Wai-Lok Yau

Pascale Pescher

Andrea Macdonald

Sonia Hem

Dorothea Zander

Silke Retzlaff

Thierry Blisnick

Brice Rotureau

Heidi Rosenqvist

++ Martin Wiese

Philippe Bastin

Joachim Clos

Gerald F Späth
email: gerald.spaeth@pasteur.fr

The Leishmania donovani chaperone cyclophilin 40 is essential for intracellular infection independent of its stage-specific phosphorylation status

Keywords: Leishmania donovani, stress protein, cyclophilin 40, null mutant analysis, intracellular survival, chaperone phosphorylation

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

promastigotes in the midgut of female phlebotomine sand flies where they undergo an environmentally triggered differentiation process termed metacyclogenesis to develop into highly infectious metacyclic promastigotes [START_REF] Sacks | Identification of an infective stage of Leishmania promastigotes[END_REF]. Following transmission by blood feeding sand flies, metacyclic parasites are engulfed by phagocytes such as macrophages,

where they differentiate into non-motile amastigotes that cause the pathology of the disease.

In vitro differentiation of infectious metacyclic parasites is induced by nutritional starvation or acidic pH, and development of amastigote-like cells in culture is triggered by pH and temperature change [START_REF] Zakai | In vitro stimulation of metacyclogenesis in Leishmania braziliensis, L. donovani, L. major and L. mexicana[END_REF][START_REF] Cunningham | Regulation of differentiation to the infective stage of the protozoan parasite Leishmania major by tetrahydrobiopterin[END_REF][START_REF] Barak | Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response[END_REF], suggesting that Leishmania stage-differentiation occurs through sensing of environmental stress signals encountered inside insect and vertebrate hosts [START_REF] Zilberstein | The role of pH and temperature in the development of Leishmania parasites[END_REF]. Recently, a role of stress signaling in parasite development has been suggested based on phosphoproteomics studies. Gel-based proteomics approaches comparing enriched phosphoprotein fractions from promastigotes and axenic amastigotes revealed a surprising divergence of the stage-specific phosphoproteome with more than 30% of proteins showing statistically significant differential phosphorylation [START_REF] Morales | Phosphoproteomic analysis of Leishmania donovani pro-and amastigote stages[END_REF][START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF]. Phosphorylation events in amastigotes were almost exclusively restricted to heat shock proteins and chaperones and occurred largely at parasite-specific phosphorylation sites [START_REF] Hem | Identification of Leishmania-specific protein phosphorylation sites by LC-MS/MS and comparative genomics analyses[END_REF], suggesting regulation of the Leishmania response to stress by post-translational rather than classical transcriptional mechanisms. This possibility has been investigated previously for the stressinduced protein 1 (STI1), a chaperone that acts as a scaffolding protein for the assembly of foldosome complexes [START_REF] Pratt | Studies with purified chaperones advance the understanding of the mechanism of glucocorticoid receptor-hsp90 heterocomplex assembly[END_REF]. Null mutant analysis of STI1 in L. donovani established the requirement of this protein for parasite survival in vitro, and complementation analysis by plasmid shuffle identified the two phosphorylation sites S15 and S481 as essential for STI1 function and parasite viability [START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF].

One group of stress proteins that showed increased phosphorylation in amastigotes in these studies is represented by immunophilins, a protein family that includes cyclophilins (CyPs) and FK506 binding proteins (FKBPs), which are characterized by a peptidyl-prolyl isomerase activity required for proper protein folding [START_REF] Barik | Immunophilins: for the love of proteins[END_REF]. In a previous study, we revealed stage-specific functions of cyclophilins using the CyP inhibitor cyclosporin A (CsA) [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF]. Inhibitor-treated promastigotes underwent non-synchronous cell cycle arrest and acquired features similar to amastigotes, including oval shape and shortening of the flagella reminiscent of L. donovani promastigotes treated with the HSP90 inhibitor Geldanamycin [START_REF] Wiesgigl | Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani[END_REF]. In contrast, treatment of axenic amastigotes resulted in parasite death, a phenomenon that was abrogated by temperature shift from 37°C to 26°C. While this pharmacological approach suggests potential roles of cyclophilins in parasite proliferation and thermotolerance and sheds new light on the stage-specific action of CsA and its lethal effects on intracellular Leishmania [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF], the pleiotropic inhibition of multiple cyclophilins and the phosphatase calcineurin by CsA-CyP complexes [START_REF] Chappell | Cyclosporin A: antiparasite drug, modulator of the host-parasite relationship and immunosuppressant[END_REF]) raise questions about the specific function of each of the 17 Leishmania cyclophilins.

The effects of CsA on L. donovani differentiation and thermotolerance, its binding to parasite cyclophilin 40 (LdCyP40) [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF], and its stage-speific phosphorylation in the axenic amastigotes prompted us to assess the role of this co-chaperone and its modification in parasite survival and infectivity combining gene deletion, phosphoproteomics, and complementation approaches. CyP40 is a highly conserved bi-functional protein that carries peptidyl-prolyl isomerase (PPIase) activity and forms dynamic complexes in yeast and mammalian cells with HSP90 through its conserved tetratricopeptide repeat (TPR) domains [START_REF] Hoffmann | Cyclophilin-40: evidence for a dimeric complex with hsp90[END_REF]. The loss-of-function study presented here identifies important roles for LdCyP40 in the development of infectious parasites in vitro and intracellular parasite survival independent of its phosphorylation status, thus establishing a novel link between L. donovani chaperone activity and stress-induced parasite differentiation relevant for host cell infection.

Results

L. donovani CyP40 is constitutively expressed but stage-specifically phosphorylated.

CyP40 belongs to the immunophilin superfamily that comprises 17 members in Leishmania [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF]. We first investigated the domain structure and sequence conservation of this protein across trypanosomatid parasites (L. major, L. infantum, L. braziliensis, T. brucei and T.

cruzi) and various higher eukaryotes, including human, mouse, cow, and yeast. The putative CyP40 protein sequences were retrieved from the UniProt (http://www.uniprot.org/) and TriTrypDB databases (http://tritrypdb.org/tritrypdb/) by Blast search using L. major cyclophilin 40 (LmjF35.4770) as a query and aligned using ClustalW 2.0.12. Leishmania CyP40 displays the characteristic two-domain structure (Fig. 1A). The N-terminal cyclophilin-like domain (CLD) spans 171 residues and is highly conserved showing 68.8% of amino acid identity across the analyzed organisms. This domain carries the enzymatic peptidyl-prolyl isomerase (PPIase) function and is the target of the cyclophilin-specific inhibitor cyclosporin (CsA). The functional residues for both PPIase activity and CsA binding are highly conserved as expected from our previously published observations that L. donovani CyP40 carries isomerase activity and can be enriched from crude parasite extracts by affinity chromatography using immobilized CsA [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF]. In addition, a cleft localized C-terminal of the CLD domain shows a series of conserved residues that have been previously implicated in CyP40 chaperone activity [START_REF] Mok | The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains[END_REF]. The C-terminal part of CyP40 contains three distinct TPR motifs that are less conserved (50.4%, 47.3% and 50.2% of identity for TPR1, 2 and 3 respectively) and together form the TPR domain known to interact with the conserved EEVD consensus sequence of HSP70 and HSP90 [START_REF] Ward | A structurebased mutational analysis of cyclophilin 40 identifies key residues in the core tetratricopeptide repeat domain that mediate binding to Hsp90[END_REF]. Together these data identify Leishmania CyP40 as a highly conserved member of the eukaryote immunophilin protein family that likely interacts with HSPs and carries chaperone function.

In our previous proteomics studies on the protein phosphorylation dynamics during Leishmania differentiation, L. donovani LdCyP40 abundance was significantly increased in the phosphoproteome of axenic amastigotes when compared to promastigotes, suggesting amastigote-specific phosphorylation of this protein [START_REF] Morales | Phosphoproteomic analysis of Leishmania donovani pro-and amastigote stages[END_REF][START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF].

However, this putative stage-specific phosphorylation has not been validated in bona fide animal-derived amastigotes, and whether the increase of abundance results from an increase in phospho-stoichiometry or simply increased protein expression has not been investigated. To distinguish between these two possibilities, we analyzed LdCyP40 stage-specific expression and phosphorylation profiles by Western blotting using total protein extracts and phosphoprotein fractions enriched by immobilized metal affinity chromatography obtained from cultured promastigotes, axenic amastigotes, and bona fide amastigotes purified from L.

donovani infected hamster spleen. As judged by α-tubulin expression used as normalization control, LdCyP40 is constitutively expressed across all stages tested, but shows a significant increase in abundance in the phosphoprotein fraction of axenic and hamster-derived amastigotes compared to promastigotes (Fig. 1B). The weaker signal observed in splenic amastigotes compared to axenic amastigotes may result from unequal loading or reflects a difference in phosphorylation levels between both samples. These results establish LdCyP40 stage-specific de novo phosphorylation and a substantial increase in the phospho-stoichiometry of this protein in amastigotes.

Establishment of cyp40-/-null mutants.

We established LdCyP40 null mutant parasites to study the biological functions of LdCyP40 and ultimately assess the physiological role of stage-specific CyP40 phosphorylation by complementation. The endogenous alleles were replaced using two targeting constructs comprising puromycin and bleomycin resistance genes flanked by the 5' and 3'UTRs of LdCyP40 (Fig. 2A and Fig. S1). Respective add-back controls re-expressing LdCyP40 from the ribosomal locus under the control of the CyP40 3'UTR were generated using a knock-in strategy (Fig. 2A and Fig. S2). The absence of the LdCyP40 ORF and corresponding protein in six independent null mutant clones and its re-expression in complemented parasites was confirmed by PCR (Fig. 2B and data not shown) and Western blot analysis (Fig. 2C and Fig. S3).

Morphological characterization of cyp40-/-null mutant and add-back clones.

Microscopic observation of the two independent null mutant clones cyp40-/-cl.4 and cl.5 and four independent add-back clones as well as add-back pools derived from each null mutant clone revealed important morphological alternations at stationary growth phase that correlated with the absence of CyP40 expression (Fig. S4A). At logarithmic growth phase, CyP40 null mutant promastigotes were morphologically identical to wild-type and add-back controls as judged by Giemsa staining of fixed parasite cultures (Fig. S4A, left panels). By contrast 70% of cyp40-/-cells at stationary growth phase of two independent null mutant clones acquired a small spherical cell shape, while wild-type parasites and add-back clones showed the spindle shaped cell body characteristic for stationary phase parasites (Fig. S4A, right panels).

Moreover, measurements of cell body and flagellum length revealed a significant reduction of the ratio between both parameters in cyp40-/-cells (Fig. S4B). Given the reproducibility of this As cell rounding is often associated with cellular stress and apoptotic cell death [START_REF] Lee | Programmed cell death in the unicellular protozoan parasite Leishmania[END_REF][START_REF] Gannavaram | Programmed cell death in Leishmania: biochemical evidence and role in parasite infectivity[END_REF], we next investigated integrity and ultra-structural organization of CyP40 null mutant cells and controls. Wild-type, cyp40-/-and cyp40-/-/+ promastigotes were fixed with 2% glutaraldehyde and analyzed by scanning electron microscopy (Fig. 3A). This analysis confirmed the atypical spherical cell shape of cyp40-/stationary phase promastigotes and provided a first indication of their cellular integrity.

Likewise, analysis by transmission electron microscopy revealed no overt alterations in cellular organization of cyp40-/-cells, which showed intact nuclei and mitochondria, and absence of apoptotic bodies or autophagosomes (Fig. 3B). Finally, direct assessment of cell death by FACS analysis following membrane exposure of phosphatidylserine with annexin V-FITC and exclusion of propidium iodide did not reveal any significant change in cell viability of stationary cyp40-/-parasites after 6 days of in vitro culture (Fig. 3C). Together these results rule out the possibility of cell death as the cause of cyp40-/-morphological alterations but rather suggest a defect in parasite development and cellular remodeling at stationary phase.

Phenotypic characterization of stationary phase cyp40-/-promastigotes.

During the infectious cycle, non-infectious procyclic promastigotes differentiate into highly infective metacyclic promastigotes in response to nutritional starvation after digestion and excretion of the blood meal. This process is mimicked in culture during stationary growth phase [START_REF] Sacks | Identification of an infective stage of Leishmania promastigotes[END_REF]da Silvaand Sacks, 1987) and is accompanied by the expression of characteristic biological and morphological markers, including expression of high molecular weight LPG and increase in SHERP mRNA abundance. The altered morphology of cyp40-/parasites primed us to investigate the status of these phenotypic markers. First, monitoring cell density of WT, cyp40-/-and add-back promastigotes with a CASY cell counter, deletion of CyP40 did not affect the rate of proliferation nor the density reached at stationary phase (Fig. 4A). Surprisingly, despite defects in stationary phase morphology, cyp40-/-promastigotes showed normal expression of higher molecular weight LPG characteristic for stationary phase parasites as revealed by Western blot analysis using the anti-LPG antibody CA7AE (Fig. 4B), and induced SHERP mRNA to levels corresponding to WT and add-back controls (Fig. 4C).

Together these data demonstrate that L. donovani CyP40 is dispensable for parasite survival in vitro under standard culture conditions, and is not required for metacyclic-specific glycolipid remodeling or gene regulation, at least for the markers investigated here. To further characterize the stationary phase defect of cyp40-/-parasites, we investigated the functional properties of infectious, stationary phase parasites, including cell motility and intracellular survival.

CyP40 null mutants show reduced motility and a loosened subpellicular tubulin network.

An important functional characteristic of stationary phase parasites is a significant increase in motility, which is of physiological relevance in the natural setting for migration of metacyclic parasites from sand fly midgut to larynx [START_REF] Walters | Ultrastructural development of Leishmania chagasi in its vector, Lutzomyia longipalpis (Diptera: Psychodidae)[END_REF][START_REF] Walters | Life cycle of Leishmania major (Kinetoplastida: Trypanosomatidae) in the neotropical sand fly Lutzomyia longipalpis (Diptera: Psychodidae)[END_REF], or the establishment of host cell infection [START_REF] Uezato | The attachment and entry of Leishmania (Leishmania) major into macrophages: observation by scanning electron microscope[END_REF][START_REF] Forestier | Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process[END_REF]. We quantified motility in at least 741 WT, cyp40-/-and add-back promastigotes obtained from logarithmic and stationary phase cultures utilizing video microscopy and in silico tracking analysis.

Qualitative observation of each movie did not reveal any significant difference in flagellar wave orientation and flagellar beating amplitude across all lines and both culture conditions (data not shown). Likewise, quantitative analysis showed no significant difference in beating frequencies and speed between WT, cyp40-/-, and add-back log phase promastigotes. As expected, WT and add-back promastigotes showed a statistically significant increase in motility speed and flagellar beat frequency at stationary phase (Fig. 5A), and moved in a uni-directional way over extended distances (Fig. 5B and Video S1). By contrast, CyP40 null mutant parasites do not increase speed at stationary growth phase (Fig. 5A) and show a defect in directional motility as they largely spin on themselves (Fig. 5B and Video S1).

As judged by transmission electron microscopy, this motility defect was not caused by alterations in flagellar structure of cyp40-/-parasites, which maintained the characteristic 9+2 organization of the axoneme (Fig. 5C). However, investigating more closely the Leishmania subpellicular tubulin network by scanning EM after removal of the parasite plasma membrane by detergent treatment, stationary phase cyp40-/-promastigotes showed a significantly loosened tubulin network, in contrast to the very dense, small-meshed network observed in wild-type parasites (Fig. 5D). Western blot analysis of WT, cyp40-/-and add-back parasites from logarithmic and stationary cultures did not reveal any significant difference in expression levels of α-and β-tubulin, with the exception of a small β-tubulin isoform at around 33 kDa, which is specific for stationary phase cells and slightly reduced in CyP40 null mutants (Fig. S5). This finding was confirmed by quantitative proteomics using 2D-DIGE analysis of total extracts of WT and cyp40-/-promastigotes from stationary growth phase that identified the small β-tubulin isoform (LinJ.21.2240, spot 928) with a highly significant, four-fold reduction of abundance in the null mutant cyp40-/-(Fig. 5E and Fig. S6). This isoform corresponds to a previously described proteolytic cleavage product of β-tubulin with a molecular weight of about 34 kDa [START_REF] Drummelsmith | Proteome mapping of the protozoan parasite Leishmania and application to the study of drug targets and resistance mechanisms[END_REF]. Similar low molecular weight isoforms have been identified in the stage-specific proteomes of L. major and T. cruzi metacyclic parasites [START_REF] Parodi-Talice | Proteomic analysis of metacyclic trypomastigotes undergoing Trypanosoma cruzi metacyclogenesis[END_REF][START_REF] Mojtahedi | Leishmania major: Identification of developmentally regulated proteins in procyclic and metacyclic promastigotes[END_REF], but their biological significance in stage-specific remodeling of the tubulin network remains to be elucidated. Together these data demonstrate non-redundant CyP40 functions in cytoskeletal remodeling of the parasite during stationary

Page 10 of 49 Molecular Microbiology growth phase, which is likely the basis for the aberrant morphology and the motility defect observed in cyp40-/-parasites.

Leishmania cyp40-/-mutants fail to establish intracellular infection.

The development of highly infectious metacyclic parasites can be mimicked by exposing promastigotes to various forms of stress, including nutritional starvation and acidification at stationary phase (da Silva and [START_REF] Da Silva | Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation[END_REF]Bate and Tetley, 1993;[START_REF] Zakai | In vitro stimulation of metacyclogenesis in Leishmania braziliensis, L. donovani, L. major and L. mexicana[END_REF][START_REF] Serafim | Leishmania metacyclogenesis is promoted in the absence of purines[END_REF]. The stationary phase-specific defects of cyp40-/-promastigotes primed us to investigate their capacity to infect host cells. Bone marrow-derived macrophages obtained from C57BL/6 mice were incubated for 2 hours with WT, cyp40-/-, and add-back stationary phase promastigotes at a multiplicity of infection of 10 parasites per host cell, free parasites were removed by washing, and intracellular parasite burden was determined by nuclear staining and fluorescence microscopy at 2, 24, and 48 hours post infection. The cyp40-/-cells were phagocytosed at levels comparable to the controls with 80% of macrophages hosting a mean of 2.5 parasites per infected host cell (Fig. 6A). While WT and add-back parasites persisted over the 48 hours post infection period and showed intracellular proliferation as documented by the increase in number of parasites per infected macrophage (Fig. 6A, lower panel), the level of cyp40-/-infection was reduced by over 90% during the first 24 hours and parasites were completely eliminated by 48 hours post infection (Fig. 6A). We next tested if intracellular elimination of cyp40-/-parasites is caused by a susceptibility to elevated temperature or acidic pH encountered inside the host cell, or results from a defect in amastigote differentiation. CyP40 null mutant promastigotes grown at 37°C did not show any significant difference to WT and add-back parasites (Fig. S7A), and axenic amastigotes developed and proliferated normally in response to both temperature and pH shift as judged by cell counting (Fig. 6B) and assessment of expression of the axenic amastigote marker protein A2 (Fig. 6C). Thus, LdCyP40 carries essential functions for successful host cell infection independent of parasite resistance to acidic pH, elevated temperature, or amastigote development.

Functional assessment of CyP40 phosphorylation.

The failure of cyp40-/-parasites to establish intracellular infection provided an interesting read out to test for the function of CyP40 phosphorylation in parasite infectivity using complementation analysis. We investigated the CyP40 amino acid residues that were phosphorylated by using LC-ESI-MS/MS analysis. Axenic amastigote protein extracts from transgenic L. donovani over-expressing Strep::CyP40 were collected and Strep::CyP40 was enriched by affinity chromatography using streptactin agarose liquid chromatography (Fig. S8).

After SDS-PAGE and Coomassie Brilliant Blue staining, the band corresponding to Strep::CyP40 was extracted and subjected to phosphopeptide analysis by LC-ESI-MS/MS. MS analysis revealed that residue serine 274 (S274) of CyP40 was the only detected phosphorylation site of CyP40 in axenic amastigotes (Fig. 7A and Fig. S9). The multiple sequence alignment of trypanosomatid CyP40 described in Fig. 1 localizes S274 to the center of the TPR motif 2 and shows that it is unique among trypanosomatids (Fig. 7B). An independent study also pointed out that S274 is phosphorylated in L. mexicana (Dr. Martin Wiese, University of Strathclyde, Glasgow, unpublished data). These data suggest that phosphorylation at S274 of CyP40 is Leishmania-specific and may have critical roles in parasite survival.

To study the relationship between CyP40 phosphorylation and parasite infectivity, an add-back line was generated expressing a serine 274 to alanine (S274A) phosphorylation site mutant version of CyP40 (Fig. 7C, left panel, and Fig. S10), which was subsequently tested for in vitro infectivity. We next assessed the accumulation of this mutant in the parasite phosphoprotein fraction to test if elimination of S274 is sufficient to abrogate phospho-specific affinity purification of CyP40. Total protein extracts and phospho-protein enriched extracts from WT treated or not with lambda phosphatase, or add-back parasites expressing CyP40-S274A were analyzed by western blotting using anti-CyP40 antibody and anti-α-tubulin antibody for normalization of the signals. As expected, robust amounts of CyP40 were detected in the wildtype phospho-protein extracts, and this signal was strongly reduced by phosphatase treatment prior to enrichment representing background levels of CyP40 enrichment likely due to nonspecific binding to the affinity matrix (Fig. 7C, right panel). The same level of background signal was obtained with CyP40-S274A confirming that this residue represents the only phosphorylation site in CyP40.

Bone marrow-derived macrophages were then infected with cyp40-/-promastigotes complemented with CyP40-S274A. The controls, WT, cyp40-/-and add-back promastigotes behaved in a similar fashion as described previously (Fig. 7D). The higher counts of infected macrophage obtained with WT parasites compared to add-back control may be due to batch-tobatch variation of parasites. The cyp40-/-/+S274A add-back promastigotes entered macrophages and established infection at similar efficiency. The parasite number decreased in the first 24 hours post infection but parasites started to multiply after 48 hours (Fig. 7D, left panel). The intracellular proliferation of CyP40-WT and CyP40-S274A add-back did not show any significant difference, as indicated by the number of parasites per 100 macrophages at 48 hours post infection (Fig. 7D, right panel). These data indicate that phosphorylation of CyP40 is either not important for the function of CyP40 in Leishmania intracellular survival, or can be compensated by other co-chaperones.

Discussion

The conserved co-chaperone CyP40 participates in the formation of highly dynamic heat shock protein complexes across various eukaryotes [START_REF] Pratt | Role of hsp90 and the hsp90-binding immunophilins in signalling protein movement[END_REF][START_REF] Ratajczak | Immunophilin chaperones in steroid receptor signalling[END_REF][START_REF] Riggs | Functional specificity of co-chaperone interactions with Hsp90 client proteins[END_REF][START_REF] Pearl | Structure and mechanism of the Hsp90 molecular chaperone machinery[END_REF], and has been linked to many tissue-and organismspecific functions, including steroid receptor signaling in mammalian cells and yeast [START_REF] Ratajczak | Cyclophilin 40: An Hsp90cochaperone associated with apo-steroid receptors[END_REF], or microRNA activity and assembly of RNA-induced silencing complexes (RISCs) in plants [START_REF] Smith | Cyclophilin 40 is required for microRNA activity in Arabidopsis[END_REF]Ilki et al., 2012). No information is available on how this cochaperone affects viability and infectivity of trypanosomatid pathogens, including Leishmania, even though parasite differentiation is triggered by environmental stress signals [START_REF] Zilberstein | The role of pH and temperature in the development of Leishmania parasites[END_REF], has been previously linked to the formation of heat shock complexes [START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF], and is regulated by HSP90 activity [START_REF] Wiesgigl | Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani[END_REF]. Here by utilizing a targeted null mutant approach we reveal non-redundant and essential CyP40 functions in Leishmania stage-specific morphogenesis, motility, and the development of infectious-stage parasites.

We recently obtained first insight into the biological roles and functional diversity of L.

donovani CyPs and FKBPs using a pharmacological approach. Treatment of L. donovani promastigotes and axenic amastigotes with CsA and FK506 revealed important, redundant functions of CyPs and FKBPs in promastigote proliferation and morphogenesis, but unique function of Leishmania CyPs in parasite thermotolerance [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF]. The direct enrichment of CyP40 from L. donovani extracts using immobilized CsA, the presence of a conserved TPR domain in this protein, and its stage-specific phosphorylation in axenic amastigotes [START_REF] Morales | Phosphoproteomic analysis of Leishmania donovani pro-and amastigote stages[END_REF][START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF] suggested important stress functions of CyP40 in Leishmania differentiation and thermotolerance that may be regulated at posttranslational levels.

However, the CyP40 loss-of-function analysis performed in this study shed a substantially different light on the protein's unique functions. In contrast to CsA treated promastigotes that underwent non-synchronous cell cycle arrest at logarithmic growth phase and showed morphological changes reminiscent of axenic amastigotes, i.e. retraction of the flagellum and oval cell body [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF], cyp40-/-promastigotes did not show any overt defects in cell growth or cell shape at this culture stage. Moreover, absence of CyP40 expression did neither alter the IC50 to CsA nor did it affect the phenotypic response to this inhibitor (Fig S7 and data not shown) suggesting that CyP40 is not a major target of CsA at this culture stage. Finally, cyp40-/-parasites did not show any overt defects in resistance to various forms of stress, including nutritional starvation, acidic pH and elevated temperatures (Fig. 6D and S7), while CsA-treated parasites underwent cell death at 37°C [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF]. Thus, in contrast to other TPR domain containing chaperones in Leishmania, such as STI1 (Morales et al., 2010;Hombach et al., 2013) or SGT (Ommen et al., 2010), which are both essential for Leishmania promastigote viability, CyP40 carries non-essential functions at this stage, which may be redundant to other PPIases of the CyP or FKBP protein families and thus compensated in the null mutant.

Various aspects of the cyp40-/-null mutant phenotype pointed at essential and nonredundant CyP40 functions that were not observed during CsA treatment and thus are likely independent of the CyP40 PPIase activity targeted by this inhibitor [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF]. First, lack of CyP40 expression caused important morphological and structural alterations of stationary phase parasites, which adopted a rounded rather then characteristic needle-shaped morphology. These defects were linked to a loosening of the subpellicular tubulin network that determines parasite morphology and provides the rigidity necessary for efficient motility (Seebeck et al., 1990). These results point to unique CyP40 functions in regulating Leishmania cytoskeletal dynamics. Microtubule elongation and shortening are highly dynamic events that are regulated by stabilizing or destabilizing interactions [START_REF] Walczak | Microtubule dynamics and tubulin interacting proteins[END_REF]. Recently, a role of the CyP40 related immunophilin FKPB52 in regulating microtubule depolymerization through direct interaction with tubulin via its TPR domain has been described in rat brain [START_REF] Chambraud | The immunophilin FKBP52 specifically binds to tubulin and prevents microtubule formation[END_REF]. Given the capacity of CyP40 and FKBP52 to compete for molecular partners [START_REF] Ratajczak | Immunophilin chaperones in steroid receptor signalling[END_REF], it is possible that CyP40 interacts with microtubules and promotes tubulin polymerization to counteract FKBP52-mediated depolymerization. Alternatively, the requirement of CyP40 for Leishmania morphological integrity at stationary growth phase may be indirect and caused by misfolding of client proteins that require CyP40 co-chaperone function. In yeast, TPR-domain containing co-chaperones make distinct and extensive contacts with HSP90 that lead to a differential modulation of HSP90 chaperone function (Ratajcak et al., 2009). For example, displacement of STI1 from HSP90 by the yeast CyP40 homolog CPR6 restores ATPase activity [START_REF] Pearl | Structure and mechanism of the Hsp90 molecular chaperone machinery[END_REF]. Based on our previous observation that L. donovani STI1 acts as a scaffolding protein to assemble highly dynamic heat shock complexes [START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF], it is possible that lack of CyP40 alters the formation and activity of foldosome complexes causing the observed phenotypic consequences of CyP40 deletion.

Second, cyp40-/-parasites showed an important, stage-specific defect in directional motility despite integrity of the 9+2 structure of the axoneme. The presence of trypanosomatid CyP40 in the flagellar proteome [START_REF] Oberholzer | Independent analysis of the flagellum surface and matrix proteomes provides insight into flagellum signaling in mammalian-infectious Trypanosoma brucei[END_REF] indicates that LdCyP40 may directly interact with motor components for proper folding. This possibility is supported by various studies of related FKBPs showing (i) direct interaction between the FKBP52 PPIase domain and the motor protein dynein via dynamitin [START_REF] Silverstein | Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor, hsp90, and cytoplasmic dynein[END_REF][START_REF] Galigniana | Evidence that the peptidylprolyl isomerase domain of the hsp90-binding immunophilin FKBP52 is involved in both dynein interaction and glucocorticoid receptor movement to the nucleus[END_REF][START_REF] Galigniana | Cyclophilin-A is bound through its peptidylprolyl isomerase domain to the cytoplasmic dynein motor protein complex[END_REF]; (ii) an essential role of FKBP52 for sperm tail development and movement [START_REF] Hong | Deficiency of cochaperone immunophilin FKBP52 compromises sperm fertilizing capacity[END_REF]; and (iii) a requirement of FKBP12 in Trypanosoma brucei motility [START_REF] Brasseur | Trypanosoma brucei FKBP12 differentially controls motility and cytokinesis in procyclic and bloodstream forms[END_REF].

Page 16 of 49 Molecular Microbiology

Finally, the most striking loss-of-function phenotype of cyp40-/-parasites is represented by the abrogation of intracellular parasite survival. Unfortunately the L. donovani strain used in our analysis failed to establish hamster infection (Fig. S11) likely due to a non-specific loss of virulence that occurs during parasite culture adaptation [START_REF] Pescher | Quantitative proteome profiling informs on phenotypic traits that adapt Leishmania donovani for axenic and intracellular proliferation[END_REF]. While we cannot rule out that loss of intracellular survival reflects a developmental defect caused by the morphological and cytoskeletal alterations observed at stationary phase culture, our observations that null mutant parasites are normal in viability and metacyclic marker expression, and convert normally into axenic amastigotes may point to different possible scenarios. Intracellular killing of cyp40-/-mutants may be associated with the requirement of LdCyP40 co-chaperone activity for proper folding of parasite survival factors, which may confer parasite resistance to macrophage cytolytic activities. Alternatively, the identification of LdCyP40 in the parasite secretome [START_REF] Silverman | Proteomic analysis of the secretome of Leishmania donovani[END_REF] opens the possibility that this cochaperone may directly interact with host phagolysosomal or cytoplasmic proteins to subvert host cytolytic activities and favor intracellular parasite survival, a scenario reminiscent to secreted T. cruzi CyP19, which inactivates the anti-microbial peptide trialysin of the reduviid insect vector by direct binding [START_REF] Kulkarni | Secreted trypanosome cyclophilin inactivates lytic insect defense peptides and induces parasite calcineurin activation and infectivity[END_REF]. A third possibility is the involvement of CyP40 in the sorting of exosome payload proteins, similar to the role played by the chaperone HSP100 [START_REF] Silverman | Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells[END_REF]. This idea would be supported by the very similar phenotypes of CyP40 (this paper) and HSP100 null mutants [START_REF] Krobitsch | A novel role for 100 kD heat shock proteins in the parasite Leishmania donovani[END_REF].

In previous phosphoproteomics investigations we identified LdCyP40 as a major amastigote-specific phosphoprotein and revealed a coordinated increase in phosphorylation of all major parasite chaperones and HSPs during axenic amastigote differentiation, which occurred largely at parasite-specific sites [START_REF] Morales | Phosphoproteomic analysis of Leishmania donovani pro-and amastigote stages[END_REF][START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF][START_REF] Hem | Identification of Leishmania-specific protein phosphorylation sites by LC-MS/MS and comparative genomics analyses[END_REF]. These results are compatible with the hypothesis that Leishmania stress protein function is regulated at post-translational levels through phosphorylation by stress-activated protein kinases, a possibility that is supported by the genetic validation of two phosphorylation sites in L. donovani STI1 that are essential for parasite viability [START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF]. The failure of cyp40-/-parasites to establish intracellular infection provided a powerful read out to further test this hypothesis and to assess the physiological role of CyP40 phosphorylation by site-directed mutagenesis of the single CyP40 phosphorylation site. Surprisingly, although CyP40 phosphorylation at S274 was highly stage-specific and observed exclusively in axenic and bona fide splenic amastigotes, expression of an LdCyP40 S274A mutant fully restored intracellular survival of cyp40-/-parasites thus ruling out an essential role of this modification in intracellular infection in vitro. The biological function of this highly conserved phosphorylation site eludes us. Phosphorylation of FKBP52 disrupts the binding activity of this chaperone to HSP90 and thus is required for the dynamic turnover of heat shock complexes [START_REF] Miyata | Phosphorylation of the immunosuppressant FK506-binding protein FKBP52 by casein kinase II: regulation of HSP90-binding activity of FKBP52[END_REF]. The S274 CyP40 phosphorylation site maps to the TPR2 subdomain and thus may play a similar role in regulating the dynamics of protein-protein interactions, but may not cause a discernible phenotype due to the expression of other, functionally redundant TPR-domain proteins in Leishmania.

In conclusion, null mutant analysis of LdCyP40 revealed important non-redundant functions of this co-chaperone in parasite morphogenesis, cytoskeletal remodeling during stationary growth phase, motility, and infectivity. How LdCyP40 carries out its parasitespecific functions, and whether they depend on CyP40 isomerase and/or (co-)chaperone activities remains to be elucidated. In other organisms, CyP40 exerts its function mainly through binding to HSP90 [START_REF] Li | Mixed Hsp90-cochaperone complexes are important for the progression of the reaction cycle[END_REF]. There are several lines of evidence that CyP40 functions in Leishmania may not follow this established model as isolation of HSP90 complexes by anti-STI1 immuno-precipitation from L. donovani promastigotes and axenic amastigotes did not reveal LdCyP40 [START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF], although both co-chaperones can bind HSP90 simultaneously in other organisms [START_REF] Li | Mixed Hsp90-cochaperone complexes are important for the progression of the reaction cycle[END_REF]. In addition, while the Page 18 of 49 Molecular Microbiology LdCyP40 PPIase domain is highly conserved, the TPR domain underwent substantial evolutionary modification [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF], which may impact on LdCyP40-HSP interactions.

Future studies applying structure/function analyses to assess LdCyP40 isomerase and chaperone activities, combined with pull down assays and native electrophoresis, will elucidate the roles of the LdCyP40 PPIase and TPR domains in L. donovani differentiation, virulence and stress protein interaction. Mice, macrophages and parasites. Wild-type C57BL/6 mice were purchased from Charles River, and were kept and treated according to the institutional guidelines for animal experiments. Bone marrow-derived macrophages (BMMs) were prepared from the femurs of 6 -8 weeks old female mice as described [START_REF] Forestier | Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process[END_REF]. Leishmania donovani strain 1SR (MHOM/SD/62/1SR) was maintained at 25°C, pH 7.4 in supplemented M199 medium [START_REF] Kapler | Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression[END_REF][START_REF] Hubel | Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite[END_REF]. Axenic differentiation of promastigotes was performed as described [START_REF] Goyard | An in vitro system for developmental and genetic studies of Leishmania donovani phosphoglycans[END_REF][START_REF] Morales | Phosphoproteomic analysis of Leishmania donovani pro-and amastigote stages[END_REF]. [START_REF] Henikoff | Performance evaluation of amino acid substitution matrices[END_REF]. Sequence alignments with

Multiple sequences analysis.

Expect (E) values less than 10 -5 were considered significant.

Page 20 of 49 Molecular Microbiology

Generation of L. donovani cyp40-/mutants by gene replacement. Null mutant parasites

were generated by sequential replacement of the endogenous CYP40 alleles using two targeting constructs comprising puromycin and bleomycin resistance genes flanked by approximately 1000 bp of the 5' and 3'UTRs of the L. infantum CYP40 gene (GeneDB accession number LinJ.35.4830) that were PCR amplified from L. infantum genomic DNA (strain MHOM/FR/91/LEM2259). Genomic DNA of parasites was extracted with Gentra Systems Puregene Tissue Core Kit A (Qiagen) according to the manufacturer's instruction.

For subsequent cloning of the 5'UTR, primer pairs CYP40-5'UTR-fwd Acc65I and SwaI, the 5'-protruding end of the digested fragment was filled-in with Klenow fragment, and cloned into XbaI/BglII digested and blunt ended vector pIRmcs3+ [START_REF] Hoyer | A Leishmania donovani gene that confers accelerated recovery from stationary phase growth arrest[END_REF] yielding the final construct pIR-CYP40 used for integration into the small ribosomal subunit (SSU) locus of the cyp40-/-genome.

The plasmids were extracted from transformed E. coli DH5α, purified using CsCl continuous gradient centrifugation and linearized with SwaI. The targeting fragments were separated by agarose electrophoresis, purified and transfected into logarithmic promastigotes by electroporation as described [START_REF] Spath | Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major[END_REF][START_REF] Ommen | One-step generation of double-allele gene replacement mutants in Leishmania donovani[END_REF]. Integration of gene replacement constructs was verified by PCR using the primer pairs: (P1, CATTTGGCGCTTTTCATGC; P2, ATTACATCAGACGTAATCTG; P3, TGGAAAGTGCTACCCTGGTACGTC; P4, GTGGGCTTGTACTCGGTC; P5, GGAACGGCACTGGTCAAC). PCR products were analyzed by agarose gel electrophoresis.

Site-directed mutagenesis. Cloned L. major CYP40 in the construct pUC-CYP40+3'UTR was used as template for site-directed mutagenesis that is based on a modified PCR (Hombach et al., 2013). The template was amplified by PCR using the following phosphorylated oligonucleotide pairs (mutation sites were underlined) to introduce the mutation: CYP40-S274A-fwd (GCAGTGGGCGGAGGCTCGCCACACGGCGTC) and CYP40-S274-rev (TGGAGCTTGATGGCGCACATTG). The PCR products were ligated and used to transform competent E. coli DH5α (Invitrogen). Plasmids were extracted and purified for DNA sequencing to verify the mutation.

Cell counting. The cell density of parasite cultures were determined every 24 hours either using a CASY cell counter (Schärfe System) or microscopically by counting 2% (w/v) glutaraldeyhde fixed cells loaded into a Neubauer-improved cell counting chamber (Marienfeld).

Macrophage infection assays.

Isolation and differentiation of BMMs were performed as described [START_REF] Forestier | Imaging host cell-Leishmania interaction dynamics implicates parasite motility, lysosome recruitment, and host cell wounding in the infection process[END_REF]. The mature BMMs were treated with 20 mM EDTA for detachment. The dislodged cells were resuspended in complete RPMI medium and plated overnight in 24-well plates containing sterile 12-mm glass coverslips at a cell density of 1 × 10 5 cells/well. L. donovani promastigotes cultured for 6 days until stationary-phase (8 -10 × 10 7 /ml), or axenic amastigotes from logarithmic growth phase (1 -5 × 10 7 /ml) were washed three times with plain RPMI medium and once with infection medium (0.7% BSA, 25 mM HEPES [pH 7.4] in plain RPMI medium), incubated with BMMs at 37ºC with 5% CO 2 for 2 hours at a multiplicity of infection of 10 parasites per host cell. Extracellular parasites were then removed by washing the coverslips extensively with PBS and transfer of the cover slip into new 24-well plates containing 1 ml of fresh complete RPMI medium per well. This washing procedure was repeated daily until 5 days post infection. The plates were incubated for up to 5 days at 37ºC with 5% CO 2 during the experiment. Briefly, the fixed cells were treated with 1% (w/v) OsO 4 and dehydrated followed by criticalpoint drying (CPD 7501, Polaron) and coated with gold powder (Ion Beam Coater 681, Gatan).

Analysis

Samples were visualized with scanning electron microscope SEM 500 (Philips). For cytoskeleton analysis, cells were treated with 1% Triton X-100 at 4ºC in PBS for 10 minutes to strip the plasma membrane and visualize the cytoskeleton. The samples were then washed twice in PBS, fixed in glutaraldehyde and processed for scanning electron microscopy in standard conditions as described [START_REF] Absalon | Flagellum elongation is required for correct structure, orientation and function of the flagellar pocket in Trypanosoma brucei[END_REF].

Transmission electron microscopy. Parasites were washed twice in ice-cold PBS and then fixed with 2% (v/v) glutaraldehyde (Sigma) in PBS with 0.1 M sodium cacodylate (pH 7.2). The fixed cells were post-fixed in 1% (w/v) OsO 4 and then dehydrated with increasing ethanol concentrations (70-100%) and embedded in EPON epoxy resin (Hexion Speciality Chemicals). Ultrathin sections (~70 nm) were prepared using an Ultramicrotome (Ultra Cut E;

Reichert/Leica) and stained with uranyl acetate and lead citrate. Sections were analyzed using the Tecnai Sprit TEM (FEI).

Annexin V-FITC assay. Parasites (2 × 10 7 cells) were washed and resuspended in 100 µl of binding buffer containing 100 µg/ml of FITC conjugated annexin V (a gift from Dr. Aude Foucher, Photeomix, Paris), 1 µg/ml of PI, 10 mM HEPES (pH 7.4), 140 mM NaCl and 5 mM CaCl 2 . The suspensions were incubated at room temperature for 10 min in the dark. The stained cells were subjected to FACS analysis (λ ex = 488 nm; λ em = 515 nm [FITC] and 617 nm [PI])

with a FACSCalibur TM (BD Biosciences). 10,000 events were analyzed using FlowJo 8.7 (Tree Star).

SYBR Green-based semi-quantitative RT-PCR. SHERP cDNA was amplified using forward primer CGACAAGATCCAGGAGCTGAAGGAC and reversed primer CCTTGATGCTCTCAACCGTGCTG). Beta actin cDNA was amplified as reference gene to calculate relative expression levels of SHERP mRNA.

Motility analysis.

For each condition, at least 3 movies were recorded (500 frames at 33 ms of exposure for 16 s movies). Samples were observed in their respective culture media (cell density of about 5 × 10 6 cells/ml in logarithmic phase or 10 7 cells/ml in stationary phase) under a 10× objective of an inverted DMI4000 microscope (Leica). Cells were filmed using a numeric camera Photometrics Evolve 512 (Photometrics). Movies were converted with the MPEG Streamclip V1.9b3 software (Squared 5) and analyzed with the ICY software (http://www.bioimageanalysis.org/). For each movie, at least 255 cells were simultaneously tracked in silico. Mean speeds (in µm/s) were calculated from instant velocity data transferred and compiled in Microsoft Excel. Statistical analyses were performed in the KaleidaGraph V4.0 software (Synergy Software). Data were first filtered (number of spot detections > 3 and mean speed < 100 µm/s) to exclude false tracks (non-Leishmania objects, dead cells and/or attached cells). An ANOVA test was then performed with Tukey ad-hoc post-tests at 0.5 for intergroup comparisons (p < 0.0001).

Leishmania phosphoprotein enrichment. Promastigotes, axenic amastigotes, and splenic amastigotes derived from Leishmania infected hamsters, were centrifuged at 1000 × g at 4ºC for 10 min. Pellets were washed twice with plain M199 medium, then resuspended in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% Triton X-100, 1% benzonase, and protease inhibitor cocktail [Roche]) and lysed at 4ºC for 30 min with vortexing every 10 min. The lysate was then sonicated on ice for 5 min (10 seconds pulse with a 20 seconds pause) using Bioruptor (Diagenode). Cell debris was removed by brief centrifugation. Protein concentration of lysates was estimated by RC DC protein assay (Bio-rad) and adjusted to 0.1 mg protein/ml with lysis buffer. Phosphoprotein was enriched using the Phosphoprotein purification kit (Qiagen) according to the manufacturer's instructions. Briefly, 2.5 mg of total protein were loaded to the equilibrated columns. Unbound proteins were removed by washing the column with 6 ml of lysis buffer. Bound phosphorylated proteins were eluted with 2 ml elution buffer and concentrated using Amicon Ultra-0.5 ml centrifugal filters with Ultracel ® 10K membrane (Millipore).

Phosphopeptide identification. Cell lysate extracted from transgenic L. donovani expressing Strep::CyP40 (refer to supporting information) was separated by SDS-PAGE. Separated

Strep::CyP40 was trypsin-digested and diluted in loading buffer (80% ACN, 5% TFA). The sample was then processed for LC-ESI-MS/MS analysis to identify phosphorylation sites, as described [START_REF] Morales | Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage[END_REF]. Briefly, digested protein was diluted in loading buffer (80% ACN, 5% TFA) and loaded on TiO 2 microcolumns. After two washing steps in 10 µl loading buffer and 60 µl buffer 2 (80% ACN, 1% TFA), phosphopeptides were eluted using 10 µl NH 4 OH (pH 12), dried, then resuspend in 10 ul of 0.1% formic acid before analyzing with a Q-TOF mass spectrometer (Maxis; Bruker Daltonik GmbH, Bremen, Germany), interfaced with a nano-HPLC U3000 system (Thermo Scientific, Waltham, USA). Samples were concentrated with a pre-column (Thermo Scientific, C18 PepMap100, 300 µm × 5 mm, 5 µm, 100 A) at a flow rate of 20 µL/min using 0.1% formic acid. After pre-concentration, peptides were separated with a reversed-phase capillary column (Thermo Scientific, C18 PepMap100, 75 µm × 250 mm, 3 µm, 100 A) at a flow rate of 0.3 µL/min using a two-step gradient (8 % to 28 % acetonitrile in 40 min then 28 % to 42 % in 10 min), and eluted directly into the mass spectrometer. Proteins were identified by MS/MS by information-dependent acquisition of fragmentation spectra of multiple charged peptides and MS/MS raw data were analysed using Data Analysis software (Bruker Daltonik GmbH, Bremen, Germany) to generate the peak lists.

The L. major database from GeneDB was searched using MASCOT software (Matrix Science, London, UK) with the following parameters: trypsin cleavage, one missed cleavage sites allowed, carbamidomethylation set as fixed modification, 10 ppm mass tolerance for MS, and 0.05 Da for MS/MS fragment ions. Phosphorylation (ST), Phosphorylation (Y) and oxidation (M) were allowed as variable modifications. All phosphorylated peptides were first checked for the presence of the major fragment ion [MH-H 3 PO 4 ]+ = MH -98 Da corresponding to the loss of the phosphate moiety and identified positively by MASCOT. In addition, all MS/MS spectra were carefully checked manually for assignment of phosphorylation sites.

Western blot analysis. Samples in 1 × Laemmli buffer were separated by 10% SDS-PAGE and then transferred to PVDF membranes. Rabbit anti-CyP40 antiserum (1:20000 dilution, [START_REF] Yau | Cyclosporin A treatment of Leishmania donovani reveals stage-specific functions of cyclophilins in parasite proliferation and viability[END_REF], mouse anti-A2 antibody (clone C9, 1:50 dilution, [START_REF] Zhang | Identification and overexpression of the A2 amastigote-specific protein in Leishmania donovani[END_REF], mouse anti-LPG antibody (clone CA74E, 1:20000 dilution, [START_REF] Tolson | Distribution of lipophosphoglycan-associated epitopes in different Leishmania species and in African trypanosomes[END_REF], mouse anti-α-tubulin antibody (clone B-5-1-2, 1:20000 dilution, Sigma), goat anti-mouse HRP conjugated antibody

(1:20000 dilution, Thermo Scientific) and goat anti-rabbit HRP conjugated antibody (1:20000 dilution, Thermo Scientific) were used to probe the membrane. SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) was added to the membranes and chemiluminescent signals were revealed on X-ray films.

2D-DIGE analysis. The analysis was performed as previously described [START_REF] Pescher | Quantitative proteome profiling informs on phenotypic traits that adapt Leishmania donovani for axenic and intracellular proliferation[END_REF].

Briefly, 50 µg of G-Dye-labeled total protein extracts from wild-type and cyp40-/promastigotes from stationary growth phase were separated in the first dimension by IEF on 18 cm pH 4 to 7 IPG strips, and the second dimension by SDS-PAGE. Three gels representing independent experimental replicates were scanned with a Typhoon imager and analyzed with the DeCyder TM software package from GE Healthcare. Gels were stained with Lava purple, revealed by scanning on a Typhoon imager, and differentially expressed proteins were subjected to MS analysis.

Statistical analysis. Numerical data were expressed as mean ± standard deviation. Mann-Whitney U test was used to compare the significance between specific groups and p < 0.05 was considered as statistically significant. All statistical analysis was performed using either Excel

  the genus Leishmania are the etiological agents of leishmaniases, severe human diseases with clinical manifestations ranging from self-curing cutaneous lesions to fatal visceral infection. During the infectious cycle, Leishmania replicate as extracellular flagellated

  All animals were handled according to institutional guidelines of the Central Animal Facility of the Institut Pasteur (Paris, France) and experiments were performed in accordance with protocols approved by the animal Experimentation Ethics Committee of the Institut Pasteur (permit #03-49) and the veterinary service of the French Ministry of Agriculture (number B-75-1159, 30 May 2011). The animals were housed and handled in accordance with good animal practice as defined by FELASA (www.felasa.eu/guidelines.php).

  added EcoRI and SwaI sites (underlined) to the 5'-end, and a KpnI site to the 3'-end. For cloning of the 3'UTR, primer pairs CYP40-3'UTR-fwd (GGGGGATCCGTTGCGCCTGCCGGACAGAAC) and CYP40-3'UTR-rev (CCCAAGCTTATTTAAATGAGTGAGACGTGCACGCAAC) added BamHI site to the 5'end, and HindIII and SwaI sites to the 3'-end. The amplified fragments were digested with EcoRI/KpnI and BamHI/HindIII and ligated into pUC19 vector digested with the same enzyme pairs yielding the constructs pUC-CYP40-5'UTR and pUC-CYP40-3'UTR. Next, the cloned 5'UTR fragment was liberated by EcoRI and KpnI digestion, ligated into pUC-CYP40-3'UTR digested with the same enzyme pair yielding the construct pUC-CYP40-5'3'UTR. For the construction of the final targeting constructs, the antibiotic resistance genes PAC and BLE were isolated from pUC-PAC and pUC-BLE after digestion with KpnI and BamHI and ligated into pUC-CYP40-5'3'UTR digested with the same enzymes, yielded the final constructs pUC-CYP40-5'PAC3' and pUC-CYP40-5'BLE3' used for Leishmania CYP40 gene replacement.For genetic complementation of the CyP40 null mutant, an "add-back" line was established using a knock-in strategy. The CYP40 open reading frame was PCR amplified using adding KpnI and BglII sites (underlined) to the 5'-and 3'-ends of the LmCYP40, respectively. The amplified fragment was digested with these enzymes, ligated into pUC-CYP40-3'UTR digested with KpnI and BamHI yielding the construct pUC-CYP40+3'UTR. The cloned CYP40+3'UTR was liberated by digestion with

  of intracellular parasite burden. Coverslips of quadruplicate experiments were collected at 0, 24 and 48 hours post infection, washed once with PBS and then fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature. After three further washes with PBS and one wash with ddH 2 O, the coverslips were mounted with Mowiol medium containing nuclear dye Hoechst 33342 (Invitrogen). At least 100 macrophages were imaged per coverslip using Axioplan 2 wide field light microscope with the Apotome module (Carl Zeiss). Number of parasites and macrophages was estimated counting host cell and parasite nuclei using ImageJ (National Institute of Health, US) using a predefined pixel range for each cell type (400 to infinity pixels, macrophages; 0 to 150 pixels, parasites; 100 to 200 pixels, CFSE labeled parasites in case of mixed infection assay).Microscopic analyses. Giemsa staining analysis. Parasites (1 × 10 6 ) were settled on poly-Llysine coated glass slides and air-dried overnight. The dried cells were fixed with methanol and stained with Giemsa solution (Sigma) for 10 min. After rinsing with water and air-drying, the slides were mounted with Immersol 518N (Carl Zeiss) and observed under an Axioplan 2 wide field light microscope (Carl Zeiss). The acquired images were analyzed by Zeiss Axiovision release 4.7 (Carl Zeiss) or Openlab v3.0 (Improvision).Scanning electron microscopy. Parasites were washed twice in ice-cold PBS and then fixed with 2% (w/v) glutaraldehyde (Sigma) in PBS with 0.1 M sodium cacodylate (pH 7.2).

  2003 (Microsoft) or Prism 5.0 (GraphPad). We thank Dirk Schmidt-Arras (University of Kiel), Gabi Ommen, Mareike Chrobak and Christel Schmetz (Bernhard Nocht Institute, Hamburg) for discussion, Eric Prina for critical reading of the manuscript, Claire Forestier and Evie Melanitou (Institut Pasteur) for advices on macrophage experiment, Emanuelle Perret from the Institut Pasteur Imaging platform for advice on fluorescence microscopy, and Abdelkader Namane, Pascal Normand, and Christine Laurent from the Institut Pasteur Proteomics platform for protein determination and quantitative analysis of the 2D-DIGE gels. This work was supported by the Institut Pasteur, Réseau International des Instituts Pasteur, Centre Nationale de Recherche Scientifique, the 7th Framework Programme of the European Commission through a grant to the LEISHDRUG Project (223414), the Fondation de Médicale Recherche and Deutscher Akademischer Austausch Dienst, and the French Government's Investissements d'Avenir program: Laboratoire d'Excellence 'Integrative Biology of Emerging Infectious Diseases' (grant no. ANR-10-LABX-62-IBEID).

Figure legends Fig 1 .

 legends1 Figure legends

Fig. 2 .

 2 Fig. 2. CyP40 null mutant parasites are viable in culture. (A) Targeted replacement and

Fig. 3 .

 3 Fig. 3. Stationary cyp40-/promastigotes show altered morphology. (A, B) Electron

Fig 4 .Fig. 5 .

 45 Fig 4. Growth characterization of Cyp40 null mutant promastigotes. (A) In vitro growth

Fig. 6 .Fig 7 .

 67 Fig. 6. Intracellular survival and axenic differentiation of the cyp40-/null mutants. (A)

Figure 22 .

 22 Figure 22. Amino acid sequence analysis of Leishmania CyP40. Multiple alignment of CyP40 amino acid sequences of mammal (human, mouse and bovine), trypanosomatid (Leishmania and Trypanosoma) and yeast was performed using ClustalW as described in Section 2.2.1. CLD/PPIase and TPR domains are indicated. Degree of residue conservation is represented by the colour code (Green, highly conserved; yellow, medium conserved; red, less conserved). *, key residues for cyclosporin A binding and PPIase activity; ^, key residues for chaperone function; !, key residues for HSP90 binding; #, phosphorylated residue in L. donovani.

re 22 .

 22 Amino acid sequence analysis of Leishmania CyP40. Multiple alignment of CyP40 amino sequences of mammal (human, mouse and bovine), trypanosomatid (Leishmania and Trypanosoma) east was performed using ClustalW as described in Section 2.2.1. CLD/PPIase and TPR domains are ated. Degree of residue conservation is represented by the colour code (Green, highly conserved; w, medium conserved; red, less conserved). *, key residues for cyclosporin A binding and PPIase ity; ^, key residues for chaperone function; ), key residues for HSP90 binding; #, phosphorylated ue in L. donovani.

Page 7 of 49 Molecular Microbiology phenotype

  , most of the subsequent experiments were performed with CyP40 null mutant clone 4 and its corresponding add-back pool if not otherwise stated.

M.mus H.sap B.tau S.cer T.cru T.bru L.bra L.mex L.inf L.maj

	M.mus H.sap
	B.tau S.cer T.cru T.bru
	L.bra L.mex L.inf L.maj
	M.mus H.sap
	B.tau S.cer T.cru T.bru
	L.bra L.mex L.inf L.maj
	M.mus H.sap
	B.tau S.cer T.cru T.bru
	L.bra L.mex L.inf L.maj

ManuscriptYau et al., 18.04.2014

Acknowledgments