Characterization of cerebrosides from the thermally dimorphic mycopathogen Histoplasma capsulatum: expression of 2-hydroxy fatty N-acyl (E)-{Delta}3-unsaturation correlates with the yeast–mycelium phase transition

Marcos S. Toledo, Steven B. Levery1,2, Erika Suzuki, Anita H. Straus and Helio K. Takahashi1

Department of Biochemistry, Universidade Federal de São Paulo/Escola Paulista de Medicina, Rua Botucatu 862, 04023–900, São Paulo, SP, Brasil, and 2The Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, 220 Riverbend Road, Athens, GA 30602–7229, USA

Received on June 7, 2000; revised on September 26, 2000; accepted on September 26, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Cerebroside (monohexosylceramide) components were identified in neutral lipids extracted from both the yeast and mycelial forms of the thermally dimorphic mycopathogen Histoplasma capsulatum. The components were purified from both forms and their structures elucidated by 1- and 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and low energy tandem collision-induced dissociation mass spectrometry (ESI-MS/CID-MS). Both components were characterized as ß-glucopyranosylceramides (GlcCers) containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base, attached to 18-carbon 2-hydroxy fatty N-acyl components. However, while the fatty acid of the yeast form GlcCer was virtually all N-2'-hydroxyoctadecanoate, the mycelium form GlcCer was characterized by almost exclusive expression of N-2'-hydroxy-(E)-{Delta}3-octadecenoate. These results suggest that the yeast—mycelium transition is accompanied by up-regulation of an as yet uncharacterized ceramide or cerebroside 2-hydroxy fatty N-acyl (E)-{Delta}3-desaturase activity. They also constitute further evidence for the existence of two distinct pathways for ceramide biosynthesis in fungi, since glycosylinositol phosphorylceramides (GIPCs), the other major class of fungal glycosphingolipids, are found with ceramides consisting of 4-hydroxysphinganine (phytosphingosine) and longer chain 2-hydroxy fatty acids. In addition to identification of the major glucocerebroside components, minor components (<5%) detectable by molecular weight differences in the ESI-MS profiles were also characterized by tandem ESI-MS/CID-MS analysis. These minor components were identified as variants differing in fatty acyl chain length, or the absence of the sphingoid 9-methyl group or (E)-{Delta}8-unsaturation, and are hypothesized to be either biosynthetic intermediates or the result of imperfect chemical transformation by the enzymes responsible for these features. Possible implications of these findings with respect to chemotaxonomy, compartmentalization of fungal glycosphingolipid biosynthetic pathways, and regulation of morphological transitions in H.capsulatum and other dimorphic fungi are discussed.

Key words: glycosphingolipid/glucosylceramide/fungus/yeast/thermal dimorphism


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Increasing occurrences of life-threatening systemic mycosis have paralleled the growth in populations of immunosuppressed or -compromised individuals, especially those with acquired immune deficiency, but also organ and tissue transplant recipients, and patients with leukemias and other cancers (Dixon et al., 1996Go; Walsh et al., 1996Go; Durden and Elewski, 1997Go; Wade, 1997Go; Lortholary et al., 1999Go). An additional cause for concern has been the emergence of fungal strains resistant to existing therapeutics. To facilitate development of new diagnostic and therapeutic agents, continuing studies directed toward dissecting the relationship between fungal life cycles, processes of mycotic infection, and factors contributing to virulence and antibiotic resistance are urgently needed. Several promising lines of research have focused on the function of fungal glycosphingolipids (GSLs), particularly of glycosylinositol phosphorylceramides (GIPCs). The synthesis of GIPCs appears to be required for the survival of fungi, but this class of GSLs has not been found in mammalian cells (Daum et al., 1998Go; Dickson, 1998Go; Dickson and Lester, 1999Go). Inhibitors of inositol phosphorylceramide (IPC) synthase are highly toxic to many fungi but exhibit low toxicity in mammals (Takesako et al., 1993Go; Mandala et al., 1997Go, 1998; Nagiec et al., 1997Go).

In addition to GIPCs, fungi also express monohexosylceramides (cerebrosides, or CMHs) having distinctive structural modifications of the ceramide moiety, some of which are also found in GSLs of plants and certain marine invertebrates, but not in those of mammals (Fujino and Ohnishi, 1976Go; Ballio et al., 1979Go; Karlsson et al., 1979Go; Fogedal et al., 1986Go; Matsubara et al., 1987Go; Sitrin et al., 1988Go; Shibuya et al., 1990Go; Jin et al., 1994Go; Natori et al., 1994Go; Sawabe et al., 1994Go; Villas Boas et al., 1994Go; Costantino et al., 1995aGo,b; Duarte et al., 1998Go). These modifications include addition of a characteristic {Delta}8-unsaturation and a branching 9-methyl group to the sphingoid base (see Scheme 1). In the case of fungi, such additional variations may have functional importance in growth, life cycle, morphogenesis, and host–pathogen interactions. For example, Kawai and Ikeda (Kawai and Ikeda, 1982Go, 1983, 1985a; Kawai et al., 1985bGo; Kawai, 1989Go) reported that fungal glucocerebrosides or structurally similar analogs exhibited fruiting-inducing activity in bioassays with Schizophyllum commune. An intact 9-methyl-4,8-sphingadienine, but not the ß-glucopyranosyl residue, was essential for activity (Kawai and Ikeda, 1983Go; Kawai et al., 1985bGo). Glucocerebrosides extracted from the rice pathogen Magnaporthe grisea were found to be highly active elicitors of defense responses, including accumulation of phytoalexins and hypersensitive cell death, when applied to rice leaves (Koga et al., 1998Go). However, although these and a number of other suggestive phenomena have been observed with fungal cerebrosides (Kawai and Ikeda, 1982Go, 1983, 1985a; Kawai et al., 1985bGo; Koga et al., 1998Go; Mizushina et al., 1998Go; Toledo et al., 1999Go), very little is known about their true functions, biosynthesis, or metabolic fate in vivo.



View larger version (16K):
[in this window]
[in a new window]
 
Scheme 1. Structures of fungal cerebrosides: (A) with 2'-hydroxy fatty N-acylation, sphingoid 9-methyl group and (E)-{Delta}8 unsaturation; (B) same with additional (E)-{Delta}3 unsaturation of fatty acid. Monosaccharide may be either glucosyl- (R1 = OH; R2 = H) or galactosyl- (R1 = H; R2 = OH) residue.

 
In mammalian systems, considerable evidence has accumulated that simple sphingolipids such as cerebrosides, sphingomyelin, ceramide, or other products of sphingolipid catabolism, have definite functional roles, for example, as activating or modulating elements of membrane-associated signaling cascades controlling major cellular events during development, morphogenesis, apoptosis, or stress response (Hakomori, 1990Go, 1996; Dobrowsky and Hannun, 1993Go; Hakomori and Igarashi, 1993Go; Hannun et al., 1993Go; Spiegel et al., 1993Go; Mathias et al., 1998Go; Villalobo and Gabius, 1998Go; Kolter and Sandhoff, 1999Go). Since a growing body of evidence now suggests that homologous signal transduction pathways regulate morphological transitions in fungi (Banuett and Herskowitz, 1994Go; Roberts and Fink, 1994Go; Aquino-Pinero and Rodriguez del Valle, 1997Go; Diez-Orejas et al., 1997Go; Gow, 1997Go; Lichter and Mills, 1997Go; Yaar et al., 1997Go; Roze and Linz, 1998Go), the possibility of similar functional involvement of cerebrosides in these processes should be considered.

A key component of our efforts to understand more about the possible functional roles of GSLs has been studies directed toward defining precisely the structural similarities and variations among different species and strains of pathogenic and related non-pathogenic fungi. Such studies of secondary product structure are an essential complement to molecular genetics for defining the function of enzymes associated with their biosynthesis. One focus of these studies has been on an important group of dimorphic mycopathogens which grow in a low temperature saprophytic phase exhibiting hyphal morphology, but are found in tissues of an infected host primarily as budding yeasts. Within this group, which includes the closely related trio Histoplasma capsulatum, Blastomyces dermatitidis, and Paracoccidioides brasiliensis, as well as the more distantly related Sporothrix schenckii, temperature is a dominant, but not exclusive, determinant of morphology. To varying extents depending on species and strain, CO2 concentration, pH, and nutritional factors also contribute to maintenance of a particular form in vitro (Szaniszlo et al., 1983Go; Travassos, 1985Go).

We have now characterized both GIPCs and CMHs of P. brasiliensis (Levery et al., 1998Go, 2000; Toledo et al., 1999Go) as well as CMHs of S. schenckii (Toledo et al., 2000Go). In a classic 1984 study, the structures of GIPCs of H. capsulatum were elucidated by Lester and his colleagues (Barr, 1984aGo,b), but the CMHs of this fungus have never been examined. In this paper we report on the detailed characterization of cerebroside components from both mycelium and yeast forms of H. capsulatum; these studies revealed a remarkable difference in ceramide structure correlating with morphological transition in this fungus.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Detection and isolation of cerebroside fractions from mycelium and yeast forms of H. capsulatum
Cerebrosides were initially detected in the neutral lipid fractions from H. capsulatum by analytical HPTLC with comparison to authentic standards (Figure 1). In lipids extracted from both the yeast and mycelium forms (lanes 1 and 2, respectively), a single putative CMH component was observed, having an Rf value identical to that of GlcCer from P. brasiliensis (lane 5), and stainable by both primulin and orcinol. Both H. capsulatum lipid fractions were subjected to sequential normal phase column chromatography and preparative-scale HPTLC to purify the components of interest to apparent homogeneity (lanes 3 and 4, respectively), and these were characterized by nuclear magnetic resonance (NMR) spectroscopy and positive ion mode electrospray ionization mass spectrometry (+ESI-MS).



View larger version (121K):
[in this window]
[in a new window]
 
Fig. 1. HPTLC analysis of crude neutral lipid fractions from H. capsulatum yeast and mycelium forms (lanes 1, 2); purified CMH components from H. capsulatum yeast and mycelium forms (lanes 3, 4); standard GlcCer previously isolated and characterized from the yeast form of P. brasiliensis (lane 5). Analysis on silica gel 60 HPTLC plates was performed using two different solvent systems: (A) chloroform–methanol–water 60:40:9 (v/v/v; containing 0.002% w/v CaCl2; solvent D); (B) chloroform–methanol–aq. ammonium hydroxide (15 N)–aq. ammonium chloride (0.83%) 50:36:8.6:7.2 (v/v/v/v; solvent E). Approximately 3–5 µg total cerebroside was applied as a 5 mm streak in each lane; further details of analysis and visualization (orcinol) are described in Materials and methods.

 
Nuclear magnetic resonance spectroscopic analysis of H. capsulatum cerebrosides
Both 1H- and 13C-NMR spectra for several fungal cerebrosides have been previously acquired in DMSO-d6/2% D2O at 35°C and all resonances assigned by homonuclear and heteronuclear 2-D correlation methods (Toledo et al., 1999Go). It was therefore sufficient for the present work to obtain 1-D 1H-NMR spectra on the two H. capsulatum cerebrosides under identical conditions in order to characterize them with respect to monosaccharide identity and key ceramide structural features, including the presence or absence of (E)-{Delta}3-unsaturation in the 2-hydroxy fatty N-acyl moiety (see below). However, in order to verify independently all resonance assignments and their corresponding connectivities, 1-D 13C, 2-D 1H-1H TOCSY (homonuclear total correlation spectroscopy) and 1H-detected 13C-1H gHSQC (heteronuclear single quantum correlation) and gHMBC (heteronuclear multiple-bond correlation) spectra were also acquired for both components (not shown) and interpreted as described previously (Toledo et al., 1999Go). The 13C and 1H NMR data and assignments for both H. capsulatum components are compiled in Table I.


View this table:
[in this window]
[in a new window]
 
Table I. Chemical shifts (p.p.m.) of hexose (Hex), sphingosine (Sph), and fatty acyl (Fa) 13C and 1H, and J (i,j) 1H-1H coupling constants for GlcCer from mycelium and yeast forms of H. capsulatum, in DMSO-d6/2% D2O at 35°C
 
The spectra of the mycelium and yeast form components (Figure 2A,B) were virtually identical with respect to resonances corresponding to the monosaccharide and sphingoid moieties, both exhibiting chemical shifts and coupling patterns characteristic for the seven-proton ß-glucopyranosyl spin system (see Scheme 1 for structures and numbering), and additional resonances identifying (4E,8E)-9-methyl-4,8-sphingadienine. A key upfield resonance for these compounds is that for the sphingadienine branching 9-methyl group (H/C-19), which was observed in both spectra as a singlet at 1.545 ± 0.003 p.p.m. (not shown). The latter forms part of a distinctive spin system, consisting of Sph C-1 through C-9 plus C-19, and Sph H-1a/b through H-8 plus H-19, whose pattern and sequence of connectivities (see Table I) can be established unambiguously via 2-D TOCSY, gHSQC, and gHMBC experiments, as described previously (Toledo et al., 1999Go). On the other hand, while the mycelium form GlcCer spectrum exhibited resonances characteristic for N-2'-hydroxy-(E)-3'-alkenoate (Toledo et al., 1999Go), the resonances characteristic for the (E)-{Delta}3 unsaturation (respectively, Fa H-2, -3, and -4 at 4.300, 5.445, and 4.685 p.p.m. [3J3,4=15.5 Hz]; Fa C-2, –3, and –4 at 72.29, 129.45, 131.69) were essentially absent in the spectrum of the yeast form GlcCer, indicating that the fatty acyl form is predominantly N-2'-hydroxyalkanoate. The proportion of (E)-{Delta}3-unsaturation, calculated from the relative integrals of the Fa-4 and Sph-5 1H resonances, was >95% (±2%) in the mycelium form glucocerebroside.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 2. Downfield sections of 1-D 1H-NMR spectra of cerebroside fractions from H. capsulatum (A, mycelium form component; B, yeast form component). Resonances from non-exchangeable protons of sphingosine (Sph), fatty acyl (Fa), and Hexose (prefix omitted) are designated by Arabic numerals.

 
Remaining GSL structural features, not conveniently assessed by NMR analysis, are the chain lengths of the fatty acyl and sphingosine moieties. These were determined unambiguously by mass spectrometric methods, which in addition provided confirmation of most of the features discussed above. Mass spectrometry was also used to characterize very minor components whose presence was not readily apparent from the NMR spectra alone.

Analysis of H. capsulatum cerebrosides by electrospray ionization mass spectrometry and tandem collision-induced dissociation mass spectrometry
Major components.
In positive ion mode ESI-MS, abundant monolithiated molecular ion adducts were observed virtually exclusively at m/z 760 and m/z 762 for the mycelium and yeast form glucocerebrosides, respectively (Figure 3A,B), corresponding to nominal molecular masses of 753 and 755 Da. These molecular masses are consistent with monohexosylceramides containing (d19:2) (4E,8E)-9-methyl-4,8-sphingadienine attached to either N-2'-hydroxy-(E)-3'-octadecenoate or N-2'-hydroxyoctadecanoate, respectively. Confirmation that the observed difference of m/z 2 is due to variation in the fatty acid moiety was provided by tandem collision induced-dissociation mass spectrometry (+ESI-MS/CID-MS) experiments. In these experiments, product ion spectra were obtained from the lithiated molecular ions, either m/z 760 or m/z 762, selected in Q1 (Figure 4A,B). All spectra were characterized by highly abundant [M + Li – acyl]+ (O) and [M + Li – HexOH – Sph-C3–C19]+ (T {equiv} Z0/G) fragments, as observed previously under these conditions for a variety of fungal cerebrosides (Levery et al., 2000Go). In both spectra, the O ion is observed at m/z 480, while the m/z 2 difference is carried by the T fragment, containing the fatty acid moiety, observed at either m/z 330 or m/z 332 depending on the mass of the pseudomolecular ion selected. Furthermore, the masses and relative abundances of additional major and minor fragments are in each case virtually identical with those previously obtained under these conditions for fungal cerebrosides containing (4E,8E)-9-methyl-4,8-sphingadienine as the long-chain base in combination with N-2'-hydroxy-(E)-{Delta}3-octadecenoate and N-2'-hydroxyoctadecanoate, respectively. A number of these fragments can be considered as diagnostic for the presence and positions of characteristic functional groups such as unsaturations and fatty N-acyl 2-hydroxylation (Levery et al., 2000Go). The assignments for these fragments are summarized in Table II.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. Molecular ion regions of +ESI-MS spectra for Li+ adducts of cerebrosides from H. capsulatum (A, mycelium form component; B, yeast form component).

 


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Tandem +ESI-MS/CID-MS product ion spectra of selected [M+Li]+ from major H. capsulatum cerebroside components. (A) product ion spectrum from m/z 760, mycelium form cerebroside profile; (B) product ion spectrum from m/z 762, yeast form cerebroside profile.

 

View this table:
[in this window]
[in a new window]
 
Table II. +ESI-MS/CID-MS data (all fragments ·Li+ except where noted) for H. capsulatum mycelium form GlcCer (major component, H1a; minor components, H1b1, H1b2, H1c); and yeast form GlcCer (major component, H2a; minor components, H2b, H2c); with proposed interpretations of fragments (all values nominal, monoisotopic m/z)
 

Minor components.
In addition to completing the structure elucidation of the major components, it was also possible by +ESI-MS/CID-MS to characterize minor components appearing in the molecular ion profiles of mycelium and yeast form GlcCer, which could correspond either to low abundance intermediates in the biosynthetic pathway or to structural variants differing, for example, in fatty acid chain length. Both of these possibilities are apparent in the product ion spectrum of m/z 746 (Figure 5A) selected from the molecular ion profile of the mycelium form GlcCer. The spectrum exhibits a single [M + Li – hexose]+ (Y0) ion at m/z 584; as expected, this is 14 Th less than that observed for the major molecular ion (Y0 at m/z 598). However, at lower m/z, product ions arising from two isobaric components are apparent, characterized by two sets of O and T ions, one at m/z 480 and 316, the other at m/z 466 and 330. These are consistent with components in which the 14 Th decrement is carried by either the fatty N-acyl group or the sphingoid moiety, respectively, present in approximately equal amounts. In contrast with the former case, which can be clearly attributed to a variant carrying an h17:1 fatty N-acyl group, the latter case most likely corresponds to a h18:1/d18:2 component in which the sphingoid 9-methyl group is missing, which may represent a biosynthetic intermediate.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Tandem +ESI-MS/CID-MS product ion spectra of selected [M+Li]+ from minor H. capsulatum cerebroside components. (A) product ion spectrum from m/z 746, mycelium form cerebroside profile; (B) product ion spectrum from m/z 748, yeast form cerebroside profile; (C) product ion spectrum from m/z 750, yeast form cerebroside profile.

 

Unlike the result with the mycelium form m/z 746 ion, products of the corresponding minor m/z 748 ion in the yeast form profile (Figure 5B) are consistent with essentially a single component in which the 14 Th decrement is carried by the sphingoid moiety, as characterized by a single set of O and T ions at m/z 466 and 332. With the exception of a very low abundance set of O and T ions at m/z 480 and 318, corresponding to the h17:0/d19:2 fatty N-acyl/sphingoid combination, the masses and relative abundances of almost all other fragments in the spectrum are consistent with a h18:0/d18:2 component lacking the sphingoid 9-methyl group.

Products of the m/z 750 component (Figure 5C) were also consistent with essentially one component lacking both the sphingoid 9-methyl group and the {Delta}8-unsaturation. In addition to the O and T fragments observed at m/z 468 and 332, respectively, which show the additional 2 Th increment to be carried by the sphingoid moiety, further evidence for the mono-unsaturated (d18:1) sphing-4-enine structure are the obvious changes in the abundance of the T and N (m/z 306) fragments relative to each other and the increase in the abundance of the Y0 (m/z 588) relative to the O fragment. A number of other characteristic changes in the spectrum are diagnostic for the 2-hydroxy fatty N-alkanoyl/sphing-4-enine combination under these conditions, and in fact the product spectrum is essentially the same as that obtained previously from a bovine brain galactocerebroside with h18:0/d18:1 ceramide (Levery et al., 2000Go). Interestingly, an analogous component is not observed in significant abundance in the mycelium form profile (at m/z 748, see Figure 3A). On the other hand, a molecular ion at m/z 774 was sufficiently abundant in the mycelium form profile for a product ion spectrum to be acquired (not shown). In this case the predominant O and T ions were observed at m/z 480 and 344, respectively, consistent with an h19:1/d19:2 fatty N-acyl/sphingoid combination. Other product ions observed in the spectrum were consistent with this structure (see Table II). An analogous molecular ion m/z 776 in the yeast form profile was not sufficiently abundant to produce a useful CID spectrum.

Comparison with results from P. brasiliensis cerebrosides
The +ESI-MS profile obtained from a yeast form GlcCer of P. brasiliensis (Levery et al., 2000Go) was very similar to that obtained here for the corresponding H. capsulatum fraction, differing mainly in the proportions of some minor components. Aside from the appearance of an abundant m/z 762 component (yielding an identical CID product spectrum), minor ions at m/z 750 and 748 were also observed in the P. brasiliensis yeast form GlcCer profile. In the case of m/z 750, in P. brasiliensis as with H. capsulatum the only product ions observed corresponded to the h18:0/d18:1 component. On the other hand, in the case of m/z 748 from P. brasiliensis yeast form isobaric h18:0/d18:2 and h17:1/d19:2 components were comparably represented (Levery et al., 2000Go), whereas the latter was barely observed in H. capsulatum (Figure 5B).

The +ESI-MS profile of the P. brasiliensis mycelium form GlcCer was also similar to that from H. capsulatum, although with P. brasiliensis the m/z 760 component was accompanied by a significant ion abundance at m/z 762 (Levery et al., 2000Go). This was in agreement with the NMR and fatty acid analysis, which showed incomplete conversion to the {Delta}3-unsaturated form (Toledo et al., 1999Go). A minor m/z 746 ion was also observed in the P. brasiliensis mycelium form GlcCer profile. Unlike the case with H. capsulatum, in which product ions from the isobaric h18:1/d18:2 and h17:1/d19:2 components could be observed in comparable amounts, the latter was only barely detectable in the P. brasiliensis m/z 746 spectrum. A number of other minor ions were observed in the profiles of P. brasiliensis glucocerebrosides, and CID data acquired (Levery et al., 2000Go), but were not sufficiently abundant in the H. capsulatum profiles for useful product spectra to be obtained; these were m/z 748 and 750 in the mycelium form profile, and m/z 734 and 790 in both profiles.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Structure elucidation of CMH components from yeast and mycelial forms of Histoplasma capsulatum showed that they are identical with respect to hexose moiety, both forms expressing GlcCer exclusively. However, detailed characterization of the ceramide moieties revealed a remarkable difference in the level of (E)-{Delta}3-unsaturation in the 2-hydroxy fatty N-acyl moiety which correlates with the phenotypic dimorphism exhibited by this fungus. The chemical dimorphism with respect to ceramide (E)-{Delta}3-unsaturation appears to be qualitatively similar to that observed in Paracoccidioides brasiliensis (Toledo et al., 1999Go), a closely related dimorphic mycopathogen also expressing GlcCer in both forms, but the quantitative differences observed with H. capsulatum are much more dramatic. With P. brasiliensis the levels of (E)-{Delta}3-unsaturation were found to be ~15% in the yeast form and ~55% in the mycelium form, while in H. capsulatum the corresponding levels were <5% and >=95%, respectively. The results together suggest that in these fungi activation of a 2-hydroxy fatty N-acyl (E)-{Delta}3-desaturase accompanies the yeast -> mycelium transition, or suppression of this activity accompanies the mycelium -> yeast transition. Given the close taxonomic relationship between P. brasiliensis, H. capsulatum and Blastomyces dermatitidis, we hypothesize that cerebrosides of B. dermatitidis should follow a similar pattern of dimorphism with respect to (E)-{Delta}3-unsaturation in the yeast and mycelium forms. In contrast, cerebrosides of mycelium and yeast forms of Sporothrix schenckii did not differ significantly in the level of (E)-{Delta}3-unsaturation but, while GlcCer was expressed in both forms, the yeast form was distinctively characterized by the appearance of GalCer as well (Toledo et al., 2000Go).

Although reports of physiological activities for fungal glucocerebrosides (Kawai and Ikeda, 1982Go, 1983, 1985a; Kawai et al., 1985bGo; Kawai, 1989Go; Koga et al., 1998Go; Mizushina et al., 1998Go) are not necessarily unambiguous demonstrations of true function, they are highly suggestive that significant functional roles exist for these compounds in vivo. In the present case, although the mechanism and functional implications of the observed ceramide structural dimorphism are unclear, the changes in levels of (E)-{Delta}3-unsaturation could have implications for the regulation of morphological transitions in H. capsulatum and P. brasiliensis (and possibly B. dermatitidis). We previously proposed that the (E)-{Delta}3 modification of P. brasiliensis GlcCer could serve a specific messenger function; with activation (or deactivation) of a desaturase responsible for this modification being one step in a signaling cascade directing the transition from yeast to mycelium (or the reverse) which is initiated by a change in temperature. It is possible that the observed changes in CMH composition in dimorphic fungi could simply be ascribed to temperature sensitivity of expression, stability, or activity for the enzymes involved, rather than regulation by more complex mechanisms, but this would not rule out their potential functional significance in morphogenesis. On the contrary, changes in CMH hexose and/or ceramide structure distribution, regulated directly by a simple temperature dependent parameter such as enzymatic activity, could constitute ideal chemical switches activating other processes following alterations in environmental temperature. As pointed out in the Introduction, the possibility that cerebrosides may be components of signal transduction pathways regulating morphological transitions in fungi should be investigated further.

Interestingly, (E)-{Delta}3-unsaturation in the 2-hydroxy fatty N-acyl moiety so far appears to be a modification found only in CMHs of Euascomycetes, having been previously observed in varying amounts in Fusicoccum amygdali Delacroix (Ballio et al., 1979Go), Pachybasium spp. (unnamed) (Sitrin et al., 1988Go), Magnaporthe grisea (Koga et al., 1998Go), Penicillium funiculosum (Kawai et al., 1985bGo), Aspergillus spp. (Ohnishi, 1976; Villas Boas et al., 1994Go; da Silva Bahia et al., 1997Go; Toledo et al., 1999Go; Levery et al., 2000Go), Fusarium spp. (Duarte et al., 1998Go), P. brasiliensis (Toledo et al., 1999Go; Levery et al., 2000Go), and S.schenckii (Toledo et al., 2000Go), but not reported in any Basidiomycete so far investigated, including Schizophyllum commune (Kawai and Ikeda, 1982Go, 1983, 1985a), Lentinus edodes (Kawai, 1989Go), Hypsizigus marmoreus (Sawabe et al., 1994Go), Ganoderma lucidum (Mizushina et al., 1998Go), Clitocybe spp. (Fogedal et al., 1986Go), and yeastlike pathogenic Cryptococcus spp. (Levery et al., 2000Go); nor in the Hemiascomycete dimorphic pathogen Candida albicans (Matsubara et al., 1987Go; Levery et al., 2000Go). It may therefore be tentatively proposed as a chemotaxonomic marker for Euascomycetes.

Finally, the results herein constitute further evidence for the partitioning of fungal ceramide catabolism into two distinct pathways for CMH and GIPC biosynthesis, as has been pointed out independently by Lester and Dickson (Dickson and Lester, 1999Go) and by us (Toledo et al., 1999Go; Toledo et al., 2000Go). For example, unlike the CMHs from Candida albicans (Matsubara et al., 1987Go), P. brasiliensis (Toledo et al., 1999Go), and H. capsulatum (this work), in which the ceramides are of the type shown in Scheme 1, the GIPCs of the same fungi are found predominantly with saturated, longer chain 2-hydroxy fatty acids (h24:0 or h26:0) attached to t18:0 4-hydroxysphinganine (phytosphingosine) (Wells et al., 1996Go; Levery et al., 1998Go; Barr and Lester, 1984bGo; Barr et al., 1984aGo). It is not known how this partitioning of ceramide types into CMH and GIPC biosynthesis is accomplished, but two possibilities which have been suggested (Dickson and Lester, 1999Go; Toledo et al., 1999Go;) are compartmentalization of their respective biosynthetic and/or transport pathways, or selective recognition of ceramide structural elements somewhat remote from the reaction site by the putative IPC synthase (Nagiec et al., 1997Go) and the as yet uncharacterized fungal cerebroside synthase(s). The latter possibility could depend, for example, on the interaction of different ceramide acceptor substrates, having either a 4-hydroxyl group or a 4-unsaturation on the sphingoid, with specific domains on the respective synthases selectively recognizing these features. On the other hand, selectivity in this step alone would not explain the difference with respect to fatty acid chain length in the two types of products. This difference implies a prior partitioning or some other discriminatory process associating a specific fatty acid chain length with its appropriate sphingoid partner.

Knowledge of the CMH biosynthetic pathway in fungi is still at a rudimentary stage, and appears to be more complex than that for GIPCs with respect to assembly of the ceramide moiety. A considerable number of associated genes and their products remain to be discovered. Some of these may be highly homologous to those found widely among eukaryotes, such as the sphingoid (E)-{Delta}4-desaturase or the GlcCer and GalCer synthases. A gene encoding a plant sphingoid (E)-{Delta}8-desaturase was recently confirmed (Sperling et al., 2000Go), and similar genes should eventually be found in fungi, sponges, and echinoderms; one or more 9-methyltransferases should also be found in the latter species. Still others, such as the Euascomycete-associated 2-hydroxy fatty N-acyl (E)-{Delta}3-desaturase, may have no homologues in other taxa. Recently, candidate GlcCer synthase homologs could be extracted from publicly accessible fungal genome databases via tBLASTn searching with mammalian UDP-glucose:ceramide glucosyltransferases (Ugcg) peptide sequences (Ichikawa et al., 1996Go, 1998). Single-exon predicted ORFs, identified in the ongoing Candida albicans (Contig4-3104 [HSX11]; Stanford DNA Sequencing and Technology Center) and Neurospora crassa (9a3.pep1152; Münchener Informazionscentrum für Proteinsequenzen—MIPS) genomic sequence databases, have stretches of significantly conserved homology encompassing the NRD2S and NRD2L motifs proposed by Kapitonov and Yu (1999)Go to be characteristic for the family of glycosyltransferases which includes the mammalian Ugcg proteins; within these motifs, the homology appears closest to those in the Ugcg group (S. B. Levery and J. K. Rose, unpublished observations). Interestingly, the fungal Ugcg sequences contain numerous extra stretches of peptide interpolated between conserved motifs; some of these could comprise additional recognition domains responsible for maintaining strict specificity against Glc residue transfer to phytosphingosine-containing ceramides. The high likelihood of the involvement of cerebrosides in a variety of key fungal cellular processes should provide considerable incentive for future investigations in this area.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Fungal isolate and growth conditions
The culture of Histoplasma capsulatum, strain 496 (originally obtained from a pulmonary lesion of an otherwise healthy individual), was provided by Dr. Olga Gompertz, Department of Cellular Biology, Universidade Federal de São Paulo/Escola Paulista de Medicina, São Paulo, SP, Brasil. Mycelium and yeast forms of H. capsulatum strain 496 were grown in brain–heart infusion (BHI; 37 g/l water), incubated at 25°C and 37°C, respectively, using 2.5 l Fernbach flasks in a shaker at 150 r.p.m.. Both forms were inactivated with 0.1% of thimerosal, and after 48 h mycelium forms were collected by filtration on Whatman no. 1 filter paper, while yeast forms were harvested by centrifugation at 5000 r.p.m. for 20 min (Toledo et al., 1995Go).

Solvents for extraction and anion exchange chromatography
Solvent A, isopropanol/hexane/water (55:20:25, v/v/v, upper phase discarded); solvent B, chloroform/methanol (2:1, v/v); solvent C, chloroform/methanol/water (30:60:8, v/v/v).

High performance thin layer chromatography
Both analytical and preparative HPTLC were performed on silica gel 60 plates (E. Merck, Darmstadt, Germany) using chloroform/methanol/water (60:40:9 v/v/v, containing 0.002% w/v CaCl2; solvent D) or chloroform–methanol–aq. ammonium hydroxide (15 N)–aq. ammonium chloride (0.83%) (50:36:8.6:7.2 v/v/v/v; solvent E) as mobile phases. Lipid samples were dissolved in solvent B and applied by streaking from 5 µl Micro-caps (Drummond, Broomall, PA). For analytical HPTLC, detection was made by Bial’s orcinol reagent (orcinol 0.55% (w/v) and H2SO4 5.5% (v/v) in ethanol/water 9:1 (v/v); the plate is sprayed and heated briefly to ~200–250°C). For preparative HPTLC, samples were streaked lengthwise on 10 x 20 cm plates; separated glycosphingolipid bands were visualized under UV after spraying with primulin (Aldrich; 0.01% in 80% aqueous acetone). Bands were marked by pencil and individually scraped from the plate. Glycosphingolipids were then isolated from the silica gel by repeated sonication in solvents A and B followed by centrifugation. Following concentration of the extract, primulin was removed by passage through a short column of DEAE-Sephadex A-25 in Solvent C.

Extraction and purification of glycosphingolipids
Extraction and purification of glycosphingolipids were carried out as described previously (Toledo et al., 1995Go, 1999). Briefly, glycosphingolipids were extracted by homogenizing yeast or mycelium forms (25–35 g wet weight) in an Omni-mixer (Sorvall Inc. Wilmington, DE), three times with 200 ml of solvent A, and twice with 200 ml of solvent B. The five extracts were pooled, dried on a rotary evaporator, dialyzed against water, lyophilized, resuspended in solvent C, and applied to a column of DEAE-Sephadex A-25 (Ac– form). Neutral glycosphingolipids were eluted with five volumes of solvent C. The neutral glycosphingolipid fraction was further purified from other contaminants by column chromatography on silica gel 60 using a step-wise gradient of chloroform/methanol from 9:1 to 1:1 (v/v) (Sweeley, 1969Go). Fractions containing ceramide monohexosides (CMHs), as assessed by analytical HPTLC, were pooled, dried, and further purified by preparative-scale HPTLC as described above. The purity of each fraction was assessed by analytical HPTLC.

1H-nuclear magnetic resonance spectroscopy
Samples of underivatized CMH (~0.5–1.0 mg) were deuterium exchanged by repeated evaporation from CDCl3/CD3OD (2:1 v/v) under N2 stream at 37°C, and then dissolved in 0.5 ml DMSO-d6/2% D2O (Dabrowski et al., 1980aGo,b; Yamada et al., 1980Go) for NMR analysis. 1-D 1H-NMR, 2-D 1H-1H-TOCSY (Braunschweiler and Ernst, 1983Go; Bax and Davis, 1985Go), 1H-detected, 13C-decoupled, phase sensitive, gradient (Davis et al., 1992Go) 13C-1H-HSQC (Bodenhausen and Ruben, 1980Go) and -HMBC (Bax and Summers, 1986Go; Bax and Marion, 1988Go) experiments were performed at 35°C on a Varian Unity Inova 600 MHz spectrometer using standard acquisition software available in the Varian VNMR software package. Proton-decoupled 1-D 13C-NMR spectra were acquired by direct detection on a Varian Unity Inova 500 MHz spectrometer under identical conditions. Proton chemical shifts are referenced to internal tetramethylsilane ({delta} = 0.000 p.p.m.), carbon chemical shifts to the center line of residual DMSO (set at {delta} = 39.82 p.p.m.).

The percentage of (E)-{Delta}3 unsaturation was calculated from the integrated ratio of the vinyl proton resonances corresponding to H-4'' of (E)-{Delta}3 unsaturated fatty acid and H-5 of the sphingosine moiety (Toledo et al., 1999Go, 2000). These resonances were chosen since they have similar splitting patterns and chemical shifts, but are completely resolved from each other in all spectra; although the chemical shift of H-5 is slightly affected by the presence or absence of (E)-{Delta}3 unsaturation, the total integral for this resonance was assumed to represent 1.00 mol, regardless of fatty acyl distribution.

Electrospray ionization mass-spectrometry
ESI-MS and tandem ESI-MS/CID-MS were performed in the positive ion mode on a PE-Sciex (Concord, Ontario, Canada) API-III spectrometer, with a standard IonSpray source (orifice-to-skimmer voltage (OR), 120–160 V; Ionspray voltage, 5 kV; interface temperature, 45°C), using direct infusion (3–5 µl/min) of CMH samples dissolved (~20 ng/µl) in 100% MeOH to which was added a solution of LiI (10 mM) in MeOH until the observed ratio of Li+ to Na+ molecular ion in +ESI-MS profile mode was >95:5 (the final concentration of LiI was generally 2–3 mM) (Levery et al., 2000Go). For +ESI-MS/CID-MS experiments, precursor ions selected in Q1 were subjected to collision induced dissociation (with argon as collision gas) in Q2, while the mass range in Q3 was scanned from m/z 100–800 in 0.2 u steps. OR was set to 120 V, the collision gas was argon (collision gas temperature (CGT) = 380–400 [x 1012 molecules/cm2]), and collision energy was 80 eV. Other parameters were set to achieve a peak width at height of 0.6–0.7 Th (measured at m/z 332), deemed sufficient to assign nominal masses to all peaks in the mass range of interest (Levery et al., 2000Go). Dwell time of 5 ms (2.5 ms for minor components), giving a total cycle time 19 s (or 9.5 s). In general, spectra represent summations of 5–10 scans for single analyzer profiles, and 10–30 scans for CID experiments (50–100 for minor components). Fragment nomenclature, illustrated in Scheme 2, is after Costello et al. (Domon and Costello, 1988Go; Costello and Vath, 1990Go; Domon et al., 1990Go) as modified and expanded by Adams and Ann (1993; see also Sullards et al., 2000Go).



View larger version (13K):
[in this window]
[in a new window]
 
Scheme 2. Fragmentation of a fungal cerebroside with nomenclature of Costello et al. (Domon and Costello, 1988; Costello and Vath, 1990; Domon et al., 1990Go) as modified by Adams and Ann (1993).

 

    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
We gratefully acknowledge the technical support of Dr. John Glushka (Complex Carbohydrate Research Center NMR Facility). This work was supported by FAPESP, CNPq, and PRONEX (Brasil; M.S.T., E.S., A.H.S., and H.K.T.); a Glycoscience Research Award from Neose Technologies, Inc. (S.B.L.); and the National Institutes of Health Resource Center for Biomedical Complex Carbohydrates (NIH #5 P41 RR05351; S.B.L.).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
CID, collision-induced dissociation; CMH, ceramide monohexoside ({equiv} cerebroside); ESI, electrospray ionization; GalCer, ß-galactopyranosyceramide ({equiv} galactocerebroside); GlcCer, ß-glucopyranosylceramide ({equiv} glucocerebroside); GSL, glycosphingolipid; GIPC, glycosylinositol phosphorylceramide; HPTLC, high performance thin layer chromatography; 1H-1H TOCSY, homonuclear total correlation spectroscopy; 13C-1H gHSQC, heteronuclear single quantum correlation; 13C-1H gHMBC, heteronuclear multiple-bond correlation; 2-D, two dimensional; Fa, fatty acyl; Sph, sphingoid.


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Adams, J., and Ann, Q. (1993) Structure determination of sphingolipids by mass spectrometry. Mass Spectrom. Rev., 12, 51–85.[ISI]

Aquino-Pinero, E.E., and Rodriguez del Valle, N. (1997) Different protein kinase C isoforms are present in the yeast and mycelium forms of Sporothrix schenckii. Mycopathologia, 138, 109–115.[ISI][Medline]

Ballio, A., Casinovi, C.G., Framondino, M., Marino, G., Nota, G., and Santurbano, B. (1979) A new cerebroside from Fusicoccum amygdali Del. Biochim. Biophys. Acta, 573, 51–60.[ISI][Medline]

Banuett, F., and Herskowitz, I. (1994) Identification of fuz7, a Ustilago maydis MEK/MAPKK homolog required for a-locus-dependent and -independent steps in the fungal life cycle. Genes Dev., 8, 1367–1378.[Abstract]

Barr, K., Laine, R.A., and Lester, R.L. (1984a) Carbohydrate structures of three novel phosphoinositol-containing sphingolipids from the yeast Histoplasma capsulatum. Biochemistry, 23, 5589–5596.[ISI][Medline]

Barr, K., and Lester, R.L. (1984b) Occurrence of novel antigenic phosphoinositol-containing sphingolipids in the pathogenic yeast Histoplasma capsulatum. Biochemistry, 23, 5581–5588.[ISI][Medline]

Bax, A., and Davis, D.G. (1985) MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson., 65, 355–360.[ISI]

Bax, A., and Marion, D. (1988) Improved resolution and sensitivity in 1H-detected heteronuclear multiple-bond correlation spectroscopy. J. Magn. Reson., 78, 186–191.[ISI]

Bax, A., and Summers, M.F. (1986) 1H and 13C assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc., 108, 2093–2094.[ISI]

Bodenhausen, G., and Ruben, D.J. (1980) Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Lett., 69, 185–189.[ISI]

Braunschweiler, L., and Ernst, R.R. (1983) Coherence transfer by isotropic mixing: application to proton correlation spectroscopy. J. Magn. Reson., 53, 521–528.[ISI]

Costantino, V., Fattorusso, E., and Mangoni, A. (1995a) Glycolipids from sponges. I. Glycosyl ceramide composition of the marine sponge Agelas clathrodes. Liebigs Ann., 1995, 1471–1475.

Costantino, V., Fattorusso, E., and Mangoni, A. (1995b) Glycolipids from sponges. III. Glycosyl ceramides from the marine sponge Agelas conifera. Liebigs Ann., 1995, 2133–2136.

Costello, C.E., and Vath, J.E. (1990) Tandem mass spectrometry of glycolipids. Methods Enzymol., 193, 738–768.[Medline]

da Silva Bahia, M.C., Vieira, R.P., Mulloy, B., Hartmann, R., and Bergter, E.B. (1997) The structures of polysaccharides and glycolipids of Aspergillus fumigatus grown in the presence of human serum. Mycopathologia, 137, 17–25.[ISI][Medline]

Dabrowski, J., Egge, H., and Hanfland, P. (1980a) High resolution nuclear magnetic resonance spectroscopy of glycosphingolipids. I. 360 MHz 1H and 90.5 MHz 13C NMR analysis of galactosylceramides. Chem. Phys. Lipids, 26, 187–196.[ISI][Medline]

Dabrowski, J., Hanfland, P., and Egge, H. (1980b) Structural analysis of glycosphingolipids by high-resolution 1H nuclear magnetic resonance spectroscopy. Biochemistry, 19, 5652–5658.[ISI][Medline]

Daum, G., Lees, N.D., Bard, M., and Dickson, R. (1998) Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast, 14, 1471–1510.

Davis, A.L., Keeler, J., Laue, E.D., and Moskau, D. (1992) Experiments for recording pure-absorption heteronuclear correlation spectra using pulsed field gradients. J. Magn. Reson., 98, 207–216.[ISI]

Dickson, R.C. (1998) Sphingolipid functions in Saccharomyces cerevisiae: comparison to mammals. Annu. Rev. Biochem., 67, 27–48.[ISI][Medline]

Dickson, R.C., and Lester, R.L. (1999) Yeast sphingolipids. Biochim. Biophys. Acta, 1426, 347–357.[ISI][Medline]

Diez-Orejas, R., Molero, G., Navarro-Garcia, F., Pla, J., Nombela, C., and Sanchez-Perez, M. (1997) Reduced virulence of Candida albicans MKC1 mutants: a role for mitogen- activated protein kinase in pathogenesis. Infect. Immun., 65, 833–837.[Abstract]

Dixon, D.M., McNeil, M.M., Cohen, M.L., Gellin, B.G., and La, M.J. (1996) Fungal infections: a growing threat. Public Health Rep., 111, 226–235.[ISI][Medline]

Dobrowsky, R.T., and Hannun, Y.A. (1993) Ceramide-activated protein phosphatase: partial purification and relationship to protein phosphatase 2A. Adv. Lipid Res., 25, 91–104.[ISI][Medline]

Domon, B., and Costello, C.E. (1988) Structure elucidation of glycosphingolipids and gangliosides using high-performance tandem mass spectrometry. Biochemistry, 27, 1534–1543.[ISI][Medline]

Domon, B., Vath, J.E., and Costello, C.E. (1990) Analysis of derivatized ceramides and neutral glycosphingolipids by high-performance tandem mass spectrometry. Anal. Biochem., 184, 151–164.[ISI][Medline]

Duarte, R.S., Polycarpo, C.R., Wait, R., Hartmann, R., and Bergter, E.B. (1998) Structural characterization of neutral glycosphingolipids from Fusarium species. Biochim. Biophys. Acta, 1390, 186–196.[ISI][Medline]

Durden, F.M., and Elewski, B. (1997) Fungal infections in HIV-infected patients. Semin. Cutan. Med. Surg., 16, 200–212.

Fogedal, M., Mickos, H., and Norberg, T. (1986) Isolation of N-2'-hydroxyhexadecanoyl-1-O-ß-D-glucopyranosyl-9-methyl-D-erythro-sphingadienine from fruiting bodies of two Basidiomycetes fungi. Glycoconj. J., 3, 233–237.[ISI]

Fujino, Y., and Ohnishi, M. (1976) Structure of cerebroside in Aspergillus oryzae. Biochim. Biophys. Acta, 486, 161–171.[ISI][Medline]

Gow, N.A. (1997) Germ tube growth of Candida albicans. Curr. Top. Med. Mycol., 8, 43–55.[Medline]

Hakomori, S. (1990) Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions. J. Biol. Chem., 265, 18713–18716.[Abstract/Free Full Text]

Hakomori, S. (1996) Sphingolipid-dependent protein kinases. Adv. Pharmacol., 36, 155–171.[Medline]

Hakomori, S., and Igarashi, Y. (1993) Gangliosides and glycosphingolipids as modulators of cell growth, adhesion, and transmembrane signaling. Adv. Carbohydr. Chem., 25, 147–162.

Hannun, Y., Obeid, L.M., and Wolff, R.M. (1993) The novel second messenger ceramide: Identification, mechanism of action, and cellular activity. Adv. Lipid Res., 25, 43–64.[ISI][Medline]

Ichikawa, S., Ozawa, K., and Hirabayashi, Y. (1998) Molecular cloning and expression of mouse ceramide glucosyltransferase. Biochem. Mol. Biol. Int., 44, 1193–1202.

Ichikawa, S., Sakiyama, H., Suzuki, G., Hidari, K.I., and Hirabayashi, Y. (1996) Expression cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first glycosylation step of glycosphingolipid synthesis [published erratum appears in Proc. Natl Acad. Sci. USA (1996), 93, 12654]. Proc. Natl Acad. Sci. USA, 93, 4638–4643.

Jin, W., Rinehart, K.L., and Jares-Erijman, E.A. (1994) Ophidiacerebrosides: cytotoxic glycosphingolipids containing a novel sphingosine from a sea star. J. Org. Chem., 59, 144–147.[ISI]

Kapitonov, D., and Yu, R.K. (1999) Conserved domains of glycosyltransferases. Glycobiology, 9, 961–978.[Abstract/Free Full Text]

Karlsson, K.-A., Leffler, H., and Samuelsson, B.E. (1979) Characterization of cerebroside (monoglycosylceramide) from the sea anemone, Metridium senile. Biochim. Biophys. Acta, 574, 79–93.[ISI][Medline]

Kawai, G. (1989) Molecular species of cerebrosides in fruiting bodies of Lentinus edodes and their biological activity. Biochim. Biophys. Acta, 1001, 185–190.[ISI][Medline]

Kawai, G., and Ikeda, Y. (1982) Fruiting-inducing activity of cerebrosides observed with Schizophyllum commune. Biochim. Biophys. Acta, 719, 612–618.[ISI]

Kawai, G., and Ikeda, Y. (1983) Chemistry and functional moiety of a fruiting-inducing cerebroside in Schizophyllum commune. Biochim. Biophys. Acta, 754, 243–248.[ISI]

Kawai, G., and Ikeda, Y. (1985a) Structure of biologically active and inactive cerebrosides prepared from Schizophyllum commune. J. Lipid Res., 26, 338–343.[Abstract]

Kawai, G., Ikeda, Y., and Tubaki, K. (1985b) Fruiting of Schizophyllum commune induced by certain ceramides and cerebrosides from Penicillium funiculosum. Agric. Biol. Chem., 49, 2137–2146.[ISI]

Koga, J., Yamauchi, T., Shimura, M., Ogawa, N., Oshima, K., Umemura, K., Kikuchi, M., and Ogasawara, N. (1998) Cerebrosides A and C, sphingolipid elicitors of hypersensitive cell death and phytoalexin accumulation in rice plants. J. Biol. Chem., 273, 31985–31991.[Abstract/Free Full Text]

Kolter, T., and Sandhoff, K. (1999) Sphingolipids—their metabolic pathways and the pathobiochemistry of neurodegenerative diseases. Angew. Chem. Int. Ed. Engl., 38, 1532–1568.[ISI]

Levery, S.B., Toledo, M.S., Straus, A.H., and Takahashi, H.K. (1998) Structure elucidation of sphingolipids from the mycopathogen Paracoccidioides brasiliensis: an immunodominant ß-galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen. Biochemistry, 37, 8764–8775.[ISI][Medline]

Levery, S.B., Toledo, M.S., Straus, A.H., and Takahashi, H.K. (2000) Comparative analysis of ceramide structural modification found in fungal cerebrosides by electrospray tandem mass spectrometry with low energy collision-induced dissociation of Li+ adduct ions. Rapid. Commun. Mass Spectrom., 14, 551–563.[ISI][Medline]

Lichter, A., and Mills, D. (1997) Fil1, a G-protein alpha-subunit that acts upstream of cAMP and is essential for dimorphic switching in haploid cells of Ustilago hordei. Mol. Gen. Genet., 256, 426–435.[ISI][Medline]

Lortholary, O., Denning, D.W., and Dupont, B. (1999) Endemic mycoses: a treatment update. J. Antimicrob. Chemother., 43, 321–331.[Abstract/Free Full Text]

Mandala, S.M., Thornton, R.A., Milligan, J., Rosenbach, M., Garcia-Calvo, M., Bull, H.G., Harris, G., Abruzzo, G.K., Flattery, A.M., Gill, C.J., Bartizal, S., and Kurtz, M.B. (1998) Rustmicin, a potent antifungal agent, inhibits sphingolipid synthesis at the inositol phosphoceramide synthase. J. Biol. Chem., 273, 14942–14949.[Abstract/Free Full Text]

Mandala, S.M., Thornton, R.A., Rosenbach, M., Milligan, J., Garcia-Calvo, M., Bull, H.G., and Kurtz, M.B. (1997) Khafrefungin, a novel inhibitor of sphingolipid synthesis. J. Biol. Chem., 272, 32709–32714.[Abstract/Free Full Text]

Mathias, S., Peña, L.A., and Kolesnick, R.N. (1998) Signal transduction of stress via ceramide. Biochem. J., 335, 465–480.[ISI][Medline]

Matsubara, T., Hayashi, A., Banno, Y., Morita, T., and Nozawa, Y. (1987) Cerebroside of the dimorphic human pathogen, Candida albicans. Chem. Phys. Lipids, 43, 1–12.[ISI][Medline]

Mizushina, Y., Hanashima, L., Yamaguchi, T., Takemura, M., Sugawara, F., Saneyoshi, M., Matsukage, A., Yoshida, S., and Sakaguchi, K. (1998) A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases. Biochem. Biophys. Res. Commun., 249, 17–22.[ISI][Medline]

Nagiec, M.M., Nagiec, E.E., Baltisberger, J.A., Wells, G.B., Lester, R.L., and Dickson, R.C. (1997) Sphingolipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cerevisiae by the AUR1 gene. J. Biol. Chem. 272, 9809–9817.[Abstract/Free Full Text]

Natori, T., Morita, M., Akimoto, K., and Koezuka, Y. (1994) Agelasphins, novel antitumor and immunostimulatory cerebrosides from the marine sponge Agelas mauritianus. Tetrahedron, 50, 2771–2784.[ISI]

Roberts, R.L., and Fink, G.R. (1994) Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev., 8, 2974–2985.[Abstract]

Roze, L.V., and Linz, J.E. (1998) Lovastatin triggers an apoptosis-like cell death process in the fungus Mucor racemosus. Fungal Genet. Biol., 25, 119–133.[ISI][Medline]

Sawabe, A., Morita, M., Okamoto, T., and Ouchi, S. (1994) The location of double bonds in a cerebroside from edible fungi (mushroom) estimated by B/E linked scan fast atom bombardment mass spectrometry. Biol. Mass Spectrom., 23, 660–664.[ISI]

Shibuya, H., Kawashima, K., Sakagami, M., Kawanishi, H., Shimomura, M., Ohashi, K., and Kitagawa, T. (1990) Sphingolipids and glycerolipids. I. Chemical structures and Ionophoretic activities of soya-cerebrosides I and II from soybean. Chem. Pharm. Bull. (Tokyo), 38, 2933–2938.[ISI][Medline]

Sitrin, R.D., Chan, G., Dingerdissen, J., DeBrosse, C., Mehta, R., Roberts, G., Rottschaefer, S., Staiger, D., Valenta, J., Snader, K.M., Stedman, R.J., and Hoover, J.R.E. (1988) Isolation and structure determination of Pachybasium cerebrosides which potentiate the antifungal activity of aculeacin. J. Antibiot. (Tokyo), 41, 469–480.[ISI][Medline]

Sperling, P., Zahringer, U., and Heinz, E. (2000) A sphingolipid desaturase from higher plants. Identification of a new cytochrome b5 fusion protein. J. Biol. Chem., 273, 28590–28596.[Abstract/Free Full Text]

Spiegel, S., Olivera, A., and Carlson, R.O. (1993) The role of sphingosine in cell growth regulation and transmembrane signaling. Adv. Lipid Res., 25, 105–129.[ISI][Medline]

Sullards, M.C., Lynch, D.V., Merrill, A.H.J., and Adams, J. (2000) Structure determination of soybean and wheat glucosylceramides by tandem mass spectrometry. J. Mass Spectrom., 35, 347–353.[ISI][Medline]

Sweeley, C.C. (1969) Chromatography on silica gel columns. Methods Enzymol., 14, 254–267.

Szaniszlo, P.J., Jacobs, C.W., and Geis, P.A. (1983) Dimorphism: morphological and biochemical aspects. In Howard, D. H., and Howard, L. F. (eds.), Fungi Pathogenic for Humans and Animals. Part A. Biology. Marcel Dekker, New York, pp. 323–436.

Takesako, K., Kuroda, H., Inoue, T., Haruna, F., Yoshikawa, Y., and Kato, I. (1993) Biological properties of Aureobasidin A, a cyclic depsipeptide antifungal antibiotic. J. Antibiot., 49, 1414–1420.

Toledo, M.S., Levery, S.B., Straus, A.H., Suzuki, E., Momany, M., Glushka, J., Moulton, J.M., and Takahashi, H.K. (1999) Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-{Delta}3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus. Biochemistry, 38, 7294–7306.[ISI][Medline]

Toledo, M.S., Levery, S.B., Straus, A.H., and Takahashi, H.K. (2000) Dimorphic expression of cerebrosides in the mycopathogen Sporothrix schenckii. J. Lipid Res., 41, 797–806.[Abstract/Free Full Text]

Toledo, M.S., Suzuki, E., Straus, A.H., and Takahashi, H.K. (1995) Glycolipids from Paracoccidioides brasiliensis. Isolation of a galactofuranose-containing glycolipid reactive with sera of patients with paracoccidioidomycosis. J. Med. Vet. Mycol., 33, 247–251.[ISI][Medline]

Travassos, L.R. (1985) Sporothrix schenckii. In Szaniszlo, P. J., and Harris, J. L. (eds.), Fungal Dimorphism. Plenum Press, New York, pp. 121–163.

Villalobo, A., and Gabius, H.-J. (1998) Signaling pathways for transduction of the initial message of the glycocode into cellular responses. Acta Anat., 161, 110–129.[ISI][Medline]

Villas Boas, M.H., Egge, H., Pohlentz, G., Hartmann, R., and Bergter, E.B. (1994) Structural determination of N-2'-hydroxyoctadecenoyl-1-O-ß-D-glucopyranosyl-9-methyl-4, 8-sphingadienine from species of Aspergillus. Chem. Phys. Lipids, 70, 11–19.[ISI][Medline]

Wade, J.C. (1997) Treatment of fungal and other opportunistic infections in immunocompromised patients. Leukemia, 11(Suppl 4), S38–S39

Walsh, T.J., Hiemenz, J.W., and Anaissie, E. (1996) recent progress and current problems in treatment of invasive fungal infections in neutropenic patients. Infect. Dis. Clin. North Am., 10, 365–400.[ISI][Medline]

Wells, G.B., Dickson, R.C., and Lester, R.L. (1996) Isolation and composition of inositolphosphorylceramide-type sphingolipids of hyphal forms of Candida albicans. J. Bacteriol., 178, 6223–6226.[Abstract]

Yaar, L., Mevarech, M., and Koltin, Y. (1997) A Candida albicans RAS-related gene (CaRSR1) is involved in budding, cell morphogenesis and hypha development. Microbiology, 143, 3033–3044.[Abstract]

Yamada, A., Dabrowski, J., Hanfland, P., and Egge, H. (1980) Preliminary results of J-resolved, two-dimensional 1H-NMR studies on glycosphingolipids. Biochim. Biophys. Acta, 618, 473–479.[ISI][Medline]