Cyclosporin A Treatment of Leishmania donovani Reveals Stage-Specific Functions of Cyclophilins in Parasite Proliferation and Viability

Background Cyclosporin A (CsA) has important anti-microbial activity against parasites of the genus Leishmania, suggesting CsA-binding cyclophilins (CyPs) as potential drug targets. However, no information is available on the genetic diversity of this important protein family, and the mechanisms underlying the cytotoxic effects of CsA on intracellular amastigotes are only poorly understood. Here, we performed a first genome-wide analysis of Leishmania CyPs and investigated the effects of CsA on host-free L. donovani amastigotes in order to elucidate the relevance of these parasite proteins for drug development. Methodology/Principal Findings Multiple sequence alignment and cluster analysis identified 17 Leishmania CyPs with significant sequence differences to human CyPs, but with highly conserved functional residues implicated in PPIase function and CsA binding. CsA treatment of promastigotes resulted in a dose-dependent inhibition of cell growth with an IC50 between 15 and 20 µM as demonstrated by proliferation assay and cell cycle analysis. Scanning electron microscopy revealed striking morphological changes in CsA treated promastigotes reminiscent to developing amastigotes, suggesting a role for parasite CyPs in Leishmania differentiation. In contrast to promastigotes, CsA was highly toxic to amastigotes with an IC50 between 5 and 10 µM, revealing for the first time a direct lethal effect of CsA on the pathogenic mammalian stage linked to parasite thermotolerance, independent from host CyPs. Structural modeling, enrichment of CsA-binding proteins from parasite extracts by FPLC, and PPIase activity assays revealed direct interaction of the inhibitor with LmaCyP40, a bifunctional cyclophilin with potential co-chaperone function. Conclusions/Significance The evolutionary expansion of the Leishmania CyP protein family and the toxicity of CsA on host-free amastigotes suggest important roles of PPIases in parasite biology and implicate Leishmania CyPs in key processes relevant for parasite proliferation and viability. The requirement of Leishmania CyP functions for intracellular parasite survival and their substantial divergence form host CyPs defines these proteins as prime drug targets.

Parasites of the genus Leishmania cause important human diseases collectively termed leishmaniasis, which range from mild, self-healing cutaneous lesions generated by L. major to fatal visceral infection of liver and spleen caused by L. donovani [16,17]. Leishmania is transmitted by infected sand flies, which harbor the proliferating flagellate promastigote form of the parasite. Highly infectious metacyclic promastigotes are inoculated into the mammalian host during sand fly blood feeding, where they are engulfed by phagocytes of the endo-reticular system and develop inside the phagolysosome into amastigotes, which subvert the host immune response and cause the immunopathologies characteristic of the various forms of leishmaniasis [18,19].
CsA has been shown to exert a leishmanicidal effect on intracellular L. tropica [12] and L. major in mouse and macrophage infection [13][14][15]. Although these findings define members of the Leishmania CyP protein family as potential important drug targets, only little is known on this protein family in trypanosomatids and the mechanisms of the anti-parasitic effects of CsA on intracellular Leishmania remain elusive. A potential role of Leishmania CyPs in amastigote differentiation and virulence can be postulated based on the role of Leishmania donovani LdCyP in disaggregation of adenosine kinase aggregates [20], an important enzyme in the Leishmania purine salvage pathway, whose activity substantially increases during the pro-to amastigote differentiation [21]. Furthermore, the amastigote-specific phosphorylation of cyclophilin 40 [22,23] suggests that activity, localization, and interaction of this protein may be regulated in a stage-specific manner by posttranslational modification.
The use of CsA for anti-leishmanial chemotherapy is limited by its suppressive action on host immunity, which leads to aggravation of experimental visceral leishmaniasis [24]. A better understanding on the biology of Leishmania CyPs, and their structural and functional differences to human CyPs is required to pave the way for the identification of new inhibitors with increased specificity for parasite CyPs. Here we initiated a first genome-wide analysis of the Leishmania CyP protein family and used the L. donovani axenic culture system [25,26] to investigate the effects of CsA on promastigotes and amastigotes in culture. Our data indicate substantial evolutionary divergence between parasite and host CyPs, which may be exploitable for drug development. We provide evidence for stage-specific functions of Leishmania CyPs in regulation of promastigote cell shape and proliferation, and amastigote thermotolerance. We demonstrate for the first time a stagespecific and direct toxic effect of CsA on host-free amastigotes, validating Leishmania CyPs as drug targets.

Cyclosporin A and FK506 treatment
Both cyclosporin A (CsA) isolated from Tolypocladium inflatum (Calbiochem) and FK506 isolated from Streptomyces tsukubaensis (A.G. Scientific) were dissolved in absolute ethanol at a final concentration of 10 mM and the stock was stored at 220uC. Logphase promastiogtes (2610 6 /ml) were cultured in medium containing either solvent, CsA or FK506 at concentrations ranging from 5 to 25 mM and incubated at 26uC, pH 7.4 for 48 hours unless otherwise specified. Axenic amastigotes were differentiated at 37uC for 72 hours and were incubated at a density of 2610 6 parasites/ml at 37uC with 5% CO 2 , pH 5.5 for 48 hours in medium containing either solvent, CsA or FK506 unless otherwise specified.

Determination of Leishmania growth and viability
The growth of solvent treated cells controls and CsA treated parasites was determined using a CASY cell counter (Schä rfe System) or determined microscopically by cell counting using 2% glutaraldeyhde fixed cells. Cell proliferation was determined by CellTiter-Blue assay (Promega) according to the manufacturer's instructions. Briefly, 20 ml of CellTiter-Blue was added to the cells in 96-well plate and incubated at 37uC for 4 hours. Fluorescence was measured (exl = 560 nm; eml = 590 nm) using a spectrometer SP-2000 (Safas). Results were expressed in % of fluorescence intensity compared to solvent treated cells control. The tests were performed in quadruplicate.

Bioinformatics analysis
The sequences of human and Leishmania cyclophilins were retrieved using the UniProt (www.uniprot.org) and GeneDB (www.genedb.org) databases, respectively, and conserved protein domains were identified by ScanProsite (www.expasy.ch/tools/ scanprosite). In order to determine the level of conservation of CLD domains across human and trypanosomatid parasites, all putative CLD containing proteins of the sequenced genomes of L. major, L. infantum, L. barsiliensis, T. brucei, and T. cruzi [28][29][30] were retrieved from the TriTrypDB database (http://tritrypdb.org/ tritrypdb/) using HUMAN_PPIA as an initial query for PSI-BLAST. After three cycles, all hits with a significant E-value (,10E-5) and more than 70% coverage of the CLD domain were selected, and their putative CLD domain was then extracted using the alignment to HUMAN_PPIA as a guide. Given the high level

Author Summary
Visceral leishmanisasis, also known as Kala Azar, is caused by the protozoan parasite Leishmania donovani. The L. donovani infectious cycle comprises two developmental stages, a motile promastigote stage that proliferates inside the digestive tract of the phlebotomine insect host, and a non-motile amastigote stage that differentiates inside the macrophages of mammalian hosts. Intracellular parasite survival in mouse and macrophage infection assays has been shown to be strongly compromised in the presence of the inhibitor cyclosporin A (CsA), which binds to members of the cyclophilin (CyP) protein family. It has been suggested that the toxic effects of CsA on amastigotes occurs indirectly via host cyclophilins, which may be required for intracellular parasite development and growth. Using a host-free L. donovani culture system we revealed for the first time a direct and stage-specific effect of CsA on promastigote growth and amastigote viability. We provided evidence that parasite killing occurs through a heat sensitivity mechanism likely due to direct inhibition of the co-chaperone cyclophilin 40. Our data allow important new insights into the function of the Leishmania CyP protein family in differentiation, growth, and intracellular survival, and define this class of molecules as important drug targets.
CsA Treatment of L. donovani www.plosntds.org of conservation of the CLD domains, it is realistic to consider this dataset as a complete set of the CLD proteins present in the species covered by the current release of TriTrypDB (Release 1.1). These sequences were aligned with T-Coffee (default mode) [31], and a Neighbor-Joining tree was computed with 500 bootstrap replicates. Positions in contact between CsA and cyclophilin A were identified on the multiple sequence alignment and the corresponding columns were extracted. The resulting functional residues were compared and clustered for similarity using UPGMA.

Structural modeling
We first identified Leishmania CyPs that are predicted to bind CsA using multiple sequence alignment of human and Leishmania major cyclophilins, and assessing the conservation of the residues known to be involved in cyclosporin A binding in known complexes. Based on these criteria, six Leishmania major cyclophilins shared the CsA binding residues with human PPIA or PPID (LmaCyP1, LmaCyP2, LmaCyP4, LmaCyP5, LmaCyP11 and LmaCyP40) and were selected for further analysis. The leishmanial cyclophilins were modelled with the automated mode of the Swiss-Model tool [32] using the following PDB structures as templates: 2 bit [33] for LmaCyp1; 3eov [34] for LmaCyP2, 2hqj (Arakaki and Merritt, unpublished), corresponding to LmaCyP11, for LmaCyP4 and LmaCyP5; 1ihg [35] for LmaCyP40. For each model or structure, the corresponding putative model complex with cyclosporin A was built based on the complex of L. donovani cyclophilin with CsA (3eov) as a template using the program insightII. Each model complex was subjected to a very limited energy refinement (100 cycles with the insightII Discover Module, steepest descent algorithm).The 3eov CsA binding residues (R78, I80, F83, M84, Q86, G95, T96, A123, N124, A125, G126, Q133, F135, W143, L144, H148) at less than 4 Å from CsA, were used for the superposition. The subsequent analyses of the 3D model complexes and evaluations of the putative interaction with the CsA were performed with the program insightII.

FACS-based approaches
Cell death was assessed by propidium iodide exclusion assay [36]. Briefly, 10 7 promastigotes or axenic amastigotes from control or CsA treated cultures were washed and resuspended in PBS containing 2 mg/ml of propidium iodide and incubated at room temperature for 15 min in the dark. The stained cells were subjected to FACS analysis (exl = 488 nm; eml = 617 nm). 10,000 events were analyzed. For cell cycle analysis, 10 7 latelog phase promastigotes were washed once with cold PBS and resuspended in pre-chilled 90% methanol in PBS and kept at 220uC overnight. The fixed cells were washed once with cold PBS and then resuspended in propidium iodide staining solution (10 mg/ml PI, 100 mg/ml RNase A in PBS) and incubated at 37uC for 30 min in the dark. The stained cells were subjected to FACS analysis as described above. Cell cycle distribution was calculated by FlowJo (Tree Star, Inc.) using the Dean-Jett-Fox model.

Morphological analysis
For Giemsa staining, 10 7 promastigotes or axenic amastigotes were immobilized on poly-L-lysine coated cover slips, fixed with methanol and stained with Giemsa reagent (Sigma) according to the manufacturer's instructions. The stained cells were mounted with Mowiol 4-88 (Sigma) [37] and observed with a Zeiss Axioplan 2 wide field light microscope.
Cells were prepared for scanning electron microscopy as described [38]. Briefly, parasites were washed in PBS, fixed with 2.5% glutaraldehyde in PBS, and treated with 1% OsO 4 . The cells were then dehydrated and critical-point dried (Emitech K850 or Balzers Union CPD30) and coated with gold (Joel JFC-1200 or Gatan Ion Beam Coater 681). Samples were visualized with scanning microscope Joel JM6700 F. Indirect immunofluorescence staining was performed with 10 7 promastigotes or axenic amastigotes that were settled on poly-Llysine coated coverslips and fixed in methanol at -20uC for 5 min. The fixed cells were rehydrated with PBS, and sequentially incubated with a mouse anti-a-tubulin antibody (Sigma, 1:2500 dilution) and an anti-mouse IgG-rhodamine antibody (Molecular Probes, 1:250 dilution). Nuclei and kinetoplasts were stained with DAPI and the slides were mounted with Prolong (Molecular Probes).

Cyclosporin A affinity chromatography
Modified CsA with a primary amine side chain was provided by the Texas A&M Natural Products LINCHPIN Laboratory, Assistant Director Dr. Jing Li [39]. The CsA-amine was coupled to the Affi-GelH10 resin (Bio-Rad) by reaction with the Nhydroxysuccinimide ester groups of the resin. Briefly, 7.5 mmol of CsA-amine was mixed with 500 ml of Affi-GelH 10 and incubated at room temperature for 6 hours. The coupling reaction was quenched by removing the CsA-amine and blocking the unreacted Affi-GelH 10 with 0.2 M ethanolamine. Logarithmic promastigotes were lysed with lysis buffer (50 mM HEPES, 100 mM NaCl, 10% glycerol, 0.5% NP-40 and 1 mM PMSF) followed by sonication on ice (30 s sonication with 15 s pause for 5 min). Insoluble debris was removed by centrifugation. The cleared cell lysate (1 mg protein/ml) was mixed with the CsA-Affi-Gel and incubated at 4uC for 3 hours. Bound proteins were eluted using hot Laemmli buffer. The elution was subjected to 10% SDS-PAGE, stained with SyproRubyHprotein gel stain (Invitrogen), and CsA-binding proteins were identified by MS analysis as described [22] and Western blotting.

Peptidyl Prolyl cis/trans isomerization assay
Measurements were performed according to [40]. Briefly, the peptidyl prolyl cis/trans isomerization reaction was initiated by diluting the peptide Abz-Ala-Ala-Pro-Phe-pNA in an anhydrous 0.5 M LiCl/TFE mixture with 35 mM HEPES pH 7.8. Inhibition of PPIase activity was measured by pre-incubating CsA with the enzyme (29.5 nM) for 5 min at 10uC before starting the reaction by the addition of the substrate. Data analysis was performed by single exponential non-linear regression using Sigma Plot Scientific Graphing System.
Based on length and domain structure, three types of L. major cyclophilins (LmaCyPs) can be distinguished. A first group of four proteins (LmaCyP1-3, 6) is characterized by a single CsA-binding domain without any significant N-or C-terminal sequence extensions ( Fig. 1 and Table 1). A second group of 11 proteins shows significant (50 or more amino acids) N-terminal (LmaCyP4, 5, 8, 10, 12, 16), C-terminal (LmaCyP7, 11), or both N-and Cterminal extensions (LmaCyP9, [13][14][15]. These extensions are unique and not conserved in human CyPs, but are mostly conserved across other trypanosomatids, including L. infantum, T. brucei and T. cruzi. Exceptions are the C-terminus of LmaCyP13 and the N-termini of LmaCyP8, 10, and 14, which are unique to Leishmania suggesting highly parasite specific functions absent in Trypanosoma. Finally, two LmaCYPs are characterized by the presence of additional functional domains, including LmaCyP5 containing a conserved prokaryotic lipid attachment domain (PLD, prosite access number PS5125), and LmaCyP40, the cyclophilin-40 homolog containing two tetratricopeptide repeat domains (TPR, prosite accession number PS50005) known to interact with HSP90 [48][49][50][51].

Bio-informatics characterization of the LmaCyP protein family
We investigated the relationship between human and trypanosomatid CyPs by multiple alignment and cluster analysis using the sequence of the conserved CLD or the functional residues implicated in PPIase function and CsA binding. The clustering tree obtained for the CLD demonstrates that all LmaCyPs have conserved homologs in L. infantum, L. braziliensis, T. brucei, and T. cruzi, which cluster together with highly significant bootstrap values ( Fig. 2A). All LmaCyPs have one homologue in the other Leishmania or Trypanosoma species, with the exception of LmaCyP5, which underwent expansion in the T. brucei genome with five sequentially arranged copies of the gene. It is interesting to speculate that the expansion of the only cyclophilin family member that contains a conserved lipid binding domain may be a reflection of the T. brucei biology, with a potential role for example for the expression of abundant gpi-anchored VSG proteins [52].
Many of the nodes support the existence of CyP subclasses across the trypanosomatids with a significant bootstrap value. In contrast, the nodes that cluster these subclasses together with their human homologues have only poor bootstrap support. This observation suggests that the various classes of CLDs encountered in trypanosomatid cyclophilins are quite distinct from one subclass to another and to their human counterparts. Substantial conservation however was observed in the cluster analysis performed with the functional CyP residues implicated in PPIase function and CsA binding (Fig. 2B). For instance, eight human CyPs and five LmaCyPs are clustering together showing a complete conservation of the canonical signature sequence characteristic for the human CsA-binding protein PPIA ( Fig. 2B and Table 2). This represents a significant conservation when considering that the overall CLD domain is only 64% conserved between the Leishmania and Human CyPs. These results indicate that a subset of Leishmania CyPs are likely functionally conserved and implicated in PPIase function, while other, less conserved LmaCyPs may carry different enzymatic activities.
In conclusion, our analysis reveals a large Leishmania CyP protein family suggesting an important role of PPIases in parasite biology, and identifies unique sequence elements in the LmaCyP CsA-binding domains that may be exploitable for drug development. Identification of 5 out of 17 LmaCyPs with a highly conserved CsA binding motif strongly suggests inhibitor-binding to multiple LmaCyPs with potentially important consequences on the biological functions of these proteins and Leishmania infectivity. In the following we investigate this possibility studying the effects of CsA on L. donovani promastigotes and amastigotes in culture.

CsA treatment interferes with parasite growth in vitro
CsA has been previously shown to reduce the intracellular growth of L. major amastigotes [13,14]. To further elucidate the mechanisms underlying this inhibition, we investigated the effects of CsA treatment on cultured L. donovani promastigotes and axenic amastigotes. Log-phase parasites from both stages (2610 6 /ml) were cultured in medium containing either ethanol (vehicle) or CsA at concentrations ranging from 5 to 25 mM, and incubated at 26uC, pH 7.4 (promastigote) or 37uC, pH 5.5 (amastigote) for 48 hours. At the time points indicated, the cells were fixed and counted microscopically or processed for CellTiter-Blue assay to test for proliferation. CsA-treated promastigotes showed a dose- CsA Treatment of L. donovani www.plosntds.org dependent, progressive reduction of growth with an IC50 at 48 hours between 15 and 20 mM and a more than 5-fold decrease in growth at the highest inhibitor concentration compared to the control ( Fig. 3A and B, left panels). Growth reduction was associated with a strong inhibition of resazurin reduction indicating reduced cell proliferation or cell viability (Fig. 3B, right  panel). CsA-mediated growth reduction was reversible, as parasite growth resumed normally after removal of the drug after 48 hours of treatment (data not shown). Likewise, CsA had a striking effect on the growth of L. donovani axenic amastigotes. The parasites showed substantially higher susceptibility to CsA at this stage with an IC50 between 5 and 10 mM (Fig. 3A, right panel, and Fig. 3B, left panel), and strongly reduced resazurin reduction (Fig. 3B, right panel). Together, our data demonstrate that CsA interferes with the in vitro growth of both L. donovani promastigotes and axenic amastigotes. In the following we used FACS-based approaches to investigate the mechanisms underlying this growth defect.
CsA shows a stage-specific effect on L. donovani viability To elucidate the mechanisms of CsA-mediated growth inhibition, we first investigated the effects of CsA on the viability of treated promastigotes and axenic amastigotes using a propidium iodide (PI) exclusion assay [36]. The percentages of PI positive, dead promastigotes and axenic amastigotes after 48 hours of CsA treatment was revealed by FACS analysis. Promastigotes did not show any significant increase in PI positive cells after incubation with CsA ranging from 5 to 15 mM (Fig. 4A), and more than 80% of cells were viable even at 25 mM CsA. In contrast, the proportion of PI positive axenic amastigotes increased dramatically with increasing CsA concentration, with a 4-fold decrease in cell viability at only 10 mM CsA (Fig. 4A). Thus, the decrease in cell number of CsA-treated promastigotes results from a slow-down in proliferation rather than parasite killing.
This result was further confirmed by cell cycle analysis. Late-log phase promastigotes were fixed with 90% methanol in PBS, stained with PI, and cell cycle phase distribution was determined by FACS analysis. Treatment of the parasites with 15 mM and 20 mM CsA did not affect the cell cycle distribution (Fig. 4B), suggesting that inhibition of parasite proliferation results from a non-synchronous slow-down in cell cycle progression.

CsA treatment induces morphological changes in L. donovani promastigotes without induction of amastigote gene expression
CsA-treatment of promastigote cultures induced a striking effect on parasite morphology. We documented these alterations by LmaCyP3 LmaCyP40 H131 Footnote. The functional amino acid residues implicated in PPIase catalytic activity and CsA binding of human CyPA (HsCyPA) (NCBI accession no. NP_066953) are shown [73,74]  CsA Treatment of L. donovani www.plosntds.org microscopic analysis using Giemsa staining and scanning electron microscopy. Treatment of promastigotes with 10 to 20 mM CsA induced morphological changes reminiscent of axenic amastigotes, including increased aggregate formation (Fig. 5A), oval cell shape (Fig. 5B), and shortened and retracted flagella (Fig. 5C). The CsA effects on L. donovani promastigotes are reminiscent to parasites treated with the HSP90 inhibitor geldanamycin, which results in amastigote differentiation [53]. We evaluated the effect of CsA on the differentiation state by following the expression of two markers, the promastigote specific surface glycoconjugates lipophosphoglycan (LPG) [54], which is lost during amastigote differentiation, and the A2 protein, which is induced during the pro-to amastigote conversion [42,55]. Logarithmic promastigotes were incubated with vehicle alone (0.15% ethanol) or 15 mM CsA at 26uC, pH 7.4 for 72 hours, and the expression profile was compared to axenic amastigotes by Western blotting using monoclonal anti-lipophosphoglycan antibody CA7AE [41] and anti-A2 antibody C9 [42]. Despite the amastigote-like morphology, CsA-treated promastigotes maintain expression of LPG, comparable to the level of solvent treated cells promastigotes, and do not show induction of the amastigote marker protein A2 (Fig. 5D). CsA treatment of promastigotes at pH 5.5 did not result in further differentiation as judged by morphology and expression of LPG, nor did it have an effect on parasite viability (data not shown). These results demonstrate that unlike geldanamycin, CsA

Investigation of the stage-specific mechanisms underlying CsA inhibition
CsA exerts its inhibitory action through binding of CyPs and inactivation of the cellular phosphatase calcineurin by CsA/CyP complexes [8,56]. In the following, we used the unrelated calcineurin inhibitor FK506 to analyze if the CsA effects on the parasite are mediated through inhibition of this phosphatase, a test that has been previously applied on Leishmania [57]. Log-phase promastigotes and axenic amastigotes (2610 6 /ml) were cultured for 48 hours in medium containing either ethanol (vehicle) or FK506 at concentrations ranging from 5 to 25 mM, and incubated at 26uC, pH 7.4 (promastigote) or 37uC, pH 5.5 (amastigote). FK506 treatment of promastigotes induced morphological changes similar to CsA treated parasites, and strongly reduced in vitro growth and cell proliferation in a dose-dependent manner ( Fig. 6A and B, left panels, and data not shown). Like CsA, FK506 did not significantly affect promastigote cell viability at the lower drug concentrations (Fig. 6B, left panel). In contrast, FK506 treatment of axenic amastigotes did not reproduce the CsA effects. First, as judged by proliferation and viability assay, amastigotes were more resistant to FK506, with an IC50 between 15 and 20 mM, compared to ca. 7 mM for CsA (Fig. 6B, right panel). Second, unlike CsA, FK506 did not induce massive cell death in amastigotes even at the highest concentration (Fig. 6C, right  panel). These data show that CsA and FK506 have different effects on L. donovani axenic amastigotes, which may be due to either stage-specific differences in inhibitor uptake or distinct intracellular cellular targets.
CsA treatment reduces L. donovani thermotolerance Based on previously published observations, Leishmania CyPs may have important amastigote-specific chaperone functions and participate in protein disaggregation [20]. We tested if CsA treatment affects thermotolerance of promastigotes and amastigotes following the number of propidium iodide stained, dead parasites as a read out. Log-phase promastigotes or amastigotes were treated with 15 mM CsA and parasites were simultaneously incubated for various time periods at either 26uC or 37uC. As expected, CsA treated amastigotes showed increased cell death in the presence of CsA during the 20 hours time course experiment (Fig. 7, right panel). Significantly, CsA-treatment of amastigotes at 26uC completely abrogated the toxic effect of the inhibitor. This data shows that CsA-mediated amastigote killing is temperature dependent. We confirmed this result using the complementary set up, incubating CsA-treated promastigotes at high temperature. Just like amastigotes, CsA-treated promastigotes underwent cell death as soon as 10 hours after temperature shift (Fig. 7, left  panel). CsA alone or heat shock alone had no significant effect on promastigote viability. Thus, CsA affects thermotolerance of both the promastigote and amastigote stages.

Identification of CsA-binding Leishmania donovani cyclophilins
The effect of CsA on parasite thermotolerance primed us to investigate the potential interaction between this inhibitor and LmaCyP40, a bifunctional cyclophilin that has both PPIase and co-chaperone function and interacts with members of the HSP protein family through TPR domains [58]. We first used a structural approach applied on six leishmanial cyclophilins selected for their similarity to the cyclosporin A binding pocket of human orthologs. We built the corresponding model complexes with CsA and evaluated their geometric fit and ability to establish inter-molecular hydrogen bonds with the ligand. The experimentally identified CsA binding residues of the L. donovani cyclophilin (3eov) and the putative binding residues of the L. major 3D model complexes, including the one for LmaCyP40, are highly conserved (Fig. 8A). All models, even if built on different templates, display a root mean square deviation of less than 0.6 angstrom on the CsA binding residues of the experimentally determined complex structure. Consequently, all models can accommodate the CsA ligand with no molecular clash and the hydrogen-bonding pattern is conserved with respect to the experimental structure (Fig. 8A,  lower panel). Furthermore, manual inspection of the model complexes revealed a good geometric complementarity between the protein and the ligand. All these evidences support the hypothesis that these L. major cyclophilins, including LmaCyP40, are good candidates for CsA binding.
We confirmed binding of the CsA ligand to LmaCyP40 by studying the proposed interaction by affinity chromatography using CsA-loaded resin. L. donovani promastigote extracts were incubated with the resin and bound proteins were separated by SDS-PAGE. One major band, specifically retained on the CsAresin, was revealed by fluorescent protein gel staining, and identified as CyP2 by MS analysis (Fig. 8B, left panel, and Dataset S1). Western blot analysis of the gel revealed cyclophilin 40 (Fig. 8B, right panel), thus confirming the CsA-CyP40 interaction suggested by the structural modelling.
We next analyzed the biochemical characteristics of the LmaCyP40-CsA interaction using GST::Strep::CyP40 purified from recombinant bacteria (Fig. S1). We first determined the k cat /K m of Leishmania major GST::Strep::CyP40 PPIase activity by evaluating the linear dependency between k enz and enzyme concentration ranging from 14.7 to 59 nM. The catalytic efficiency of Leishmania major GST::Strep::CyP40 for Abz-Ala-Ala-Pro-Phe-pNa was found to be k cat /K M = (3.72560.16)610 5 M 21 s 21 (Fig. 8C, upper panel). We then tested direct inhibition of the LmaCyP enzymatic activity by CsA using the substrate Abz-Ala-Ala-Pro-Phe-pNA and increasing amounts of inhibitor. The IC50 value of CsA was determined to be 162646 nM CsA (Fig. 8C, lower panel) and thus similar to human CyP40 with an IC50 value of 195 nM [59]. Axenic amastigotes were prepared as described in experimental procedure. 10 7 cells were fixed with either methanol for Giemsa staining (A), or 2.5% glutaraldehyde for scanning electron microscopy (B). The bar corresponds to 1 mm (B) and 5 mm (A). Two independent experiments were performed and representative fields are shown. (C) Flagellum length measurement. CsA-treated and solvent treated cells L. donovani promastigotes were fixed in methanol and stained with anti-tubulin monoclonal antibody. Flagellum length was measured from a total of 180 cells each for control and CsA-treated samples. Only cells with a single flagellum that was completely visible and fully in focus were taken into account. Samples were observed with a DMR Leica microscope and images were captured with a Cool Snap HQ camera (Roper Scientific). Images were analysed using the IPLab Spectrum 3.9 software (Scanalytics & BD Biosciences) and flagellum length was measured using ImageJ (NIH). (D) Immunoblot analysis of CsA treated parasites. Parasites were treated with solvent or 15 mM CsA for 72 hours, lysed in 16 Laemmli buffer, and lysates equal to 2610 7 cells were analyzed by immunoblotting. Promastigote specific marker LPG (upper), amastigote specific marker A2 (middle) and a-tubulin (lower) were analyzed. Two independent experiments which gave identical results were performed. doi:10.1371/journal.pntd.0000729.g005 CsA Treatment of L. donovani www.plosntds.org

Discussion
The leishmanicidal activity of CsA has been first demonstrated in L. tropica infected BALB/c mice, which showed a dose-dependent inhibition of parasite burden and reduction in lesion formation [12]. This anti-parasitic activity has been subsequently confirmed for L. major in mouse and macrophage infection assays, and various modes of CsA action have been proposed [13,14,57]. The observation that CsA has no overt anti-microbial activity against L. major promastigotes in culture, but efficiently kills amastigotes in infected macrophages, provided support to the idea that the toxic effect of CsA on intracellular parasites depends on inhibition of host rather than Leishmania CyPs. This hypothesis was further supported by findings showing that the phosphatase calcineurin, the prime target of the inhibitory CsA/CyP complex, is expressed at very low levels and is not recognized by Leishmania LmaCyP19 (corresponding to LmaCyP1 according to our nomenclature), although this protein efficiently bound CsA [60,61]. In contrast to these previous reports, our data provide several lines of evidence for a direct action of CsA on Leishmania CyPs.
A first line of evidence resulted from the bio-informatics analysis and structural modeling of Leishmania CyPs. Blast search of the L. major and L. infantum genome databases (www.genedb.org) identified a surprisingly large family of 17 CyP-like proteins in these protozoan, compared to yeast, Drosophila, and human with 8, 14 and 19 CyPs, respectively (Table 1, Fig. 2) [62][63][64]. Multiple sequence alignment of trypanosomatid and human CyPs, cluster analysis of the functional residues implicated in PPIase catalytic activity and CsA binding of the CLD, and structural modelling revealed the presence of six Leishmania CyPs that showed conservation of the functional residues (Table 2, Figs. 2 and 8A) and were predicted to form a complex with CsA. This remarkable conservation indicates that multiple Leishmania CyPs are likely binding to CsA, a fact that we subsequently confirmed by affinity chromatography and Western blotting, revealing direct interaction of the inhibitor with Leishmana CyP2 and CyP40 (Fig. 8 B).
The effects of CsA on L. donovani promastigotes and axenic amastigotes further support this possibility and provided a second line of evidence for a direct action of CsA on Leishmania CyPs in vitro. We showed that inhibitor treatment of L. donovani promastigotes leads to dose-dependent, reversible inhibition of proliferation (Figs. 3A and B), without significant effects on cell viability (Fig. 4A) and cell cycle distribution (Fig. 4B). These results confirmed previous observations that CsA does not exert a toxic effect on Leishmania promastigotes, but revealed a strong effect on promastigote in vitro growth that escaped previous analysis, likely due to the lower CsA concentration (4 mM) used in these studies [13,14]. In contrast to promastigotes, CsA showed a direct toxic effect on L. donovani axenic amastigotes with more than 50% of parasite death in the presence of 10 mM inhibitor (Fig. 4A). This result demonstrates for the first time that the observed antileishmanial effect on intracellular amastigotes in mouse and macrophage infection [13,14,57] may rely mainly on direct inhibition of parasite CyPs by CsA, although a participation of host CyPs can not be excluded. We further investigated the mechanisms underlying the stage-specific effects of CsA using the unrelated antifungal macrolide inhibitor FK506. FK506 binds to FKBPs, a second class of PPIases (Table 1), which similar to the CsA/CyP complexes inhibit calcineurin [8]. FK506 treatment reproduced the effects observed in CsA-treated promastigotes, suggesting inhibition of calcineurin as one of the mechanisms underlying the observed growth defect of this parasite stage (Fig. 6). To our surprise, unlike CsA, FK506 did not exert a toxic effect on axenic amastigotes at concentrations between 5 and 15 mM (Fig. 6B), a fact previously observed in intracellular L. major amastigotes [57]. These data indicate that the toxic effect of CsA on amastigotes occurs likely through calcineurin-independent mechanisms, which may be directly linked to inhibition of stagespecific enzymatic functions of Leishmania CyPs.
Cyclophilins are protein chaperones with PPIase activity, which catalyzes the cis-trans isomerization of peptidyl-prolyl bonds, affecting stability, activity, and localization of client proteins [2,65]. Thus, inhibition of CyP functions by CsA may provoke pleiotropic downstream effects that may lead to the observed growth inhibition and loss of viability. In the context of the current literature, two pathways may be singled out with potential   (2), or resin coupled with CsA (+), bound proteins were analyzed by SDS-PAGE and SyproRuby staining, and identified by relevance for the CsA-dependent toxicity. First, L. donovani adenosine kinase aggregates have been identified as clients for CyP2, which disaggregates complexes of this protein [20,66], thereby playing an important function in the purine salvage pathway [67]. Inhibition of this important CyP2 chaperone function may limit the intracellular concentration of adenosine and affect DNA synthesis with consequences for promastigote growth and amastigote viability. Second, cyclophilins have been reported to participate in the response to heat stress in other microbial pathogens. In the human pathogenic fungi Cryptococcus neoformans, CsA treatment prevents growth at elevated temperatures [68,69] and the CyP-related protein Cp1a is required for full expression of fungal virulence [70]. Our data indeed established a direct link between the sensitivity of Leishmania to CsA and the parasite thermotolerance. We demonstrated that CsA-treated amastigotes are insensitive to the drug when incubated at 26uC, while CsA-resistant promastigotes are efficiently killed by the inhibitor at 37uC (Fig. 7). A second observation linked Leishmania CyPs with the response to increased temperature. We observed a striking effect of CsA on promastigote morphology, which acquired an oval cell shape and shortened their flagella, thus showing some (but not all) features characteristic for amastigote differentiation (Fig. 5). A similar morphogenic effect has been previously observed on promastigotes treated with the HSP90 inhibitor geldanamycin [53]. It is possible that both CsA and geldanamycin target different proteins are part of the same heat shock complex implicated in Leishmania differentiation and thermotolerance, such as cyclophilin 40, a multifunctional protein that interacts with various members of the HSP family through conserved TPR domains [58]. Indeed, our data identified LmaCyP40 as a direct target for CsA as judged from the direct interaction between the enzyme and the inhibitor (Fig. 8B) and CsA-dependent inhibition of LmaCyP40 PPIase activity (Fig. 8C). It is interesting to speculate that the temperature-dependent CsA effect on Leishmania viability is the result of CyP40 inhibition. Future studies employing LmaCyP40 conditional null mutants with the aim to dissociate the PPIase and chaperone functions of this enzyme may allow testing this hypothesis and shed important new light on the function of LmaCyP40 in parasite thermotolerance and infectivity.
In conclusion, our data revealed for the first time a direct cytostatic and cytotoxic effect of CsA on L. donovani in culture. We provided evidence that the stage-specific effects of CsA are governed by independent mechanisms linked to inhibition of calcineurin phosphatase activity in promastigotes, and inhibition of CyP functions relevant for thermotolerance in amastigote. We identified unique sequence elements in Leishmania CyPs and documented a considerable evolutionary expansion of this protein family, compared to other organisms, emphasizing the importance of this class of molecules for trypanosomatid-specific biology. The requirement of Leishmania CyP functions for intracellular parasite survival and their substantial divergence from host CyPs defines these proteins as prime drug targets. The suppressive action of CsA on host immunity and its exacerbating effects on murine toxoplasmosis, trypanosomiasis, and visceral leishmaniasis [24,71,72] obviously eliminates this drug for anti-parasitic intervention. Hence, the focus of future research should lie on the identification of novel CyP inhibitors that specifically target parasite CyPs without altering the host immune status.

Supporting Information
Dataset S1 MALDI-ToF-ToF mass spectrometry analysis of promastigote lysate bound to CsA-coupled resin.