FEBS Letters 585 (2011) 635–640

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Characterization of inositol phospho-sphingolipid-phospholipase C 1 (Isc1)in Cryptococcus neoformans reveals unique biochemical features

Jennifer Henry a, Aimee Guillotte a, Chiara Luberto a, Maurizio Del Poeta a,b,c,d,⇑a Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USAb Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USAc Department of Craniofacial Biology, Medical University of South Carolina, Charleston, SC, USAd Division of Infectious Diseases, Medical University of South Carolina, Charleston, SC, USA

a r t i c l e i n f o

Article history:Received 18 November 2010Revised 5 January 2011Accepted 7 January 2011Available online 21 January 2011

Edited by Sandro Sonnino

Keywords:Phospholipase CInositol sphingolipidsSphingomyelinPhytoceramidePlasma membrane ATPaseCryptococcus neoformans

0014-5793/$36.00 � 2011 Federation of European Biodoi:10.1016/j.febslet.2011.01.015

⇑ Corresponding author at: Department of BiochemMedical University of South Carolina, 173 Ashley Ave29425, USA.

E-mail address: delpoeta@musc.edu (M. Del Poeta

a b s t r a c t

In this work, we biochemically characterized inositol phosphosphingolipid-phospholipase C (Isc1)from the pathogenic fungus Cryptococcus neoformans. Unlike Isc1 from other fungi and parasiteswhich hydrolyze both fungal complex sphingolipids (IPC-PLC) and mammalian sphingomyelin(SM-PLC), C. neoformans Isc1 only exerts IPC-PLC activity. Genetic mutations thought to regulatesubstrate recognition in other Isc1 proteins do not restore SM-PLC activity of the cryptococcalenzyme. C. neoformans Isc1 regulates the level of complex sphingolipids and certain species of phy-toceramide, especially when fungal cells are exposed to acidic stress. Since growth in acidic environ-ments is required for C. neoformans to cause disease, this study has important implications forunderstanding of C. neoformans pathogenicity.� 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

In the past several years, it has become increasingly clear thatsphingolipid metabolism is intimately linked with the virulenceof the fungal pathogen Cryptococcus neoformans (Cn), a medicallyimportant microorganism [1]. Cn is a facultative intracellular path-ogen and it can grow and replicate within the phagolysosome ofphagocytic cells, such as alveolar macrophages (AMs), as well asin the extracellular spaces, such as in the alveoli or in the blood-stream [2–6].

Our laboratory has previously showed that cryptococcal sphin-golipids regulates signaling events leading to the production of vir-ulence factors [7,8] and to the regulation of phagocytosis [9,10].Interestingly, sphingolipid not only regulate the internalization ofCn by macrophages but also promote fungal growth in these envi-ronments (intracellular and extracellular). Intracellular fungalgrowth is controlled by Ipc1 [11] and inositol phosphosphingoli-

chemical Societies. Published by E

istry and Molecular Biology,nue, BSB 503, Charleston, SC

).

pid-phospholipase C 1 (Isc1) [12], whereas extracellular growthis controlled by glucosylceramide synthase (Gcs1) [13,14] (Fig. 1).The regulation of these processes significantly affects the interac-tion of Cn with macrophages, particularly in the lung environmentwith an important effect on the outcome of the disease.

In Saccharomyces cerevisiae (Sc) the enzyme which hydrolyzescomplex sphingolipids, such as inositol phosphorylceramide(IPC), generating phytoceramide and inositol phosphate was iden-tified as Isc1 by Sawai et al. [15]. In vitro, Sc Isc1 metabolizes notonly IPC and its mannosylated derivatives mannosylinositolphosphorylceramide (MIPC) and mannosyldiinositol phosphoryl-ceramide (M(IP)2C), thus exerting IPC-PLC activity, but it alsometabolizes the mammalian complex sphingolipid sphingomyelin(SM), thus exerting SM-PLC activity, in spite of the fact that fungalcells do not contain SM [15].

In the parasite Leishmania major, the Isc1 homolog (ISCL) doespossess both IPC-PLC and SM-PLC activity, even though the para-site does not produce SM and, the breakdown of host SM is anessential process for L. major to cause disease [16].

In Cn, deletion of Isc1 generated a Cn mutant (Disc1) that ishypersensitive in vitro to nitrogen oxide, hydrogen peroxide andacidic stresses and, thus, it cannot survive within the phagolyso-some of macrophages. In this study, we examined the biochemical

lsevier B.V. All rights reserved.

Fig. 1. Ipc1 and Isc1 biosynthetic pathway. Ipc1 consumes phytoceramide and generates diacylglycerol (DAG) in the synthesis of IPC, which is subsequently transformed intoMIPC and M(IP)2C. Isc1 generates phytoceramide in the hydrolysis of IPC, MIPC or M(IP)2C. The mammalian homologs, sphingomyelin synthase (SMS1 and SMS2) andsphingomyelinase (SMases), which consume and generate ceramide, respectively, are shown for comparison. In fungal cells, the fatty acid in the phytoceramide species canbe hydroxylated (in gray) in position 2 (dotted line). Bracket’s numbers represent the length of the sphingosine backbone and fatty acids. PI, Phosphatidylinositol; DAG,diacylglycerol; PC, phosphatidylcholine.

636 J. Henry et al. / FEBS Letters 585 (2011) 635–640

characteristics of the Cn Isc1 enzyme and analyzed the biochemicalfeatures of the Cn strain lacking Isc1 compared to the wild type(WT) and a reconstituted strain (Disc1REC) in acidic and neutralconditions.

2. Materials and methods

2.1. Strains and growth media

Cn var. grubii serotype A strain H99 wild type (WT); Cn var.neoformans serotype D strain JEC21; Cn var. gattii serotype B (strainMMRL 1336) and serotype C (strain MMRL 1343) [the latter twostrains were a kind gift from Wiley Schell, Duke University MedicalCenter, Durham, NC, USA]; Candida albicans (Ca) wild type strainA39; Schizosaccharomyces pombe strains 972 (h�) and 975 (h+);S. cerevisiae wild type strain (Jk9-3Da); Sc Disc1 strain [15]; CnIsc1 deletion strain (Cn Disc1) and the reconstituted strain(Disc1REC) were used in this study. For in vitro growing condition,please see Supplementary materials.

2.2. In vitro IPC and sphingomyelin SM phospholipase C (IPC-PLC andSM-PLC) activity

Phospholipase C activity against IPC (IPC-PLC) was performed aspreviously described [15]. Briefly, cell lysates containing 100 lgprotein were incubated at 30 �C for 30 min in 100 ll of buffer con-taining 50 mM Tris (pH 7.5), 5 mM MgCl2, 5 mM dithiothreitol,0.1% Triton X-100, 10 nmol of phosphatidylserine (Avanti PolarLipids), 2 nmol of unlabeled IPC, and 30 000 dpm of myo-[2-3H]ino-sitol-labeled IPC. After the incubation, 0.8 ml of chloroform, 0.4 mlof methanol, and 0.2 ml of 1% perchloric acid were added [17], andthe radioactivity in a portion (300 ll) of the upper (aqueous) phasewas measured by liquid scintillation counting. Phospholipase Cactivity against SM (SM-PLC) was tested in a similar manner, ex-cept that 2 nmol SM and 30 000 dpm of [choline-methyl-14C]-labeled SM were used in place of unlabeled and labeled IPC, respec-tively. After incubation, 0.8 ml of chloroform, 0.4 ml of methanol,and 0.2 ml of distilled water were added and the radioactivity ina portion (300 ll) of the upper phase was measured by liquid scin-tillation counting. As a negative control, the Sc Disc1 + pYES empty

Fig. 2. Cryptococcus neoformans (Cn) and C. albicans (Ca) lack SM-PLC Activity. Crudecell lysates from fungi were assessed for neutral phospholipase C (PLC) activityagainst either inositol phosphorylceramide (IPC) or sphingomyelin (SM). The fungitested included Sc (Sc), Cn var. grubii serotype A strain H99 (Cn A), Cn var. gattiiserotype B (Cn B), Cn var. gattii serotype C (Cn C), Cn var. neoformans serotype D (CnD), Ca, and S. pombe (Sp).

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strain was assayed for Isc1 activity and its value subtracted fromvalues obtained with Isc1 wild-type or mutated forms. Experi-ments were repeated at least three times. Radiolabeled IPC wasprepared by incubating WT cultures with [2-3H] inositol for 4 hand extracting sphingolipids as previously described. Non-radioac-tive labeled C26-IPC was custom-made by Avanti Polar Lipids, Ala-baster, AL, USA.

2.3. Mutagenesis studies

The Cn Isc1 cDNA from wild-type of mutated forms were sub-cloned from pCR-TOPO-Cn-Isc1 [12] into the pYES vector (Invitro-gen) and the Xpress tag was inserted at the 50 of the Cn Isc1 gene tomonitor protein expression in Sc Disc1 cells. Mutagenesis studieswere performed as described in the Supplementary materials andthe primers used are illustrated in the Supplementary Table 1.For molecular docking studies, coordinates for phosphocholinewere generated and docked manually into the Bc Isc1 structureusing the program O version 11 [18].

2.4. In vivo labeling with 3H-inositol or 3H dihydrosphingosine

For quantitative measurements of IPC-B, IPC-C/D, MIPC andM(IP)2C in Cn wild-type, Disc1 and Disc1REC strain, cells were incu-bated for 30 min with 25 lCi of myo-[2-3H] inositol (20 Ci/mmol),and the lipid were extracted and loaded onto a TLC on silica gel 60plates (EM Science) using the solvent system chloroform/metha-nol/water (65:25:4). The separated radioactive lipids were scrapedfrom the TLC and quantified using a liquid scintillation counter[15]. The mass of each species was normalized to phosphorus lev-els of each sample. Further details are illustrated in the Supple-mentary materials.

2.5. Extraction and mass spectrometry analysis of yeast sphingolipids

Lipids for mass spectrometry analysis were extracted by stan-dard methods (please see Supplementary materials). An aliquotof the extraction (300 ll) was used for phosphorous determination.Internal standards were added to the remaining aliquots, andsphingolipids were extracted in a one-phase neutral organic sol-vent (propan-2-ol/water/ethyl acetate, 30:10:60, by vol.). Sampleswere then analyzed by a Surveyor/TSQ 7000 liquid chromatogra-phy–MS system.

2.6. Statistical analysis

The two-way analysis of variance (ANOVA) was used. For allstatistical tests, P-values less than 0.05 were considered significant.

Fig. 3. Expression of C. neoformans (Cn) Isc1 in S. cerevisiae (Sc) Disc1 does notrestore SM-PLC activity. (A) IPC-PLC and SM-PLC activity using protein lysatesobtained from Sc Disc1 + pYES Sc Isc1 mixed with 50, 100 or 150 lg of Cn proteinlysates. (B) IPC-PLC and (C) SM-PLC activity of Sc wild-type (WT) + pYES empty, ScDisc1 + pYES empty, Sc Disc1 + pYES Sc Isc1, and Sc Disc1 + pYES Cn (Cn) Isc1. Dataare average ± standard deviation of three separate experiments.

3. Results and discussion

3.1. Cn lacks sphingomyelinase activity

Our first interest was to investigate whether Cn cell protein ly-sates would hydrolyze the mammalian complex sphingolipidsphingomyelin (SM), as was observed for the Sc protein lysates.Unexpectedly, we found that Cn cannot metabolize SM (Fig. 2),suggesting that the cryptococcal Isc1 does not have SM-PLC activ-ity. In addition, we did not detect any SM-PLC activity in other ser-otypes (B, C and D) of Cn, nor in another pathogenic yeast, such asC. albicans (Ca). As expected, we did detect SM-PLC activity in Scand in Saccharomyces pombe protein lysates. We confirmed theseresults by in vivo labeling with NBD-labeled C6-SM, and foundthat, whereas Sc could generate NBD-labeled ceramide, Cn couldnot (data not shown). These results suggest that the cryptococcal

Isc1 enzyme may possess different biochemical properties thanthose exerted by Sc.

Because we observed that Cn protein extracts do not possessSM-PLC activity (Fig. 2), we wondered whether a cryptococcal fac-tor(s) may inhibit such activity. To address this hypothesis, differ-ent concentrations of protein lysates isolated from Cn Disc1 mutantwere mixed with protein lysates isolated from Sc Disc1 + pYES ScIsc1, and IPC-PLC and SM-PLC activities were measured. We foundthat presence of Cn proteins did not inhibit SM-PLC or IPC-PLCactivities of Sc Isc1 (Fig. 3A). Next, we wondered whether a fac-tor(s) from Sc would be necessary for SM-PLC activity. Thus, theCn Isc1 cDNA was expressed into the Sc Disc1 strain and bothIPC- and SM-PLC activity were measured. Sc Isc1 cloned into pYESwas used as a positive control, whereas the pYES empty plasmidwas used as a negative control. As additional controls for Isc1protein expression, we also included the Sc wild-type JK-3Da

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transformed with pYES empty vector. We found that IPC-PLC activ-ity is promptly restored in the Sc Disc1 strain when over-express-ing either Sc Isc1 or Cn Isc1 (Fig. 3B). However, only the expressionof Sc Isc1 and not Cn Isc1 restores SM-PLC activity in the Sc Disc1strain (Fig. 3C). These results suggest that Cn Isc1 is biochemicallydifferent from Sc Isc1 in terms of substrate specificity.

3.2. Functional analysis of Cn Isc1 by site-directed mutagenesis of theP-loop-like domain

Studies on the Sc Isc1 [19,20] and Bacillus cereus SMase [21]have identified distinct domains within the enzyme which areessential for substrate binding and hydrolysis. One such domain,the P-loop-like domain, shows homology among the Isc1 super-family, including bacterial and human SMases. The Cn Isc1 P-loopdomain is contained between G113 and A120 and is illustratedin Fig. 4A. Okamoto et al. have shown that substituting the second

Fig. 4. Effect of mutations of the C. neoformans Isc1 P-loop domain on IPC-PLC andSM-PLC activity. (A) Alignment of amino acid sequences of P-loop-like wild-typedomain and mutant constructs. Amino acids sequences are given for residues 113–120 of Cn Isc1. The residue(s) mutated from the wild-type is underlined. (B) Effect ofmutations in the P-loop on Isc1 activity. Isc1 activity was measured using SM (SM-PLC) or IPC (IPC-PLC) as a substrate, as described. Inset in B shows a representativeWestern blot of P-loop-like domain mutants of Cn Isc1 with anti-Xpress antibody.Shown are two different clones (Cn D114A) and Cn F115W) of each over expressedcells compared to Cn Isc1 wild-type.

glycine of the P-loop with an alanine decreased Sc Isc1 activityagainst SM nearly 100-fold, though activity against IPC was not as-sayed [20]. Of interest, Cn has a lysine instead of a glycine at thisposition and additional differences between the two Isc1 homologexist (Fig. 4A). Thus, we wondered whether the differences in ami-no acid residues found in the Cn Isc1 are responsible for the lack ofSM-PLC activity. We expressed Cn Isc1 in S. cerevisiae Disc1 and as-sessed the role of the P-loop residues on both SM-PLC and IPC-PLCactivities. The inset in Fig. 4B is representative Western blot of theexpressed proteins showing that the level of expression was foundto be comparable. We found that D114A and K119A mutations lostIPC-PLC activity completely Fig. 4B, suggesting that these two ami-no acids are essential for catalysis of Cn Isc1. In contrast to Sc, nei-ther of the remaining mutated forms lost IPC-PLC activity nor theydid restore SM-PLC activity, even when the Cn Isc1 P-loop wasidentical to the Sc Isc1 P-loop (Fig. 4B) [20]. These results suggestthat either the P-loop is not involved in the substrate recognitionor that it is not sufficient to discriminate between IPC and SM.

We therefore performed docking studies of Cn Isc1 protein inthe attempt to identify key residues that would interact with IPCand not with SM based on the available structure of B. cereus SMase[22] and found none. Amino acids predicted to interact with thecholine phosphate of SM, such as D195, N197, W232, D295,W284 and F285, are either conserved in Cn Isc1 or absent in othereukaryotic SMase. Thus, which amino acid(s) are most likely to beresponsible for substrate (IPC or SM) recognition or exclusion by CnIsc1 is still unknown.

The observation that Cn Isc1 is not able to breakdown host SMas other microbes do is intriguing. It is possible that, since SM is anessential sphingolipid for mammalian cell viability [23,24], oppor-tunistic pathogenic yeasts, such as Cn, may have lost SM-PLC activ-ity to prevent mammalian cell death due to their close interactionwith their host. This hypothesis is supported by the observationthat Ca, an opportunistic and commensal fungal pathogen, alsodoes not display SM-PLC activity. In non-opportunistic pathogens,such as L. major, the ability to breakdown host SM is maintained bytheir Isc1 homolog ISCL and, importantly, the authors elegantlyshowed that this activity is required for virulence [16].

3.3. Isc1 regulates the level of complex sphingolipids and specificphytoceramide species

Since Isc1 generates phytoceramide during the hydrolysis ofcomplex sphingolipids, we next investigated how the loss of Isc1modulated the level of complex sphingolipids and phytoceramide.Complex sphingolipids were measured by pulse chase labelingusing [3H]-inositol because mass spectrometry analysis could notbe performed due the lack of IPC, MIPC and M(IP)2C standards.Cn Disc1 cells showed a significantly increased of IPC C/D andM(IP)2C compared to Cn WT or Disc1REC strains when cells weregrown at either pH 4.0 (Fig. 5) or 7.0 (data not shown). Similar re-sults were obtained when cultures were labeled with tritiateddihydrosphingosine.

Interestingly, deletion of Isc1 causes the accumulation of twocomplex sphingolipids more polar than M(IP)2C. In other fungi,such as Ca, a new inositol containing sphingolipid, dimmannoseinositol phosphorylceramide (M2IPC), has been identified [25]. Incontrast to IPC, MIPC and M(IP)2C that are anchored to the plasmamembrane, M2IPC can ‘‘diffuse’’ from the plasma membrane to thecell wall. In the dimorphic fungus Histoplasma capsulatum, in addi-tion to M2IPC another sphingolipid has been identified as galacto-syldimmannose inositolphosphorylceramide (GalM2IPC) whichexists in two forms depending on the addition of a galactofuranoseresidue at the 6-position of mannose (yeast form specific) or theaddition of galactopyranose residue at the 4-position of mannose(hyphal form specific) [26,27]. Although not described thus far,

Fig. 5. In vivo labeling of WT, Disc1, and Disc1REC strains of C. neoformans. Cultureswere labeled with 3H-inositol for 30 min, and lipids were extracted and resolved ina TLC (A) as described in Section 2. Panel A is a representative of three separateexperiments. (B) Quantitative analysis of scraped lipids from the TLC normalized tophosphorus (Pi) content. �P < 0.05, Disc1 versus WT or Disc1REC. Data are aver-age ± standard deviation of three separate experiments.

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most likely these new complex sphingolipid species are also pres-ent in C. neoformans, and, since they are more polar than M(IP)2C,they would run below M(IP)2C on a TLC plate. Presumably,M2IPC and GalM2IPC are breakdown by Isc1 and, thus, they wouldaccumulate in condition in which Isc1 is deleted. Further studiesare clearly needed to investigate such hypotheses.

When we measured the level of phytoceramide using massspectrometry, we found that, except at 12 h of growth, at pH 7.0the WT or Disc1REC strain contained approximately the same levelof total phytoceramide than Disc1 mutant (Fig. 6A) but not at pH4.0, in which the level of phytoceramide at 12 and 24 h of growthis significantly lower (Fig. 6B). Interestingly, the Disc1 strain grownat pH 7.0 recovered its total phytoceramide pools very quickly andby 24 h its level is similar to the one measured in the WT or

Fig. 6. Isc1 regulates phytoceramide levels in C. neoformans. Mass spectrometry quphytoceramide (C and D) of WT, Disc1, and Disc1REC grown at pH 7.0 (A and C) or pH 4.0 (BDisc1REC at pH 4; #P < 0.05, WT at pH 4.0 versus pH 7.0. Data are average ± standard dev

Disc1REC strain. In contrast, the Isc1 mutant grown at pH 4.0 doesrecover its phytoceramide pool to a level similar to the WT strainsat 48 h of growth.

An advantage of mass spectrometry analysis is the ability toquantify the different species of lipids. Using this method, wefound that the level of C26 phytoceramide in WT or Disc1REC strainis significantly higher when the strains are grown at low comparedto neutral pH (Fig. 6C and D). Importantly, the Disc1 strain dis-played a significantly lower level of C26 phytoceramide comparedto WT and Disc1REC strains, especially at low pH (Fig. 5D). This sug-gests that Isc1 may directly regulate the level of C26 phytocera-mide in Cn. The lack of C26-phytoceramide in the Disc1 mutantis intriguing, as it suggests a possible involvement of the plasmamembrane ATPase (Pma1), a proton-extruding plasma membranepump, in the regulation of Cn growth when cells are exposed tolow pH [28], which has also been proposed in Sc [29–31].

Although the involvement of sphingolipids in the fungal stressresponse appears to be conserved, our study adds a new dimensionbecause Cn is a facultative intracellular pathogen. Clinically treat-ing cryptococcal infections is often hampered by the persistenceof fungal cells in infected organs. Similar to Mycobacterium tubercu-losis, cryptococcal persistence may be a result of its intracellularability to survive within macrophages [3]. This hypothesis is sup-ported by studies suggesting that Cn may lay dormant in the lungsof individuals many years before disease occurs [32,33], and bystudies in M. tuberculosis that, peculiarly, accumulates C26 triacyl-glycerides during hypoxia, an in vitro condition that mimics thein vivo dormancy prior re-activation [34]. Thus, through the pro-duction of C26-phytoceramide, Cn may be protected against acidicstresses so that it can survive within the hostile phagolysosome ofmacrophages.

In conclusion, we show that Cn Isc1 is biochemically differentthan Sc Isc1 and it synthesizes mainly C26 phytoceramide espe-cially in acidic environments. Since the intracellular niche of hostcells in which Cn grows is characteristically acidic, these results

antification of total phytoceramide pools (A and B) and very long chain (C26)and D). ⁄P < 0.05, Disc1 versus WT or Disc1REC at pH 7; �P < 0.05, Disc1 versus WT or

iation of three separate experiments.

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may have important implications for the interaction of Cn with thehost.

Acknowledgments

We thank John Shea and Talar Kechichian for technical helpwith this work. We also thank Christopher Davies for docking stud-ies. We thank Dr. Alicja Bielawska and Dr. Jacek Bielawski for thelipid analysis. This work was supported in part by the BurroughsWellcome Fund, the National Institutes of Health (AI56168 andAI71142 to M.D.P.), and the Centers of Biomedical Research Excel-lence Program of the National Center for Research Resources(Grant RR17677 Project 2 to M.D.P. and Project 6 to C.L.). The lipidanalysis was partially supported by the National Institutes ofHealth, Grant number C06 RR015455 from the Extramural Re-search Facilities Program of the National Center for Research Re-sources. M.D.P. is a Burroughs Wellcome New Investigator inPathogenesis of Infectious Diseases.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.febslet.2011.01.015.

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