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

Wai-Lok Yau, Pascale Pescher, Andrea Macdonald, Sonia Hem, Dorothea
Zander, Silke Retzlaff, Thierry Blisnick, Brice Rotureau, Heidi Rosenqvist,
Martin Wiese, et al.

► To cite this version:

Wai-Lok Yau, Pascale Pescher, Andrea Macdonald, Sonia Hem, Dorothea Zander, et al. The Leishmania donovani chaperone cyclophilin 40 is essential for intracellular infection independent of its stage-specific phosphorylation status. Molecular Microbiology, 2014, 93 (1), pp.80-97. 10.1111/mmi.12639. hal-01054664

HAL Id: hal-01054664

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Submitted on 13 Feb 2019

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

3 Wai-Lok Yau¹,², Pascale Pescher¹, Andrea MacDonald², Sonia Hem³, Dorothea Zander², Silke

4 Retzlaff³⁺, Thierry Blisnick⁴, Brice Rotureau⁴, Heidi Rosenqvist⁵⁺⁺, Martin Wiese⁵, Philippe

5 Bastin⁴, Joachim Clos², and Gerald F. Späth¹,*

6
7 Institut Pasteur and Centre National de la Recherche Scientifique URA 2581, ¹Unité de

8 Parasitologie Moléculaire et Signalisation, and ⁴Unité de Biologie Cellulaire des Trypanosomes,

9 25 rue du Dr Roux, F-75015 Paris, France; ²Clos Group (Leishmanasis) and ³Electron

10 Microscopy Service, Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Str. 74, D-

11 20359 Hamburg, Germany ³Plate-forme de spectrométrie de masse protéomique - MSPP,

12 Biochemie et Physiologie Moléculaire des Plantes, UMR 5004 CNRS/UMR 0386

13 INRA/Montpellier SupArgo/Université Montpellier II, F-34060 Montpellier, France;

14 ⁵Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde,
15 Glasgow, Scotland, UK

16
17 * Correspondence: Gerald Späth

18 Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France

19 Phone: +33.1.40.61.38.58; Fax: +33.1.45.68.83.33

20 E-mail: gerald.spaeth@pasteur.fr

21 Short title: LdCyP40 null mutant analysis

22 Key words: *Leishmania donovani*, stress protein, cyclophilin 40, null mutant analysis,
23 intracellular survival, chaperone phosphorylation

24 +current address: Altona Diagnostics, Moerkenstr. 12, 22767 Hamburg, Germany

25 ++current address: Novo Nordisk, Novo Nordisk Park 1, DK-2760 Maaloev, Denmark
## 26 Summary

During its life cycle, the protozoan pathogen *Leishmania donovani* is exposed to contrasting environments inside insect vector and vertebrate host, to which the parasite must adapt for extra- and intracellular survival. Combining null mutant analysis with phosphorylation site-specific mutagenesis and functional complementation we genetically tested the requirement of the *Leishmania donovani* chaperone cyclophilin 40 (LdCyP40) for infection. Targeted replacement of LdCyP40 had no effect on parasite viability, axenic amastigote differentiation, and resistance to various forms of environmental stress in culture, suggesting important functional redundancy to other parasite chaperones. However, ultra-structural analyses and video microscopy of *cyp40-/-* promastigotes uncovered important defects in cell shape, organization of the subpellicular tubulin network and motility at stationary growth phase. More importantly, *cyp40-/-* parasites were unable to establish intracellular infection in murine macrophages and were eliminated during the first 24 hours post infection. Surprisingly, *cyp40-/-* infectivity was restored in complemented parasites expressing a CyP40 mutant of the unique S274 phosphorylation site. Together our data reveal non-redundant CyP40 functions in parasite cytoskeletal remodeling relevant for the development of infectious parasites *in vitro* independent of its phosphorylation status, and provides a framework for the genetic analysis of *Leishmania*-specific phosphorylation sites and their role in regulating parasite protein function.

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46 **Introduction**

47 Parasitic protozoa of the genus *Leishmania* are the etiological agents of leishmaniases, severe
48 human diseases with clinical manifestations ranging from self-curing cutaneous lesions to fatal
49 visceral infection. During the infectious cycle, *Leishmania* replicate as extracellular flagellated
50 promastigotes in the midgut of female phlebotomine sand flies where they undergo an
51 environmentally triggered differentiation process termed metacyclogenesis to develop into
52 highly infectious metacyclic promastigotes (Sacks *et al.*, 1984). Following transmission by
53 blood feeding sand flies, metacyclic parasites are engulfed by phagocytes such as macrophages,
54 where they differentiate into non-motile amastigotes that cause the pathology of the disease.

55 *In vitro* differentiation of infectious metacyclic parasites is induced by nutritional
56 starvation or acidic pH, and development of amastigote-like cells in culture is triggered by pH
57 and temperature change (Zakai *et al.*, 1998; Cunningham *et al.*, 2001; Barak *et al.*, 2005),
58 suggesting that *Leishmania* stage-differentiation occurs through sensing of environmental stress
59 signals encountered inside insect and vertebrate hosts (Zilberstein *et al.*, 1994). Recently, a role
60 of stress signaling in parasite development has been suggested based on phosphoproteomics
61 studies. Gel-based proteomics approaches comparing enriched phosphoprotein fractions from
62 promastigotes and axenic amastigotes revealed a surprising divergence of the stage-specific
63 phosphoproteome with more than 30% of proteins showing statistically significant differential
64 phosphorylation (Morales *et al.*, 2008; Morales *et al.*, 2010). Phosphorylation events in
65 amastigotes were almost exclusively restricted to heat shock proteins and chaperones and
66 occurred largely at parasite-specific phosphorylation sites (Hem *et al.*, 2010), suggesting
67 regulation of the *Leishmania* response to stress by post-translational rather than classical
68 transcriptional mechanisms. This possibility has been investigated previously for the stress-
69 induced protein 1 (STI1), a chaperone that acts as a scaffolding protein for the assembly of
70 foldosome complexes (Pratt and Dittmar, 1998). 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 (Morales *et al.*, 2010).

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 (Barik, 2006). In a previous study, we
revealed stage-specific functions of cyclophilins using the CyP inhibitor cyclosporin A (CsA)
(Yau *et al.*, 2010). 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
(Wiesgigl and Clos, 2001). 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* (Yau *et al.*, 2010), the pleiotropic inhibition of multiple
cyclophilins and the phosphatase calcineurin by CsA-CyP complexes (Chappell and Wastling,
1992) 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) (Yau *et al.*, 2010), and its stage-specific 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
(Hoffmann and Handschumacher, 1995). 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* (Yau *et al.*, 2010). 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 (Yau *et al.*, 2010). 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 (Mok *et al.*, 2006). 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 (Ward et al., 2002). 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 (Morales *et al.*, 2008; Morales *et al.*, 2010).
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.

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146 *Establishment of cyp40-/- null mutants.*

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

157

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

159 Microscopic observation of the two independent null mutant clones *cyp40-/-* cl.4 and cl.5 and
160 four independent add-back clones as well as add-back pools derived from each null mutant
161 clone revealed important morphological alternations at stationary growth phase that correlated
162 with the absence of CyP40 expression (Fig. S4A). At logarithmic growth phase, CyP40 null
163 mutant promastigotes were morphologically identical to wild-type and add-back controls as
164 judged by Giemsa staining of fixed parasite cultures (Fig. S4A, left panels). By contrast 70% of
165 *cyp40-/-* cells at stationary growth phase of two independent null mutant clones acquired a
166 small spherical cell shape, while wild-type parasites and add-back clones showed the spindle
167 shaped cell body characteristic for stationary phase parasites (Fig. S4A, right panels).
168 Moreover, measurements of cell body and flagellum length revealed a significant reduction of
169 the ratio between both parameters in *cyp40-/-* cells (Fig. S4B). Given the reproducibility of this
170 phenotype, most of the subsequent experiments were performed with CyP40 null mutant clone
171 4 and its corresponding add-back pool if not otherwise stated.

172 As cell rounding is often associated with cellular stress and apoptotic cell death (Lee *et al.*, 2002; Gannavaram and Debrabant, 2012), we next investigated integrity and ultra-structural
173 organization of CyP40 null mutant cells and controls. Wild-type, *cyp40-/-* and *cyp40-/-/+*
174 promastigotes were fixed with 2% glutaraldehyde and analyzed by scanning electron
175 microscopy (Fig. 3A). This analysis confirmed the atypical spherical cell shape of *cyp40-/-*
176 stationary phase promastigotes and provided a first indication of their cellular integrity.
177 Likewise, analysis by transmission electron microscopy revealed no overt alterations in cellular
178 organization of *cyp40-/-* cells, which showed intact nuclei and mitochondria, and absence of
179 apoptotic bodies or autophagosomes (Fig. 3B). Finally, direct assessment of cell death by
180 FACS analysis following membrane exposure of phosphatidylserine with annexin V-FITC and
181 exclusion of propidium iodide did not reveal any significant change in cell viability of
182 stationary *cyp40-/-* parasites after 6 days of *in vitro* culture (Fig. 3C). Together these results
183 rule out the possibility of cell death as the cause of *cyp40-/-* morphological alterations but
184 rather suggest a defect in parasite development and cellular remodeling at stationary phase.

186

187 *Phenotypic characterization of stationary phase cyp40-/- promastigotes.*

188 During the infectious cycle, non-infectious procyclic promastigotes differentiate into highly
189 infective metacyclic promastigotes in response to nutritional starvation after digestion and
190 excretion of the blood meal. This process is mimicked in culture during stationary growth
191 phase (Sacks *et al.*, 1984; da Silva and Sacks, 1987) and is accompanied by the expression of
192 characteristic biological and morphological markers, including expression of high molecular
193 weight LPG and increase in SHERP mRNA abundance. The altered morphology of *cyp40-/-*
194 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 (Walters *et al.*, 1989; Walters *et al.*, 1993), or the establishment of host cell infection (Uezato *et al.*, 2005; Forestier *et al.*, 2011). 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 (Drummelsmith *et al.*, 2003). Similar low molecular weight isoforms have been identified in the stage-specific proteomes of *L. major* and *T. cruzi* metacyclic parasites (Parodi-Talice *et al.*, 2007; Mojtahedi *et al.*, 2008), 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
244 growth phase, which is likely the basis for the aberrant morphology and the motility defect
245 observed in *cyp40-/-* parasites.

246

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

248 The development of highly infectious metacyclic parasites can be mimicked by exposing
249 promastigotes to various forms of stress, including nutritional starvation and acidification at
250 stationary phase (da Silva and Sacks, 1987; Bate and Tetley, 1993; Zakai et al., 1998; Serafim
251 et al., 2012). The stationary phase-specific defects of *cyp40-/-* promastigotes primed us to
252 investigate their capacity to infect host cells. Bone marrow-derived macrophages obtained from
253 C57BL/6 mice were incubated for 2 hours with WT, *cyp40-/-*, and add-back stationary phase
254 promastigotes at a multiplicity of infection of 10 parasites per host cell, free parasites were
255 removed by washing, and intracellular parasite burden was determined by nuclear staining and
256 fluorescence microscopy at 2, 24, and 48 hours post infection. The *cyp40-/-* cells were
257 phagocytosed at levels comparable to the controls with 80% of macrophages hosting a mean of
258 2.5 parasites per infected host cell (Fig. 6A). While WT and add-back parasites persisted over
259 the 48 hours post infection period and showed intracellular proliferation as documented by the
260 increase in number of parasites per infected macrophage (Fig. 6A, lower panel), the level of
261 *cyp40-/-* infection was reduced by over 90% during the first 24 hours and parasites were
262 completely eliminated by 48 hours post infection (Fig. 6A). We next tested if intracellular
263 elimination of *cyp40-/-* parasites is caused by a susceptibility to elevated temperature or acidic
264 pH encountered inside the host cell, or results from a defect in amastigote differentiation.
265 CyP40 null mutant promastigotes grown at 37°C did not show any significant difference to WT
266 and add-back parasites (Fig. S7A), and axenic amastigotes developed and proliferated normally
267 in response to both temperature and pH shift as judged by cell counting (Fig. 6B) and
268 assessment of expression of the axenic amastigote marker protein A2 (Fig. 6C). Thus,
269 LdCyP40 carries essential functions for successful host cell infection independent of parasite
270 resistance to acidic pH, elevated temperature, or amastigote development.

271

272 Functional assessment of CyP40 phosphorylation.

273 The failure of *cyp40-/-* parasites to establish intracellular infection provided an interesting read
274 out to test for the function of CyP40 phosphorylation in parasite infectivity using
275 complementation analysis. We investigated the CyP40 amino acid residues that were
276 phosphorylated by using LC-ESI-MS/MS analysis. Axenic amastigote protein extracts from
277 transgenic *L. donovani* over-expressing Strep::CyP40 were collected and Strep::CyP40 was
278 enriched by affinity chromatography using streptactin agarose liquid chromatography (Fig. S8).
279 After SDS-PAGE and Coomassie Brilliant Blue staining, the band corresponding to
280 Strep::CyP40 was extracted and subjected to phosphopeptide analysis by LC-ESI-MS/MS.
281 analysis revealed that residue serine 274 (S274) of CyP40 was the only detected
282 phosphorylation site of CyP40 in axenic amastigotes (Fig. 7A and Fig. S9). The multiple
283 sequence alignment of trypanosomatid CyP40 described in Fig. 1 localizes S274 to the center
284 of the TPR motif 2 and shows that it is unique among trypanosomatids (Fig. 7B). An
285 independent study also pointed out that S274 is phosphorylated in *L. mexicana* (Dr. Martin
286 Wiese, University of Strathclyde, Glasgow, unpublished data). These data suggest that
287 phosphorylation at S274 of CyP40 is *Leishmania*-specific and may have critical roles in
288 parasite survival.

289 To study the relationship between CyP40 phosphorylation and parasite infectivity, an
290 add-back line was generated expressing a serine 274 to alanine (S274A) phosphorylation site
291 mutant version of CyP40 (Fig. 7C, left panel, and Fig. S10), which was subsequently tested for
292 *in vitro* infectivity. We next assessed the accumulation of this mutant in the parasite phospho-
293 protein 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 wild-type phospho-protein extracts, and this signal was strongly reduced by phosphatase treatment prior to enrichment representing background levels of CyP40 enrichment likely due to non-specific 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-to-batch 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.

315

316
317 **Discussion**

318 The conserved co-chaperone CyP40 participates in the formation of highly dynamic heat shock
319 protein complexes across various eukaryotes (Pratt et al., 2004; Ratajczak et al., 2003; Riggs et
320 al., 2004; Pearl and Prodromou, 2006), and has been linked to many tissue- and organism-
321 specific functions, including steroid receptor signaling in mammalian cells and yeast (Ratajczak
322 et al., 2009), or microRNA activity and assembly of RNA-induced silencing complexes (RISCs)
323 in plants (Smith et al., 2009; Ilki et al., 2012). No information is available on how this co-
324 chaperone affects viability and infectivity of trypanosomatid pathogens, including Leishmania,
325 even though parasite differentiation is triggered by environmental stress signals (Zilberstein
326 and Shapira, 1994), has been previously linked to the formation of heat shock complexes
327 (Morales et al., 2010), and is regulated by HSP90 activity (Wiesgigl and Clos, 2001). Here by
328 utilizing a targeted null mutant approach we reveal non-redundant and essential CyP40
329 functions in Leishmania stage-specific morphogenesis, motility, and the development of
330 infectious-stage parasites.

331 We recently obtained first insight into the biological roles and functional diversity of *L.
332 donovani* CyPs and FKBPs using a pharmacological approach. Treatment of *L. donovani*
333 promastigotes and axenic amastigotes with CsA and FK506 revealed important, redundant
334 functions of CyPs and FKBPs in promastigote proliferation and morphogenesis, but unique
335 function of *Leishmania* CyPs in parasite thermotolerance (Yau *et al.*, 2010). The direct
336 enrichment of CyP40 from *L. donovani* extracts using immobilized CsA, the presence of a
337 conserved TPR domain in this protein, and its stage-specific phosphorylation in axenic
338 amastigotes (Morales *et al.*, 2008; Morales *et al.*, 2010) suggested important stress functions of
339 CyP40 in *Leishmania* differentiation and thermotolerance that may be regulated at post-
340 translational 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 (Yau *et al.*, 2010), *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 (Yau *et al.*, 2010). 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 non-redundant CyP40 functions that were not observed during CsA treatment and thus are likely independent of the CyP40 PPIase activity targeted by this inhibitor (Yau *et al.*, 2010). First, lack of CyP40 expression caused important morphological and structural alterations of stationary phase parasites, which adopted a rounded rather than 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
Manuscript Yau et al., 18.04.2014

are regulated by stabilizing or destabilizing interactions (Walczak, 2000). 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 (Chambraud et al., 2007). Given the capacity of CyP40 and FKBP52 to compete for molecular partners (Ratajczak et al., 2003), 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 (Pearl and Prodromou, 2006). Based on our previous observation that *L. donovani* STI1 acts as a scaffolding protein to assemble highly dynamic heat shock complexes (Morales et al., 2010), 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 (Oberholzer *et al.*, 2011) indicates that LdCyP40 may directly interact with motor components for proper folding. This possibility is supported by various studies of related FKBP52 showing (i) direct interaction between the FKBP52 PPIase domain and the motor protein dynein via dynamitin (Silverstein *et al.*, 1999; Galigniana *et al.*, 2001; Galigniana *et al.*, 2004); (ii) an essential role of FKBP52 for sperm tail development and movement (Hong *et al.*, 2007); and (iii) a requirement of FKBP12 in *Trypanosoma brucei* motility (Brasseur *et al.*, 2013).
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 (Pescher et al, 2011). 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 (Silverman *et al.*, 2008) opens the possibility that this co-chaperone 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 (Kulkarni *et al.*, 2013). A third possibility is the involvement of CyP40 in the sorting of exosome payload proteins, similar to the role played by the chaperone HSP100 (Silverman *et al.*, 2010). This idea would be supported by the very similar phenotypes of CyP40 (this paper) and HSP100 null mutants (Krobitsch and Clos, 1999).

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 (Morales *et al.*, 2008; Morales *et al.*, 2010; Hem *et al.*, 2010). 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 (Morales *et al.*, 2010). 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 (Miyata *et al.*, 1997). 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 parasite-specific 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 (Li *et al.*, 2011). 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 (Morales *et al.*, 2010), although both co-chaperones can bind HSP90 simultaneously in other organisms (Li *et al.*, 2011). In addition, while the
441 LdCyP40 PPIase domain is highly conserved, the TPR domain underwent substantial
442 evolutionary modification (Yau *et al.*, 2010), which may impact on LdCyP40-HSP interactions.

443 Future studies applying structure/function analyses to assess LdCyP40 isomerase and
444 chaperone activities, combined with pull down assays and native electrophoresis, will elucidate
445 the roles of the LdCyP40 PPIase and TPR domains in *L. donovani* differentiation, virulence and
446 stress protein interaction.

447

448
449 **Experimental procedures**

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

456

457 **Mice, macrophages and parasites.** Wild-type C57BL/6 mice were purchased from Charles
458 River, and were kept and treated according to the institutional guidelines for animal
459 experiments. Bone marrow-derived macrophages (BMMs) were prepared from the femurs of 6
460 – 8 weeks old female mice as described (Forestier *et al.*, 2011). *Leishmania donovani* strain
461 1SR (MHOM/SD/62/1SR) was maintained at 25°C, pH 7.4 in supplemented M199 medium
462 (Kapler *et al.*, 1990; Hubel *et al.*, 1997). Axenic differentiation of promastigotes was performed
463 as described (Goyard *et al.*, 2003; Morales *et al.*, 2008).

464

465 **Multiple sequences analysis.** The putative cyclophilin 40 protein sequences of human, mouse,
466 bovine, yeast and several trypanosomatids, including *L. major*, *L. infantum*, *L. braziliensis*, *T.
brucei* and *T. cruzi*, were retrieved from the NCBI (http://www.ncbi.nlm.nih.gov/protein/) and
467 TriTrypDB databases (http://tritrypdb.org/tritrypdb/) by Blast search with algorithm blastp
468 using *L. major* cyclophilin 40 (LmjF35.4770) as a query. The retrieved sequences were aligned
469 with ClustalW 2.0.12 using the built-in programs of Geneious version 4.8 (Larkin *et al.*, 2007;
470 Kearse *et al.*, 2012; http://www.geneious.com). Matrix Blosum62 was applied as the scoring
471 rule for amino acid substitution (Henikoff and Henikoff, 1993). Sequence alignments with
472 Expect (E) values less than 10⁻⁵ were considered significant.
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**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.

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For subsequent cloning of the 5'UTR, primer pairs CYP40-5'UTR-fwd (GGGGAATTCATTTAAATGAGATGCGGTAGATGTCCTG) and CYP40-5'UTR-rev (GGGGGTACCTGCGTGCGTGTAGGTCTAC) 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.

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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
Manuscript Yau et al., 18.04.2014

primers CYP40-5'Kpn (GGGGGTACCATGCCGAACACATACTGC) and CYP40-3'BglII (GGGAGATCTTCACGAGAACATCTTCTTG), 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 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+ (Hoyer *et al.*, 2004) 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 (Spath *et al.*, 2000; Ommen *et al.*, 2009). 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) glutaraldehyde fixed cells loaded into a Neubauer-improved cell counting chamber (Marienfeld).

**Macrophage infection assays.** Isolation and differentiation of BMMs were performed as described (Forestier *et al.*, 2011). 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 $1 \times 10^5$ cells/well. *L. donovani* promastigotes cultured for 6 days until stationary-phase ($8 - 10 \times 10^7/ml$), or axenic amastigotes from logarithmic growth phase ($1 - 5 \times 10^7/ml$) 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 37°C with 5% CO₂ 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 37°C with 5% CO₂ during the experiment.

**Analysis 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 ddH₂O, 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 ($1 \times 10^6$) were settled on poly-L-lysine 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). Briefly, the fixed cells were treated with 1% (w/v) OsO₄ and dehydrated followed by critical-point drying (CPD 7501, Polaron) and coated with gold powder (Ion Beam Coater 681, Gatan). Samples were visualized with scanning electron microscope SEM 500 (Philips). For cytoskeleton analysis, cells were treated with 1% Triton X-100 at 4°C 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 (Absalon *et al.*, 2008).

*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) OsO₄ 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 × 10⁷ 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, Photoemix, Paris), 1 µg/ml of PI, 10 mM HEPES (pH 7.4), 140 mM NaCl and 5 mM CaCl₂. The suspensions were incubated at room temperature for 10 min in the dark. The stained cells were subjected to FACS analysis (λ<sub>ex</sub> = 488 nm; λ<sub>em</sub> = 515 nm [FITC] and 617 nm [PI]) with a FACSCalibur™ (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 × 10⁶ cells/ml in logarithmic phase or 10⁷ 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 4°C 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 4°C 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 (Morales et al., 2010). Briefly, digested protein was diluted in loading buffer (80% ACN, 5% TFA) and loaded on TiO₂ 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 NH₄OH (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 Å) 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 Å) 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-H₃PO₄]⁺ = 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, Yau *et al.*, 2010), mouse anti-A2 antibody (clone C9, 1:50 dilution, Zhang *et al.*, 1996), mouse anti-LPG antibody (clone CA74E, 1:20000 dilution, Tolson *et al.*, 1994), 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 (Pescher et al, 2011). 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™ 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 2003 (Microsoft) or Prism 5.0 (GraphPad).

**Acknowledgments**
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.** *L. donovani* CyP40 is a conserved chaperone and shows amastigote-specific phosphorylation. (A) 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 experimental procedures. CLD/PPIase (cyclophilin-like domain/PPIase) and TPR domains are indicated. Degree of residue conservation is represented by the colour code (green, 80-100% conserved; yellow, 30-80% conserved; red, 10-30% conserved). *, key residues for cyclosporin A binding and PPIase activity; ^, key residues for chaperone function; Δ, key residues for
Manuscript Yau et al., 18.04.2014

698 HSP90 binding. Accession numbers: M.mus, NP_080628.1; H.sap, NP_005029.1; B. tau,
699 NP_776578.1; S.cer, NP_013317.1; T.cru, TcCLB.506885.400; T.bru, Tb927.9.9780; L.bra,
700 LbrM34.4730; L.mex, LmxM34.4770; L.inf, LinJ35.4830; L.maj, LmjF35.4770. (B) Western
701 blot analysis. Phosphoproteins of in vitro cultivated L. donovani promastigote (pro), axenic
702 amastigote (ax. ama, ax), and amastigotes harvested from infected hamster spleen (sp) were
703 enriched using a Phosphoprotein purification kit (Qiagen). Total and phosphoprotein extracts
704 were analyzed by Western blotting using anti-CyP40 antiserum and anti-α-tubulin antibody for
705 normalization.

706

707 **Fig. 2. CyP40 null mutant parasites are viable in culture.** (A) Targeted replacement and
708 add-back strategies. Targeting constructs comprising bleomycin (BLE) and puromycin (PAC)
709 resistance genes flanked by the *CYP40* 5' and 3' untranslated regions (UTRs) were used for
710 deletion of the genomic *CYP40* alleles. A targeting construct containing the nourseothricin
711 resistance gene (*SAT*) and the *Leishmania major* *CYP40* open reading frame (ORF) flanked by
712 the *L. major* small ribosomal subunit homology regions (*LmSSU*) were used for restoring
713 CyP40 expression in the *cyp40-/-* parasites. Primers specific for amplifying *CYP40* (P1/P4), the
714 resistance genes *PAC* (P2/P4) and *BLE* (P3/P4), and the *SSU* locus (P1/P5) are indicated. (B)
715 *Validation of homologous recombination events by PCR analysis.* Genomic DNA of wild-type
716 (WT), heterozygous CyP40 null mutant (-/+), homozygous CyP40 null mutant (-/-) and CyP40
717 add-back (-/-/+) was subjected to diagnostic PCR analysis using the indicated primer pairs
718 (*PₓPᵧ*) to amplify the endogenous CyP40 ORF (*CYP*), the *CYP40* ORF integrated into the
719 ribosomal locus (*SSU*), and the bleomycin (*BLE*) and puromycin (*PAC*) resistance genes. (C)
720 *Western blot analysis.* Total protein from wild-type (WT) and CyP40 null mutant (-/-) and add-
721 back (-/-/+) were extracted and analyzed by western blotting using anti-CyP40 (CyP, upper
panel) and anti-α-tubulin antibodies (Tub, lower panel). The molecular weight of marker proteins in kDa is indicated.

**Fig. 3. Stationary cyp40-/- promastigotes show altered morphology.** (A, B) Electron microscopy analysis. Promastigotes from stationary growth phase were fixed and processed for either scanning (A) or transmission EM (B) as described in Materials and Methods. Scale bar represents 10 µm in the scanning EM panels and 500 nm in the transmission EM panels. n, nucleus; m, mitochondria; k, kinetoplast; f, flagellum; fp, flagellar pocket; l, lipid droplet. (C) Cell viability determination. Stationary promastigotes of wild-type (WT) and cyp40-/- (-/-) from 6 days cultures were stained with propidium iodide (PI, 1 µg/ml) and annexin V-FITC (1 µg/ml) for 10 min, and respective fluorescence intensities were analyzed by FACS. Numbers at the lower-right quadrant represent percentage of apoptotic cells (PI-, Annexin V-FITC+), while numbers at the upper-right quadrant represent percentage of dead cells (PI+, Annexin V-FITC+).

**Fig 4. Growth characterization of Cyp40 null mutant promastigotes.** (A) *In vitro* growth curve of cyp40-/- mutants. Promastigotes of wild-type (WT), cyp40-/- (-/-) and cyp40-/-/+ (-/-/+). were cultured at 25°C, pH 7.4 for 120 hours. Aliquots of culture were collected every 24 hours and cell density was measured with a CASY cell counter. (B) Western blot analysis of LPG expression. 5 × 10⁶ wild-type or cyp40-/- parasites from logarithmic (log) or stationary (stat) cell culture were subjected to western blot analysis using anti-LPG antibody. (C) RT-PCR of SHERP expression. Total RNA of the wild-type (black), cyp40-/- (white) and cyp40-/-/+ (gray) were extracted, reverse transcribed, and amplified with SHERP specific primer. β-tubulin was amplified with specific primers as reference gene. Relative expression levels of SHERP mRNA was analyzed using semi-quantitative RT-PCR.
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**Fig. 5. Analyses of motility and cytoskeleton of stationary phase *cyp40-/-* promastigote. (A, B) Motility analysis. Motility tracks were established for logarithmic (log) and stationary phase (stat) wild-type (WT), *cyp40-/-* (-/-) and *cyp40-/-/+* (-/-/+). For each condition, at least 3 movies were recorded for the simultaneous *in silico* tracking of at least 255 cells per movie. Mean speed (in µm/s) was calculated from instant velocity data and graphically represented in left panel with SD bars. *ANOVA test, p < 0.0001. n=735 for WT Log and 737 for WT Stat, n=746 for -/- Log and 747 for -/- Stat, n=741 for -/-/+ Log and 742 for -/-/+ Stat. Characteristic tracks are shown in Fig. 5B. (C) Flagellar ultrastructure of stationary WT and *cyp40-/-* promastigotes. Scale bar represents 100 nm. (D) Cytoskeleton analysis. Wild-type (WT) and *cyp40-/-* (-/-) stationary promastigotes were treated with 1% Triton X-100 at 4°C in PBS for 10 minutes to strip the plasma membrane and visualize the parasite cytoskeleton. Samples were washed twice in PBS, fixed and processed for scanning electron microscopy as described (Absalon *et al.*, 2008). Scale bar represents 100 nm. (D) 2D-DIGE analysis. The readout of the DeCyder Biological Variation Analysis (BVA) module is shown for a small β-tubulin isoform that shows four-fold reduced abundance in the null mutant abundance. The upper panels represent 3D images of the volumes of the spots from wild-type (WT) and *cyp40-/-* (-/-). The bottom panel shows a graphic representation of differences in abundance across three independent experiments. The numbers indicate the fold difference in abundance of *cyp40-/-* to WT.

**Fig. 6. Intracellular survival and axenic differentiation of the *cyp40-/-* null mutants. (A) Macrophage infection assay.** BMMs from C57BL/6 mice were infected with stationary phase promastigotes of *L. donovani* wild-type (WT, O), *cyp40-/-* (clone 4, □, and 5, ■) or *cyp40-/-/+* (Δ) for 2 hours, and parasite burden was analyzed at 0, 24 and 48 hours post-infection by
nuclear staining with Hoechst 33342 and fluorescence microscopy. Results are representative of three triplicate experiments with standard deviation denoted by the bars is shown. (B) In vitro growth curve of cyp40-/- axenic amastigotes. Promastigotes of wild-type (WT), cyp40-/- (-/-) and cyp40-/-/+ (-/-/+) were induced for axenic differentiation at 37°C, pH 5.5 for 72 hours, and amastigote growth was monitored following inoculation of 2 × 10⁶ axenic amastigotes into fresh medium and daily determination of cell density with a CASY cell counter. (C) A2 marker protein expression analysis. Extracts from wild-type (WT), cyp40-/- (-/-) and cyp40-/-/+ (-/-/+) axenic amastigotes 72 hours after incubation under differentiation-inducing conditions were subjected to western blot analysis using an axenic amastigote marker A2-specific monoclonal antibody (upper panel). Equal loading was controlled by Coomassie staining of an acrylamide gel loaded with equivalent quantity of the samples (lower panel). The molecular weight of marker proteins in kDa is indicated.

**Fig 7. Effect of phosphorylation of CyP40 on Leishmania intracellular survival. (A, B)**

Phosphorylation site identification. (A) Peptide 270LQQWSEAR277 (m/z 1098.49) was identified in LC-ESI-MS/MS analysis of *L. major* Strep::CyP40 peptides after tryptic digestion and TiO₂ enrichment and Ser274 was identified as a phosphorylated residue.*, fragment ions arising from loss of ammonia (-17 Da); pS, phosphorylated serine. (B) Multiple sequence alignment of *L. major* CyP40 phospho-residue. Sequence segments encompassing CyP40 phosphorylation site of mouse, human, bovine, yeast, two *Trypanosoma* (*T. cruzi*, *T. brucei*), four *Leishmania* species (*L. braziliensis*, *L. mexicana*, *L. infantum*, *L. major*) were analyzed with ClustalW. The phospho-residue Ser274 of *L. major* CyP40 is marked by an asterisk (*). (C) Analysis of the CyP40-S274A add-back line by western blotting. Total protein (left panel) from wild-type (WT), CyP40 null mutant (-/-), CyP40 add-back lines Cyp40-WT and CyP40-S274A (-/-/+ and S274A, respectively), and enriched phosphoprotein extracts (right panel) of
797 axenic amastigotes of *L. donovani* wild-type (WT) and *cyp40-/-/+S274A* (S274A) were
798 analyzed. As background control of the phosphoprotein enrichment, 2 mg of the WT total
799 extract were first treated with 8000 units of lambda phosphatase (APP) for 2 hours at 30°C. The
800 APP-treated sample was then subjected to phosphoprotein enrichment. The total and
801 phosphoprotein (Phospho) extracts of the parasites were analyzed by Western blotting using
802 anti-CyP40 antiserum and anti-α-tubulin antibody. The molecular weight of marker proteins in
803 kDa is indicated. (D) *Macrophage infection assay*. BMMs from C57BL/6 mice were infected
804 for 2 hours with stationary phase promastigotes of *L. donovani* wild-type (WT, O), *cyp40-/-* (-/-,
805 □) or *CyP40-WT* and *CyP40-S274A* add-backs (respectively -/-/+, Δ and -/-/+S/A, ▼) and
806 parasite burden was at 0, 24 and 48 hours post-infection by nuclear staining with Hoechst
807 33342 and fluorescence microscopy. Result is presented as percentage infected BMMs (left
808 panel, one representative triplicate experiment with standard deviation denoted by the bars is
809 shown), and number of parasites per 100 BMMs at 48 hours post-infection (right panels, results
810 of one representative triplicate experiment with standard deviation denoted by the bars is
811 shown).

812

813

814

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1016

1017

1018
<figure><img src="image_2.png" /><figcaption>A</figcaption></figure>

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_5.png" /></figure>

total protein

phospho protein

CyP40

α-tub
A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>

C

<figure><img src="image_8.png" /></figure>
<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>
A

<figure><img src="image_13.png" /></figure>

B

<figure><img src="image_14.png" /></figure>

C

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

D

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

E

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>
A

<figure><img src="image_22.png" /></figure>

Post-infection time (h)

<figure><img src="image_23.png" /></figure>

Post-infection time (h)

<figure><img src="image_24.png" /></figure>

Post-infection time (h)

B

<figure><img src="image_25.png" /></figure>

Cells/ml

C

<figure><img src="image_26.png" /></figure>

anti-A2 WB

<figure><img src="image_27.png" /></figure>

Coomassie
A

<figure><img src="image_28.png" /></figure>

B

<figure><img src="image_29.png" /></figure>

C

[kDa] WT -/- -/-/+ S274A

<figure><img src="image_30.png" /></figure>

<figure><img src="image_31.png" /></figure>

Total

Phospho

D

<figure><img src="image_32.png" /></figure>

Post-infection time (h)

<figure><img src="image_33.png" /></figure>

Parasites/100 BMMs