼2-fold) the level of HSP70s (Fig 3E). Results presented in Figure 3C–E clearly demonstrate that on SbIII exposure, Leishmania cells respond by increasing their HSP70s.

Fig 3.

Fig 3.

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 Analysis of HSP70 expression on shock with potassium antimonyl tartrate (SbIII). (A) Western blots showing expression of HSP70 in Leishmania tarentolae transfected with pSP72α-NEO-α (ctrl), pSP72α-NEO-α-HSC70 (HSC70), or pSP72α-NEO-α-HSP70 (HSP70). (B) Western blots showing the increase in expression of HSP70 in the X6 transfectant with the integrated cosmid. (C–E) HSP70 and α-tubulin immunoblots of Leishmania after an SbIII exposure. Wild-type L tarentolae (C), L infantum in the promastigote stage (D), or in the amastigote stage (E) were incubated in the presence of 1 or 2 times the EC50 of SbIII for 0, 2, 12, or 24 hours. The EC50 for L tarentolae is 0.1 μM, 225 μM for L infantum promastigotes, and 60 μM for L infantum amastigotes. Total proteins were extracted and then reacted with monoclonal antibodies directed against HSP70 or to α-tubulin. The approximate fold increase in HSP70 expression was adjusted with the α-tubulin signal. One experiment representation of at least 3 replicates is shown

HSP70 and SbIII resistance

We next tested the status of HSP70s in metal-resistant mutants of Leishmania. We first studied L tarentolae selected for either AsIII or SbIII resistance. These analyses were done with cultures that were passaged at least twice without drugs to discriminate between an induction of HSP70 due to the presence of the drug and a stable HSP70 overexpression correlated with resistance. The HSP70s of L tarentolae cells selected for arsenite or antimonite resistance were increased compared with wild-type cells (Fig 4, lanes 1, 3, and 4). In the mutants As50.1 and Sb400.4, HSP70s were increased more than 4-fold compared with wild-type cells. Remarkably, HSP70s were still higher in the L tarentolae mutant As20.3rev (Fig 4, lane 2) (∼4-fold compared with wild-type cells), a mutant grown in the absence of arsenite for several hundreds of passages. This revertant has an EC50 of 5 μM for arsenite, which is considerably higher than that for wild-type cells (0.4 μM) but nonetheless lower than that for the parent mutant (50 μM). The overexpression of HSP70s thus appears to be a stable phenotype in these revertant cells. Similarly, HSP70s were slightly increased in the L infantum mutant selected for SbIII resistance (Fig 4, lanes 5 and 6).

Fig 4.

Fig 4.

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 HSP70 overexpression in Leishmania metal-resistant mutants. All mutants were grown without drugs for at least 2 passages, and then total proteins were extracted, resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel (SDS-PAGE) gels, transferred and then reacted with monoclonal antibodies directed against HSP70 or to α-tubulin. 1, Leishmania tarentolae wild-type; 2, L tarentolae As20.3rev; 3, L tarentolae As50.1; 4, L tarentolae Sb400.4; 5, L infantum wild-type; 6, L infantum Sb4000.3. The approximate fold increase in HSP70 expression was adjusted with the α-tubulin signal. One representative experiment of 3 independent experiments is shown

HSP70s are increased on SbIII shock and in SbIII-resistant mutants. To test further the link between HSP70s and resistance and in an attempt to discriminate whether the observed phenotype is due to either HSP70 or HSC70, we cloned individually the 2 genes in Leishmania expression vectors. These constructs were transfected independently, and both HSP versions were overexpressed as determined by Western blot analysis (Fig 3A). We then performed growth curves in the presence of varying concentrations of SbIII but found no increase in resistance compared with control cells transfected with the vector alone (result not shown). Similar negative results were obtained when AsIII was tested. We also cotransfected both genes (as part of NEO and HYG-containing vectors) in a wild-type cell and also found no increase in SbIII resistance. We thus looked for more subtle ways by which HSP70s could contribute to metal tolerance. To do so, we compared the viability of transfected cells overexpressing or not overexpressing HSP70s after a brief shock with SbIII. In this assay, we found that the L tarentolae HSP70 and HSC70 protein conferred a low but significant protection to an SbIII shock compared with control cells (Fig 5). When transfected in L tarentolae, HSC70 and HSP70 conferred 21–29% increased viability after 12 hours of SbIII exposure to either 1 or 2 times the EC50 of SbIII (0.1 and 0.2 μM) compared with the control cells. Despite the higher basal level of HSP70 expression in L infantum, overexpression of both HSP70 and HSC70 conferred a low (10–25%) but significant protection against a SbIII shock (Fig 5). HSP70s are expected to provide heat tolerance (reviewed in Kiang and Tsokos 1998), although this has never been formally reported for Leishmania. We found that transfectants overexpressing HSP70 and HSC70 had increased viability after a heat shock at 42°C, with a 60–100% increase in survival compared with control cells (Fig 5).

Fig 5.

Fig 5.

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 The role of the Leishmania HSC70 and HSP70 proteins in heat and metal protection. Viability of Leishmania cells transfected with the control plasmid pSP72α-NEO-α (in black), by pSP72α-NEO-α-HSC70 (in gray), and by pSP72α-NEO-α-HSP70 (in white) after an incubation of 2 hours at 42°C, or a 12-hour incubation to SbIII at a concentration corresponding to 1 or 2 times the EC50 for SbIII (0.1 and 0.2 μM for Leishmania tarentolae and 450 μM for L infantum). The standard error is represented for each experimental condition; 100% corresponds to the viability of cells without treatment. The viability was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) method as described in Materials and Methods. Each experiment was done at least 4 times. Results that are significantly different (P < 0.05 as calculated by the Fisher's protected least significant difference test) are marked with asterisks. Bars with different number of asterisks are statistically different among each other

DISCUSSION

By functional cloning we have isolated a cosmid that harbors 2 copies of HSP70 and 1 copy of HSC70 (Fig 1G). The copy number of HSP70 genes varies considerably among the various Leishmania species. In L infantum, a cluster of 6 HSP70 genes has been described, with the most distal gene expressed constitutively at high levels (Quijada et al 1997). The L major genome sequence (www.genedb.org) has revealed a multitude of HSP70-like proteins and other homologs. It is possible that the HSP70 cluster of L tarentolae is larger, but this is unlikely because the L major sequence on chromosome 28 has also revealed 2 copies of HSP70 and 1 copy of an HSC70. Interestingly, however, the gene order between L major and L tarentolae seems to differ (Fig 1G). The reason why the copy number of HSP70 differs greatly in different Leishmania species is unknown, from 1 in L Viannia braziliensis (Zurita et al 2003) to 6 in L infantum (Quijada et al 1997), but does not seem to relate to the temperature that Leishmania species are likely to encounter. Despite some differences in copy number and organization, the Leishmania HSP70 members, including the new L tarentolae sequences presented here, are highly conserved with more than 96% identity (Fig 2). The HSC70 proteins form a separate phylogenetic branch and are highly homologous among one other but show only 40–45% identity with the various Leishmania HSP70 members (Fig 2). In systems in which it was studied, the HSC70 gene and protein are expressed constitutively, although this was difficult to assess in L tarentolae using the antibody available. Most sequence differences between HSP70 and HSC70 were localized in the last 10 kDa of the protein, a region known in HSC70 to be involved in the stability of the bound unfolded polypeptides and possibly in self-association (Chou et al 2003).

The HSP70 and HSC70 proteins are ATP-dependent molecular chaperones assuming diverse essential cellular functions, including the assembly, folding, and translocation of oligomeric proteins (reviewed in Hartl and Hayer-Hartl 2002). Under a variety of stresses, including heat, radiation, infection, chemical or biochemical stress, induction of HSP70s can provide cytoprotection, possibly by binding to misfolded proteins (Morimoto and Santoro 1998; Hartl and Hayer-Hartl 2002). HSP70s were also found to be immunodominant molecules of many microbial pathogens and are a major target of the humoral immune response during Leishmania infections (Quijada et al 1998). Although the inducible expression of HSP70s can lead to cross-resistance to a selected number of toxic substances, this has been less studied in Leishmania. Leishmania cycles from an insect vector (25°C) to its mammalian host (37°C), and this temperature shift is a likely triggering factor of their developmental program (Zilberstein and Shapira 1994). Several of the Leishmania HSP70 transcripts are increased on heat exposure (Quijada et al 1997), and in contrast to most other organisms, this control is at the posttranscriptional level and is controlled by the 3′–untranslated region of the gene (Hunter et al 1984; Brandau et al 1995; Quijada et al 2000). Similar regulation has also been described in the related parasite T brucei (Lee 1998). It is likely that an increase in HSP70 amounts leads to heat protection, although this appears to be shown formally for the first time using transfected genes in Leishmania in Figure 5. Interestingly, both HSP70 and HSC70 can provide this increased heat tolerance. It has been shown previously that transfection of HSP70 leads to a heat-inducible increase in resistance to hydrogen peroxide, suggesting that HSP70 can also be implicated in oxidant stress in Leishmania (Miller et al 2000). Although arsenite is a known potent inducer of HSPs in general (reviewed in Del Razo et al 2001) and has been shown to induce some HSPs in Leishmania (Lawrence and Robert-Gero 1985), much less is known about the role of SbIII as an inducer of HSPs (Snawder et al 1999).

In this study we isolated a cosmid by functional cloning. In all subsequent retransfection, this cosmid was found to be integrated in the genome (Fig 1A), but its presence was associated with a very high level of resistance. Only part of the cosmid seems to integrate as the copy number of HSP70s does not change notably (Fig 1D), although some weakly hybridizing extra bands (Fig 1D, lanes 4 and 6) are visible. Instead, on integration and subsequent complex rearrangements, the vector portion of the cosmid is mostly retained, and this is consistent with the multiple HYG and plasmid hybridization bands (Fig 1 E,F). This complex rearrangement was likely selected using hygromycin B, and the multimers of vector sequences did increase the size of the L tarentolae chromosome 28 equivalent (Fig 1 B,C, lane 1). More importantly, this complex integration event led to an increase in the expression of the HSP70 and or HSC70 proteins (Fig 3B). Additional work is required to understand how the cosmid has exactly integrated and rearranged and to understand how these events led to increase in HSP70 expression. In the initial transfectant, the cosmid was found both integrated and as part of an episome. Characterization of this cosmid revealed that it contains HSP70 and HSC70 genes. Our inability to directly link HSP70 to metal resistance suggests that HSP70s play a secondary role, which could serve as a first non-specific stress response that could allow the cells to develop more specific and efficient resistance mechanisms. Indeed, HSP70 proteins were induced in cells subjected to a short pulse of low SbIII concentration. Moreover, we were able to show that in some Leishmania mutants resistant to either AsIII or SbIII, the HSP70s were increased constitutively. This increase seems stable because cells grown in the absence of drugs for several hundreds of passages still had an increase in HSP70 expression compared with susceptible cells (Fig 4, lane 2). Transfection of either HSP70 or HSC70 showed that these proteins can directly confer both heat tolerance and protection against an SbIII shock (Fig 5). The effects are small but statistically significant and under stressful conditions may confer a sufficient advantage to allow the cell to develop more specific resistance mechanisms. These data show a novel role for HSP70s in Leishmania and for the first time experimentally link HSC70 with a function. Overall, our data show that HSP70s are not directly involved in resistance to antimony but clearly give some protection to the cell against this metal. This may explain how we were able to select an HSP70-encoding cosmid in the first place using a functional screen with SbIII. The stable overexpression of HSP70s in mutant cells (Fig 4) suggests that HSP70s are indeed required during the genesis of the metal resistance phenotype in the protozoan parasite Leishmania.

Acknowledgments

We thank Dr Eric Leblanc for his help with the phylogenetic analysis, Pr Steve Beverley for the gift of cLHYG, and our colleagues from the Ouellette lab for useful comments on the manuscript. This work was supported by the Canadian Institute of Health Research group and operating grants and through a Wellcome Trust–Burroughs Wellcome Fund new initiative in infectious diseases program and a FQRNT Center for host-parasite interactions grants to M.O. C.B. was the recipient of a CIHR studentship. M.O. is a Burroughs Wellcome Fund Scholar in Molecular Parasitology and holds a Canada Research Chair in Antimicrobial Resistance.

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