Leishmania donovani 90 kD Heat Shock Protein – Impact of Phosphosites on Parasite Fitness, Infectivity and Casein Kinase Affinity

Leishmania parasites are thought to control protein activity at the post-translational level, e.g. by protein phosphorylation. In the pathogenic amastigote, the mammalian stage of Leishmania parasites, heat shock proteins show increased phosphorylation, indicating a role in stage-specific signal transduction. Here we investigate the impact of phosphosites in the L. donovani heat shock protein 90. Using a chemical knock-down/genetic complementation approach, we mutated 11 confirmed or presumed phosphorylation sites and assessed the impact on overall fitness, morphology and in vitro infectivity. Most phosphosite mutations affected the growth and morphology of promastigotes in vitro, but with one exception, none of the phosphorylation site mutants had a selective impact on the in vitro infection of macrophages. Surprisingly, aspartate replacements mimicking the negative charge of phosphorylated serines or threonines had mostly negative impacts on viability and infectivity. HSP90 is a substrate for casein kinase 1.2-catalysed phosphorylation in vitro. While several putative phosphosite mutations abrogated casein kinase 1.2 activity on HSP90, only Ser289 could be identified as casein kinase target by mass spectrometry. In summary, our data show HSP90 as a downstream client of phosphorylation-mediated signalling in an organism that depends on post-transcriptional gene regulation.

of immune modulatory secreted vesicles known as exosomes. Nearly all major chaperones, including HSP90, HSP70, CPN60.2 and CPN60.3, STIP1 and HSP100 are released into the host cell cytoplasm, with the latter playing a crucial role in the sorting of exosome content. Lack of chaperones in the exosomes abrogates their immune modulatory potential 9,10 .
Chaperone proteins of the HSP90 family play well known roles in the maturation and activation of a multitude of regulatory proteins, such as transcription factors, protein kinases, hormone receptors and cytoskeletal proteins 11,12 and can even silence mutations in regulatory proteins 13 .
Consequently, HSP90 is an essential protein in all eukaryotic organisms precluding the viability of HSP90 null mutants in any known system. Moreover, HSP90 is a highly abundant protein species, accounting for up to 3% of a cell's total protein 3,14 . Therefore, reverse genetics approaches, i.e. creating null mutants, to unravel the role and function of HSP90 are likely to fail. The discovery of the HSP90-specific inhibitor geldanamycin (GA) 15 allowed the pharmacological inactivation of this protein family and the study of the phenotypical consequences in tumor cell lines [16][17][18][19] , but also in parasitic microorganisms such as Leishmania donovani, Trypanosoma cruzi, and Plasmodium falciparum [20][21][22] . Geldanamycin has a specific affinity for the HSP90 ATP-binding pocket, an affinity it shares with another specific inhibitor, radicicol (RAD) 23,24 . Both inhibitor classes cause a cell cycle arrest in logarithmically growing culture cells and indirectly inhibit the maturation and function of HSP90-dependent client proteins, initially offering great promise as anti-cancer drugs 25 . In Leishmania donovani, pharmacological inhibition of HSP90 can induce morphological and biochemical promastigote-to-amastigote differentiation 20,26,27 , mimicking the known environmental triggers, i.e. heat shock and low pH, and indicating a pivotal role for HSP90 in environmental sensing and life cycle control.
HSP90 is usually found as a dimer in large complexes known as foldosomes. In their function they depend on ATP hydrolysis and on a cohort of so-called co-chaperones that assist in the recruitment of client proteins and in the regulation of the HSP90 reaction cycle 28,29 . The HSP90 foldosome can vary in composition depending on the client proteins, but usually includes subunits such as HSP70/40, P23, AHA1 and STIP1 (HOP) 29 . HSP90, HSP70 and STIP1 are also known as substrates of protein kinases [30][31][32] , affecting function and subcellular localisation of these chaperones.
Leishmania spp also possess an almost complete set of co-chaperones, with the notable absence of CDC-37, but including the essential co-chaperones STIP1, SGT 27,29,[33][34][35] and others with less impact on the viability, such as HIP, P23, and AHA1 [36][37][38] . The HSP90 and HSP70 chaperones, but also co-chaperones such as STIP1 and cyclophilin 40, have been shown to be phosphorylated during amastigote stage conversion 34,39 . Mutagenesis of two phosphorylation sites (P-sites) in L. donovani STIP1 caused either general lethality or no phenotype at all 34 . Mutation of the single P-site in cyclophilin 40 had no phenotypic consequences 40 .
Essential protein kinases are discussed as attractive drug targets against cancer but also against infectious diseases, such as leishmaniasis. In Leishmania, a variety of kinases have already been identified as potential drug targets, such as mitogen-activated protein kinases (MAPKs), cdc-related kinase 3 (CRK3) and GSK3 [46][47][48][49][50] . The members of the Leishmania casein kinase 1 (CK1) family have also emerged as potential drug targets 51 . The CK1 family consists of multifunctional Ser/Thr protein kinases, characterised by a highly conserved kinase domain and a specific C-terminal domain responsible for kinase regulation and localisation 52 . CK1 isoform 2 (CK1.2) of L. donovani was identified as exokinase released via immune-modulatory exosomes into the host cell cytosol, where it may phosphorylate and modulate host cell proteins 9,10,[53][54][55] . This kinase was shown to be essential for intracellular parasite survival.
Thus far, Leishmania kinases and phosphatases involved in HSP90 modification are poorly understood. Nevertheless, a recent study demonstrated that the L. donovani MAPK1, whose L. mexicana orthologue is involved in parasite viability and drug resistance 56 , interacts with HSP90 and HSP70, affects the expression of HSP90 and HSP70 and phosphorylates both chaperones in vitro 57 . Given the co-localisation of HSP90 and other major chaperones with CK1.2 in the exosome export pathway 10 , this kinase may also act as upstream kinase for HSP90 regulation.
Thus far, three phosphorylation sites have been identified in the L. donovani HSP90, Thr 223 , Ser 526 and Thr 216 34,58 , all showing increased modification during promastigote-to-amastigote differentiation. However, an analysis of the impact of phosphorylation on HSP90, a key regulator of the Leishmania life cycle 20 , was hindered so far by the high copy numbers of HSP90-coding genes in Leishmania 59 and the essential nature of HSP90. However, the introduction of an inhibitor-resistant sequence variant of L. donovani HSP90, allowing a conditional mutagenesis of this protein 27 , has paved the way for an analysis of HSP90 phosphorylation sites and their impact on fitness, morphology and infectivity. Also, the ability of CK1.2 to phosphorylate HSP90 in vitro was tested on a panel of phosphorylation site mutants, identifying at least one CK1.2-specific site.

Results
Identification of HSP90 P-sites and mutagenesis. The P-sites at Thr 223 and Ser 526 were identified in L.
To identify further phosphorylation sites within the Leishmania HSP90, phosphoproteomics analyses were performed on cultured promastigotes, in vitro differentiated axenic amastigotes and mouse-lesion-derived amastigotes of L. mexicana. This led to the identification of seven previously unknown phosphorylation sites at Thr 211 , Thr 216 , Ser 289 , Ser 526 , Ser 594 and Ser 595 . Fragmentation spectra can be found in the supplementary material www.nature.com/scientificreports www.nature.com/scientificreports/ (Fig. S1). All sites are conserved in L. donovani (Fig. 1A). The Thr 223 P-site of L. donovani is not conserved in L. mexicana.
We also identified putative P-sites by literature search and sequence alignments. Two P-sites at Thr 22 and Thr 101 are located in the ATP-binding pocket of the S. cerevisiae HSP90, and Thr to Ile exchanges affect the ATPase activity of ScHSP90 43,60 . In L. donovani HSP90, these residues are conserved at positions Thr 21 and Thr 100 and were mutated identically, namely Thr 21 to Ile 21 and Thr 100 to Ile 100 .
The putative P-sites at Thr 693 and Ser 694 were inferred from the known P-sites Thr 725 and Ser 726 in the human cytosolic αHSP90 32. A list of the serine and threonine residues targeted for replacement with non-modifiable alanine and/or phosphomimetic aspartate is shown in Table 1 and schematically in Fig. 1.
We have previously established a system for a conditional phenotype analysis of HSP90 mutants in vitro. Briefly, a Leu33Ile mutant of HSP90, dubbed HSP90rr, was created which is resistant to the HSP90-specific inhibitor RAD. Ectopic expression of HSP90rr from episomal transgenes has no effect under normal culture conditions, but under RAD challenge, HSP90rr facilitates normal growth, morphology and infectivity. Any mutation introduced into HSP90rr only shows a phenotype under RAD challenge when the endogenously coded HSP90 is inactivated 27 .  Table 1 Table 1. List of the Ser and Thr residues in HSP90 that were subjected to mutagenesis. Three-letter amino acid codes apply.
www.nature.com/scientificreports www.nature.com/scientificreports/ Using a non-commercial procedure for targeted mutagenesis 27 and specific oligonucleotide primer pairs for each target sequence (Table S1), we introduced the desired codon changes into the HSP90rr coding sequence and placed the mutated and verified genes in the shuttle expression plasmid pTLv6 27 . The various mutated HSP90rr transgenes were then stably transfected under G418 selection in L. donovani strain 1SR. Faithful transcription from the transgenes was verified by transgene-specific RT qPCR (not shown).
The growth kinetics of parasites expressing the transgenes for HSP90wt, HSP90rr and mutants thereof were monitored in the absence of RAD and found to be indistinguishable (not shown), indicating that the expression of HSP90wt from the chromosomal gene copies masks the mutant phenotypes, leading us to conclude that none of the mutations has a dominant negative effect.

Effect of P-site mutations on in vitro growth.
To assess the effect of the P-site mutations in HSP90rr on in vitro growth, we seeded promastigotes in medium without selective antibiotic and added RAD at 90% growth-inhibitory concentration (IC 90 ). Cell density was then monitored over 4 days. The density of HSP90rr cells after 96 h was then defined as 100% growth and parasites expressing the non-RAD-resistant HSP90wt transgene were taken as standard for the loss of HSP90 function.
Mutations in the N-terminal ATPase domain of HSP90rr at Thr 21 and Thr 100 both had deleterious effects on the proliferation under RAD inhibition ( Fig. 2A). Interestingly, both Thr to Ile (non-phosphorylatable) and Thr to Asp (phosphomimetic exchange) had the same effect, suggesting that the phosphomimetic exchange was not functional.
All three known or suspected P-sites in the charged linker domain (CLD), Thr 211 , Thr 216 and Thr 223 , were mutated into alanine or aspartate, respectively. All mutations caused a significant ~30% reduction of the in vitro growth rates compared to the parental HSP90rr transgene, but no differences between alanine and aspartate exchanges were observed. Combined mutations of both Thr 211 and Thr 216 had significant additive effects, reducing growth by up to 65% (Fig. 2B).
Both mutations in the middle domain (MD) of HSP90rr caused a significant growth retardation of 55% for Ser 289 Ala and 25% for Ser 526 Ala (Fig. 2C). Since the MD is thought to specify client protein affinity, phosphorylation-mediated changes may affect client protein recognition.
Two pairs of adjacent P-sites were targeted in the C-terminal domain which is important for the interaction with co-chaperones 27 and for HSP90 dimerisation. For this analysis, the P-sites Ser 594 , Ser 595 , Thr 693 and Ser 694 were mutated into Ala or Asp, respectively. While the Ser 594 Ala exchange caused a significant (~50%) reduction of in vitro growth, the Ser 594 Asp exchange abolishes parasite growth entirely. The Ser 595 Ala exchange also caused a significant but weaker (~25%) reduction of the in vitro growth, but the Ser 595 Asp exchange facilitates parasite proliferation at ~100%. The Thr 693 Ala but not the Thr 693 Asp mutation reduced growth by ~20%. A strong effect was observed for Ser 694 Asp where a permanent negative charge completely abrogates promastigote growth under RAD challenge. By contrast, the Ser 694 Ala exchange had no significant effect (Fig. 2D).
Effect of phosphosite mutations on promastigote morphology. HSP90 plays a pivotal role in L.
donovani life cycle control. Inhibition of HSP90 by geldanamycin or radicicol induces promastigote-to-amastigote conversion in the absence of elevated temperature and/or acidic pH 20,26,27,61 . Morphologically, this leads to a change of the cell shape from spindle-shaped to ovoid and to the shortening of the flagellum 27 . We therefore analysed length and width of the cell body and length of the flagellum of L. donovani transfected with the P-site-mutated HSP90rr variants 72 h after RAD treatment, using either scanning electron microscopy (SEM, Fig. 3A) 27 or anti-tubulin labelling and fluorescence microscopy ( Fig. 3B) 38 . The lengths of the mutants were compared to the lengths of HSP90rr and HSP90wt over expressing parasites (Fig. 3A,B).
Changing the two N-terminal P-sites Thr 21 and Thr 100 to Ile or to Asp abrogates the ability of HSP90rr to promote the promastigote shape under RAD challenge. For all these mutants, we observed a significant reduction of the cell body length to ~5-6 µm, an ovoid cell shape with an increase of the cell body width to ~3 µm and a significant reduction of the flagellum to ~2 µm. The Thr 100 Asp exchange in particular resulted in damaged cells under RAD treatment (Fig. 3C) and non-viable promastigotes. Therefore, it was impossible to obtain measurements of this mutant.
Due to the closeness of the positions Thr 211 and Thr 216 we created double Ala-mutants (Thr 211 /Thr 216 Ala) and analysed only the morphology of the double mutant due to its indistinguishable growth phenotype from the single mutants ( Fig. 2A). The simultaneous Ala-exchange of these two Thr-residues (Thr 211 /Thr 216 Asp) in the CLD reduces the cell body length significantly to ~6 µm and the flagellum length to ~2.5 µm under RAD. The cell body width is also significantly increased.
Peculiar morphologies correlate with the mutations at Thr 223 . Thr 223 Ala causes a shortening of the cell body (~6 µm), but normal cell body width and flagellar length (~4 µm), reminiscent of metacyclic promastigotes 62 . The cell body length phenotype is reversed in the phosphomimetic Thr 223 Asp mutant (8-9 µm), which however shows a shortened flagellum. This result hints at an involvement of Thr 223 phosphorylation in the maintenance of the procyclic promastigote.
The impact of the mutations at the C-terminus of HSP90rr were analysed by fluorescence microscopy using anti-tubulin antibodies. This showed identical effects when comparing HSP90wt and HSP90rr over expressing cells (Fig. 3B). The Ser 594 Ala mutation slightly reduces cell body length (~8 µm) and the length of the flagellum (~5.5 µm). The cell body width is slightly increased (~3 µm). The mutation Ser 594 Asp in contrast has a strong and significant impact on the promastigotes morphology, with a shortening of the cell body length to ~7 µm and the flagellum to ~4 µm. The cell body width of these mutants is significantly enlarged to ~4 µm. The mutations Ser 595 Ala and Ser 595 Asp have no significant effects on the cell body lengths, but increased the cell body widths slightly and decreased the length of the flagella to ~6 µm. Also for Thr 693 Ala and Thr 693 Asp we observed an intermediate impact of the mutations on the cell morphology. Ser 694 Ala has no impact on the cell body length www.nature.com/scientificreports www.nature.com/scientificreports/ (~10 µm) with a slight increase of the cell body width. The length of the flagellum is slightly but not significantly reduced. These morphological promastigote-like features are reversed by the phosphomimetic exchange Ser 694 Asp. The mutation Ser 694 Asp reduces the cell body length (~7 µm) and the length of the flagellum (5 µm). The cell body width is significantly enlarged to ~5 µm.
The observed impact on the morphologies of the HSP90 variant-expressing parasites largely reflects their growth rates under RAD. One exemption to this correlation is the Thr 223 site where lack of phosphorylation appears to drive the mutant towards metacyclogenesis.
Effect of phosphosite mutations on in vitro infectivity. Several P-sites in HSP90 were identified previously 34 or in this paper as being preferentially phosphorylated in amastigotes by comparative phosphoproteomics of promastigote and axenic amastigote lysates (see Results, 1st section). While axenic amastigotes are convenient models for stage conversion, we were interested in the impact of phosphorylation on the intracellular survival of true L. donovani amastigotes. Testing the sensitivity of primary mouse bone marrow-derived macrophages against RAD, we found that the concentrations required to render L. donovani dependent on the episomal HSP90rr gene variants already affected the ability of the macrophages to support a Leishmania infection (not shown). We therefore decided to pre-treat the parasites bearing the HSP90rr transgenes with RAD for 48 h prior to macrophage infection to avoid exposure of the host cells to RAD, as described previously 27 . The effects of the various HSP90rr mutants on the in vitro infectivity were analysed 48 h post-infection and compared with the recombinant cells bearing the resistant HSP90rr transgene and the HSP90wt over expressing cells (Fig. 4). We applied a duplex qPCR approach to quantify the ratio of parasite actin to macrophage actin DNA as described previously 63 .
Under these conditions, over expression of HSP90rr prevents a loss of infectivity due to the RAD treatment, resulting in a significantly higher median infection ratio compared with HSP90wt over expressing parasites, with differences slightly varying between individual experiments ( Fig. 4A-F).
The Thr 21 Ile mutation resulted in ~50% reduced parasite load, albeit twice as high as the HSP90wt control, while the phosphomimetic exchange Thr 21 Asp reduced the parasite load even below HSP90wt levels. The mutations Thr 100 Ile and Thr 100 Asp both lower the parasite load by ~60% (Fig. 4A). Obviously, the putative P-sites Thr 21 and Thr 100 that are located in the ATP-binding domain are not only critical for promastigote growth but also for infection of macrophages. Surprisingly, the Thr 100 mutants support increased intracellular persistence compared with the HSP90rr control, in spite of their inability to support growth of the promastigote (Fig. 2).
The Ala-exchanges at the P-sites in the charged linker region, Thr 211 Ala and Thr 216 Ala, resulted in non-significantly reduced parasite loads (Fig. 4B). The phosphomimetic exchange Thr 211 Asp further lowered the parasite to host cell ratio, arguing against a simple ionic effect of phosphorylated Thr 211 . Thr 216 Asp by contrast restored infectivity significantly over the Thr 216 Ala mutant (Fig. 4B). Phosphorylation of Thr 216 therefore appears to boost intracellular persistence of L. donovani.
The Thr 223 Ala exchange also affected infectivity, reducing parasite loads in macrophages to 57% (Fig. 4C, Table 2). However, the phosphomimetic Thr 223 Asp exchange cannot restore infectivity significantly, also arguing against simple ionic effects of phosphorylated Thr 223 .
The phenotypes of Ser 289 Ala and Ser 526 Ala mutations, which are located in the middle domain, are not significant and suggest a rather limited effect on virulence (Fig. 4D).
The Ser 594 Ala, Ser 595 Ala and Ser 595 Asp mutations in the C-terminal region of HSP90rr reduce infectivity slightly but significantly by ~20-30% (Fig. 4E). By contrast, the cells expressing the phosphomimetic Ser 594 Asp mutant were unable to establish an infection (Fig. 4E). This is presumably caused by the inability of these transgenic parasites to grow under RAD in vitro (Fig. 2D).
We also tested the effects of the mutated putative P-sites Thr 693 and Ser 694 located directly in front of the STIP-1 recognition motif 27 at the extreme C-terminus of HSP90. The exchanges Thr 693 Ala, Thr 693 Asp and Ser 694 Ala do not affect infection ratios. However, the phosphomimetic Ser 694 Asp exchange abrogates intracellular survival completely (Fig. 4F). Like Ser 594 Asp, the Ser 694 Asp mutant of HSP90rr is unable to promote promastigote proliferation under RAD (Fig. 2D), suggesting an inability to tolerate the pre-treatment before infection.

Identification of HSP90 as in vitro target of casein kinase1.2.
To gain insight into the mechanisms involved in HSP90 regulation, we investigated whether kinases that have been described to target mammalian HSP90 may also target Leishmania HSP90. In particular, we focused on three protein kinases, GSK-3, CK1.2 and DYRK1. Of these, GSK-3 and CK1.2 have been shown to phosphorylate the C-terminal domain of mammalian HSP90. This phosphorylation is particularly important to prevent the binding of HSP90 to CHIP, a co-chaperone containing an ubiquitin ligase activity and thus to prevent the client protein from being degraded 32 . We also selected DYRK1 as it has been shown to be a priming kinase for GSK-3; it phosphorylates the substrates on a specific site which can be recognised and phosphorylated by GSK-3 64 . Therefore, we expressed and purified recombinant L. major CK1.2 51, L. major GSK-3 65 and L. infantum DYRK1 to perform in vitro kinase assays using recombinant L. donovani HSP90wt.
As shown in Fig. 5B lane 4, we detected a signal at 80 kDa revealing the transfer of 32 P onto HSP90 by CK1.2 but not by GSK-3 or DYRK1 (lanes 5 and 6). In contrast, all three kinases were able to phosphorylate myelin basic protein (MBP), a canonical kinase substrate (lanes 1-3). Since we can exclude MBP autophosphorylation 51 , these data suggest that HSP90 is a substrate of CK1.2, which is consistent with previous findings on mammalian HSP90, but not a substrate of GSK-3 or DYRK1. This is different from mammalian HSP90 where it has been shown that mammalian GSK-3 phosphorylates HSP90 at its C-terminus 32 . This may be due to the use of the L. major (GSK3 and CK1.2) and L. infantum (DYRK1) orthologues in our in vitro assay; however, the sequence identity between the GSK3 of L. major and L. donovani is 98%, and the sequence identity between the DYRK1 of L. infantum and L. donovani is 99%, making a complete change of client specificity very unlikely.
www.nature.com/scientificreports www.nature.com/scientificreports/ Identification of the P-site(s) targeted by CK1.2. Among the P-sites that we identified, the residues Thr 211 , Thr 216 , Thr 223 , Thr 693 and Ser 694 , match the canonical consensus site for CK1 family members: S/T(X) 2-3 S/T or D/E(X) 2-3 S/T 66 . To identify which of the previously studied P-sites may be targeted by CK1.2, we expressed HSP90 with serine or threonine to alanine mutations to test which mutation abrogates HSP90 phosphorylation by CK1.2. We tested mutants for all known or suspected P-sites since members of the CK1 family can phosphorylate canonical as well as non-canonical sites such as SLS-Xn-(E/D)n 67 or K/R-X-K/R-(X) 2 -S/T 68,69 . We performed in vitro kinase assays using recombinant CK1.2 with HSP90wt or HSP90 phosphosite mutants as substrates. As shown in Fig. 5D, HSP90-Thr 693 Ala, -Ser 694 Ala and -Thr 693 Ala/Ser 694 Ala mutants are still phosphorylated by CK1.2 at levels comparable with the wild type control, although we observe that the levels of Ser 693 Ala and www.nature.com/scientificreports www.nature.com/scientificreports/ Ser 693 Ala/Thr 694 Ala are slightly lower than those of Thr 694 Ala. By contrast, phosphorylation by CK1.2 was abrogated in Thr 21 Ala, Thr 100 Ala, Thr 211/216 Ala, Thr 223 Ala, Thr 289 Ala, Ser 526 Ala and Ser 594 Ala and Ser 595 Ala mutants of HSP90 (Fig. 5D,F).
We also performed a "cold", i.e. non-radioactive in vitro kinase assay with recombinant HSP90wt and CK1.2 in the presence or absence of 10 µM D4476, a specific CK1 inhibitor 51 . The samples of three independent kinase assays were analysed by mass spectrometry to identify the sites phosphorylated by CK1.2. We found only one site, Ser 289 (Fig. 5G), which is phosphorylated in the presence of CK1.2 but not in the presence of CK1.2 + D4476, suggesting that CK1.2 targets Ser 289 . No phosphorylation was detected at Thr 211 , Thr 216 , Ser 526 , Ser 594 , Ser 595 and Thr 223 in our dataset (data not shown), suggesting that they are not targeted by CK1.2. We cannot exclude that the sites Thr 21 , Thr 100 , Thr 693 and Ser 694 may be true CK1.2 sites as the peptides containing those sites were not detected in our mass spectrometric analyses. However, those sites are only presumed phosphorylation sites based on analogies with model HSP90s. Moreover, only Thr 693 and Ser 694 resemble canonical CK1 sites.
In summary (Table 2), mutations at the established and putative phosphorylation sites in the L. donovani HSP90 result in a variety of phenotypic manifestations. The most drastic and general effects are observed after introducing mutations at sites within the highly conserved ATP-binding domain. These HSP90rr variants apparently cannot support growth or infectivity under RAD challenge. Mutation at sites in the charged linker domain show moderate effects on growth and infectivity. Middle domain site mutations result in moderate to strong growth inhibition, morphologic changes and a moderate loss of infectivity, even though one of them is established as the only CK1.2 phosphorylation site. In the C-terminal domain we observe the strongest effects for two of the phosphomimetic Ser-Asp exchanges with no or moderate impact of Ser/Thr to Ala mutations. Clearly, permanent negative charges at these sites are not conducive to parasite fitness. At other sites, Ser/Thr to Asp restore growth, morphology and/or infectivity to a various extent compared with the Ser/Thr to Ala mutations.

Discussion
Trypanosomatida and perhaps the entire phylum Euglenozoa lack inducible transcription as a means for gene expression control 70,71 . The existing genome databases contain no confirmed genes for transcriptional regulators and cis-regulatory DNA sequence elements such as gene promoters and enhancer elements known from crown group Eukaryota. Moreover, the established mode of multicistronic transcription precludes individual transcriptional control of gene expression 71,72 . Cis-splicing is very rare but trans-splicing has been discussed as point of regulation 73 . This leaves mRNA processing and stability, translation, protein folding and post-translational protein modifications as conceivable targets of gene expression control.   www.nature.com/scientificreports www.nature.com/scientificreports/ The major chaperone protein HSP90 is subject to various post-translational modifications such as phosphorylation, acetylation, s-nitrosylation, oxidation, SUMOylation or ubiquitination 31,74 . Post-translational modifications of HSP90 are known to be involved in the fine-tuning of the chaperone cycle which includes conformational changes, ATPase activity, interaction with co-chaperones and the recruitment and activation of various client proteins 31,32,42,75 . Differences in this fine-tuning processes also adapt HSP90 regulation and function to the unique intracellular environments of different organisms or to various physiological states within different tissues of metazoans 29,76 .
Previous work showed that HSP90, HSP70 and the co-chaperone STIP1 are subject to increased phosphorylation during the promastigote-to-amastigote differentiation of L. donovani 34 . These data suggested that the function of HSP90 is adaptable to the different environments of the distinct life cycle stages. It was previously described that a heat shock of yeast cells decreases the phosphorylation level of HSP90, while an opposite effect was observed looking at the turnover of phosphate groups in heat-shocked HeLa cells 77,78 . Phosphorylation of this essential chaperone is one possible mechanism to adapt its function to various environmental conditions and in a species-specific manner.
This paper focuses on the impact of HSP90 phosphorylation by introducing amino acid exchange mutations into the radicicol-resistant HSP90rr variant 27 and monitoring their impact on in vitro promastigote proliferation, morphology, in vitro infectivity and casein kinase 1.2 substrate properties.
We observed that amino acid exchanges at the two putative phosphorylation sites Thr 21 and Thr 100 which are implicated in the activity of the ATPase domain from previous work in Saccharomyces cerevisae 44,60,79 , severely inhibit growth of the promastigote stage. Even phosphomimetic exchanges to Asp in both positions cannot restore the proliferation capacity. We can offer three explanations for this observation, (i) the exchanges may render HSP90 non-functional as chaperone, (ii) the structural changes caused by the amino acid exchanges in the nucleotide binding pocket may impair ATP turnover, or (iii) the changes may render HSP90rr sensitive to RAD again. The latter view is supported by the finding that expression of these four mutant HSP90rr variants results in phenotypes indistinguishable from the cells that are over expressing the RAD sensitive HSP90wt (Table 2). While phosphorylation by casein kinase 2 at the residue Thr 22 , equivalent to Thr 21 in Leishmania HSP90, was established for the yeast HSP90 43 , it was also shown that Thr 22 Ala and Thr 22 Glu amino acid exchanges impact on ATPase activity and interaction of HSP90 with co-chaperones such as AHA1 and increase the sensitivity against RAD and GA 44 . However, we could recently demonstrate that lack of AHA1 in Leishmania has no impact on RAD sensitivity 38 , arguing against an impaired HSP90-AHA1 interaction as the cause for the observed, severe phenotypes. Testing the mutants in vitro for ATPase activity, RAD sensitivity and AHA1 binding may shed light on this phenomenon in the future.
Ala and Asp exchanges at Thr 211 and Thr 216 in the highly diverged charged linker domain have opposing effects on the parasites' infectivity. A stable negative charge at position 211 reduces parasite loads by almost 50%, while the effect of a non-phosphorylatable Ala at 216 is reverted by the phosphomimetic Asp exchange. Although neither site could be identified as CK1.2 phosphorylation site, a double Thr-Ala exchange at 211 and 216 abrogates CK1.2 phosphorylation of HSP90 and causes a reduction of the flagellum. Another site, Thr 223 , also in the CL domain and specific for Trypanosomatida HSP90s, moderately affects growth and infectivity, as well as CK1.2-dependent phosphorylation, but is no CK1.2 site itself. Of note are the effects on the morphology where abrogation of phophorylation (Thr to Ala) reduces cell body length, but not flagellar length, thereby resembling the shift from procyclic to metacyclic promastigotes 80 . This is a first hint at a possible role of HSP90 in the transition from proliferative procyclic promastigotes to the infective metacyclic form. The Thr 223 Asp exchange restores cell body length, but increases diameter and reduces flagellar length, resembling early amastigote development. This fits with the identification of Thr 223 as an amastigote-specific phosphorylation site 34 .
In the middle domain, which in model HSPs has a function in client protein recognition 60,81 , we analysed two sites, Ser 289 and Ser 526 . Ser 289 has an impact on proliferation and the maintenance of promastigote morphology. It was also identified as the only CK1.2 site known so far in HSP90, and its replacement with Ala reduces in vitro infectivity by 24%. Ser 526 , an amastigote-specific site 34 moderately affects growth, virulence, flagellar length and CK1.2 phosphorylation, either at Ser 289 or at Ser 526 , since this site was not detected in mass spectrometry. It is conceivable that the effects of Thr 211/216 , Thr 223 and Ser 526 are at least in part due to their impact on Ser 289 phosphorylation by CK1.2. The question remains open how mutations at Thr 21 , Thr 100 , Thr 211,216 , Thr 223 ,but also Ser 526 , Ser 594 , and Ser 595 can affect phosphorylation at Ser 289 .
The C-terminal domain of HSP90 chaperones is known to facilitate dimerisation and interaction with co-chaperones STIP1 27 and prolyl-peptidyl isomerases such as cyclophilin 40 40,82 and others. Four phosphorylation sites were detected or inferred by analogy. Replacement of Ser 594 caused a ~40% reduction of in vitro growth and infectivity while the Ser 694 to Ala change had little impact. However, the phosphomimetic Ser-to-Asp exchange in these positions completely abrogated HSP90-dependent growth and infectivity. We conclude that permanent negative charges in these positions have a deleterious effect on the HSP90 functionality. The HSP90 reaction cycle is known to include the ordered assembly and disassembly of HSP90-co-chaperone complexes at the C-terminal domain 83 , possibly requiring frequent reversal of post-translational modifications, such as phosphorylation. A permanent negative charge may therefore impede the ordered transitions of the HSP90 foldosome complex. The effects observed for mutation in two other sites, Ser 595 and Thr 693 , are moderate by comparison.
One lesson from our data is the strong deleterious effects of presumed phosphomimetic Asp mutations. Unlike MAP kinases where Asp replacements in the activation sites render downstream kinases permanently active, the situation in HSP90 with its multiplicity of active phosphorylation sites and a complex reaction cycle with multiple partnering chaperones and co-chaperones may be too complicated for simplistic approaches. Another problem (2019) 9:5074 | https://doi.org/10.1038/s41598-019-41640-0 www.nature.com/scientificreports www.nature.com/scientificreports/ resides in our experimental set-up where we express RAD-resistant, mutated HSP90rr before a strong background of RAD-sensitive, endogenous HSP90. A treatment with RAD is necessary to express the mutant phenotypes, yet one cannot exclude interactions and even heterologous complexes of HSP90rr and HSP90 monomers. For example, the HSP90-STIP1 interaction is not affected by RAD treatment 27 , opening up the possibility that phenotypes of mutations outside the ATP binding pocket are "watered down" by interaction with endogenously coded HSP90wt. To rectify this problem, we hope for a future implementation of advanced reverse genetics such as CRISPR/cas9 based gene editing 84,85 to eliminate the entire cluster of ~17 tandemly arranged HSP90 gene copies and to express the mutant HSP90s before a null mutant background.
So far, not much is known about the upstream and downstream players in the presumed signal transduction pathways. In the eukaryotic model organisms, from yeasts to humans, signal transduction pathways govern the activity of many gene expression regulators through reversible phosphorylation 86 . Many of these regulators are trans-acting transcription factors, a protein class that is almost entirely absent from the Leishmania proteome 71 . Therefore, post-transcriptional regulation, in particular on the protein synthesis and protein modification levels, are important for Trypanosomatida 49,87 . How these processes are linked to the signal transduction pathways remains one of the most important questions in the field. Since HSP90 and its co-chaperones are integral parts of the signal transduction pathways governing the Leishmania life cycle 20,27,61 and are targets of stage-specific phosphorylation events 34 , the post-translational modification of HSP90 is likely to have multiple effects on Leishmania viability and fitness, some of which were uncovered in our analysis.

Materials and Methods
L. donovani culture, genetic complementation and in vitro infection model. L. donovani 1SR 88 were cultivated as described 27 . Batch-specific IC 90 were determined for radicicol (Sigma-Aldrich, München, Germany) prior to each experiment. Concentration range was 0.5-1.25 μg/ml. Electrotransfection and antibiotic selection of L. donovani promastigotes was carried out as described 27 .

In vitro infection model. In vitro infection of murine bone marrow-derived macrophages with RAD-treated
L. donovani promastigotes was performed as described earlier 27 with two modifications: (i) bone marrow macrophage precursor cells were differentiated using 10-30% supernatant of LADMAC cells 89 , and (ii) quantification of infections were performed by quantitative PCR 63 . Adherent BMMs were infected at a multiplicity of infection of 10 parasites per macrophage. After 4 hours of incubation at 37 °C in DMEM/F-12, supplemented with GlutaMAX, free parasites were removed by 3 washing steps with PBS, and the infected cells were incubated at 37 °C and 9% CO 2 for another 44 hours. At 48 h post-infection, free cells in the culture supernatant and attached cells were pooled by sedimentation and lysed. Genomic DNA (gDNA) was isolated from the infected cells using the ISOLATE II Genomic DNA Kit (Bioline, Luckenwalde, Germany). Parasites were then quantified by semi-quantitative real-time PCR (qPCR) targeting host cell and parasite actin-coding genes with double labeled probes and using total parasite and host gDNA as the template 63 . The relative parasite load was defined as the ratio of parasite actin DNA against mouse actin DNA.
The derivative of the plasmid pJC45 91 , pJC45:HSP90, was also used for site-directed mutagenesis. The same set of primers (Table S1) was used. PCR, ligation, transformation of E. coli and CsCl purification of plasmids were performed the same as for the site-directed mutagenesis of pUC:HSP90rr.
Construction and preparation of recombinant DNA. The expression plasmid pTLv6 has been described 27 . Mutated HSP90 coding sequences derived from pUC:HSP90rr were excised with enzymes KpnI and BamHI (compatible with BglII sticky ends) and ligated into linearised pTLv6 to create the pTLv6:HSP90rr expression plasmids coding for the various HSP90rr variants with P-site codon exchanges.
Scanning electron microscopy (SEM). Leishmania cells were cultivated for 72 h without RAD or with the IC 90 of RAD. The treated and non-treated cells were then washed twice in PBS, fixed in 2% glutaraldehyde in sodium cacodylate buffer and postfixed with 1% osmium. Samples were dehydrated at increasing ethanol concentrations (30-100%). After critical point drying, samples were treated with gold and analysed on a Philips SEM 500 electron microscope. Images were taken using a conventional 35 mm camera, and the developed black-and-white films were digitalised using a HAMA 35 mm film scanner. separation, an Easy-nLC ™ -system (Thermo Fisher Scientific (previously Proxeon), Odense, Denmark) was coupled directly to the mass spectrometer. The Easy-nLC ™ was fitted with a home-made analytical column (50 µm Mascot searches were conducted with 5 ppm peptide tolerance and 0.6 Da fragment ion tolerance. For database searches, cysteine carbamidomethylation was included as a fixed modification, while oxidation of methionine, Gln → pyro-Glu (N-term Q), Glu → pyro-Glu (N-term E), pyrocarbamidomethyl (N-term C) and phospho (S, T, and Y) were added as variable modifications. Data sets were compiled in Scaffold (version 3.3.1, Proteome Software Inc., Portland, OR, USA) with a protein threshold of minimum 95% and a peptide threshold of minimum 95% for at least 1 peptide. The phosphorylated proteins fulfilling these criteria were further evaluated in Scaffold PTM (version ScaffoldPTM_1.1.3, Proteome Software Inc., Portland, OR, USA).
Phosphorylation sites were validated by a semi-automatic approach, applying Ascore 97 in combination with Mascot Delta Score 98 and manual inspection of selected spectra. Phosphorylation sites with Ascores >19 were accepted unconditionally.
Phosphoproteomic identification of CK1.2 phosphosites in HSP90. For identification of CK1.2 phosphosites, we performed three independent cold kinase assays that were then transferred into fresh tubes to perform in-solution digestion, according to standard protocols. Briefly, after reduction (to a final concentration of 5 mM DTT) and alkylation (to a final concentration of 10 mM iodoacetamide) samples were diluted 10-fold with ammonium bicarbonate and incubated overnight with 0.2 µg trypsin/LysC (Promega) at 37 °C. Samples were then loaded onto a homemade C 18 stage tip for desalting. Desalted samples were reconstituted in 2% MeCN/0.3% TFA and analysed by nano-LC-MS/MS by using an RSLCnano system (Ultimate 3000, Thermo Scientific) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Peptides were www.nature.com/scientificreports www.nature.com/scientificreports/ analysed using HCD fragmentation with normalised collision energy of 30, in a top speed mode with 45 seconds exclusion time. Spectra were interrogated by Sequest TM through Proteome Discoverer TM 2.1 (Thermo Scientific) with the in-house database containing the sequences of the Leishmania HSP90, the Leishmania casein kinase 1 (E9AHM9, E9AHM8 and A4IAZ8) and the most abundant contaminants (244 protein sequences). Enzyme specificity was set to trypsin and a maximum of two missed cleavage site were allowed. Oxidised methionine, N-terminal acetylation, phosphorylation of Ser, Thr and Tyr and carbamidomethyl cysteine were set as variable modifications. Maximum allowed mass deviation was set to 10 ppm for monoisotopic precursor ions and 0.6 Da for MS/MS peaks. The resulting files were further processed using myProMS 99 . FDR calculation used Percolator and was set to 1% at the peptide level for the whole study. We validated phosphorylated peptides by combining the phosphoRS information and by manually inspecting the peak assignment.
To quantify the phosphorylated S289 peptide, we extracted from the MS survey of the nanoLC-MS/MS raw files the extracted ion chromatogram (XIC) signal by using the retention time and m/z values of the well characterised tryptic peptide ions using the Xcalibur softwares (manually). XIC areas were integrated in Xcalibur under the QualBrowser interface using the ICIS algorithm. Areas were normalised by using the non-phosphorylated ion's signal 100 . Mean values and standard deviation were calculated from three independent experiments.
Recombinant expression of the mutated HSP90 variants. The constructs for the expression of recombinant wild type or mutated HSP90 were introduced into E. coli BL21(DE3) [pAPlacI Q ] and expression was induced using 0.4 mM IPTG at 37 °C for 2 h 101 . The His-tagged proteins were purified using a Ni-NTA column (Qiagen), as per the manufacturer's instructions. Purity of the proteins was verified by SDS-PAGE and Coomassie Brilliant Blue staining. The purified proteins were used for the kinase assays.
In silico procedures. DNA and protein sequence analysis was performed using the MacVector ® software (versions 12 to 16). Numerical data were analysed using the Prism ® software (version 5). Greyscale and colour images were optimised for contrast using Photoshop ® CS3 (Adobe). Composite figures were assembled using the Intaglio ® software (Purgatory). For the length measurements of cells, the digital images were imported into the ImageJ 1.42q software (Wayne Rasband, National Institute for Health, USA) analysed using the measurement line tool. Measurements in centimetres were then normalised using the integrated size bars. For the analysis of the cell body lengths 50 randomly selected cells were measured. For the analysis of the cell body widths and flagella length 25 cells were analysed. Significance was determined using the two-sided U-test 104 . All greyscale images were cropped from contiguous parts of the original image; no recombination of lanes was performed. Digital enhancements, using Adobe Photoshop CS3, were performed over the entire greyscale images and restricted to tonality (curve) optimisation and size adjustments.

Data Availability
All data and materials generated for this study may be obtained from the corresponding author upon written request with the stipulation that any work derived from the data and materials will cite the source.