Glucosylceramide synthase is an essential regulator of pathogenicity of Cryptococcus neoformans
http://www.100md.com
《临床调查学报》
1Departments of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA.
2School of Biology, Georgia Institute of Technology, Atlanta, Georgia, USA.
3Department of Molecular Biology, Scripps Research Institute, La Jolla, California, USA.
4Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA.
Abstract
The pathogenic fungus Cryptococcus neoformans infects humans upon inhalation and causes the most common fungal meningoencephalitis in immunocompromised subjects worldwide. In the host, C. neoformans is found both intracellularly and extracellularly, but how these two components contribute to the development of the disease is largely unknown. Here we show that the glycosphingolipid glucosylceramide (GlcCer), which is present in C. neoformans, was essential for fungal growth in host extracellular environments, such as in alveolar spaces and in the bloodstream, which are characterized by a neutral/alkaline pH, but not in the host intracellular environment, such as in the phagolysosome of macrophages, which is characteristically acidic. Indeed, a C. neoformans mutant strain lacking GlcCer did not grow in vitro at a neutral/alkaline pH, yet it had no growth defect at an acidic pH. The mechanism by which GlcCer regulates alkali tolerance was by allowing the transition of C. neoformans through the cell cycle. This study establishes C. neoformans GlcCer as a key virulence factor of cryptococcal pathogenicity, with important implications for future development of new antifungal strategies.
See the related Commentary beginning on page 1481
Introduction
Cryptococcus neoformans is an environmental yeast distributed worldwide and is the most common cause of fungal meningoencephalitis in immunocompromised hosts (1). Infection is initiated upon inhalation of spores or desiccated yeast. In the lung, the fungus proliferates in the alveolar space, and in immunocompetent subjects the infection is normally contained in this organ. However, in immunocompromised subjects, dissemination of the yeast cells from the lung to the brain can occur intracellularly (within host cells) and extracellularly (in the bloodstream), leading to the development of a life-threatening disease (2-4). Since C. neoformans is a facultative intracellular pathogen, many studies have addressed how the fungus survives and grows within host cells (2, 5-9), but how fungal cells survive and proliferate extracellularly is not well understood.
One of the cell surface molecules of this fungus is glucosylceramide (GlcCer), an antigenic glycosphingolipid that elicits an antibody response in patients affected with cryptococcosis (10) and in mice (11). GlcCer is synthesized by uridine diphosphate–glucose:ceramide (UDP-glucose:ceramide) glucosyltransferases encoded by GlcCer synthase (GCS) genes, which catalyze the first step in glycosphingolipid biosynthesis leading to the formation of the membrane lipid GlcCer. Very little is known about the role and function of GCS in fungal cells. The synthesis of GlcCer is absent in the yeast Saccharomyces cerevisiae, but it has been demonstrated in other fungi, such as Pichia pastoris; in fungi pathogenic to plants, such as Magnaporthe grisea (12); in fungi pathogenic to humans, such as C. neoformans (10), Candida albicans (12-14), Aspergillus fumigatus (15, 16), Histoplasma capsulatum (17), Paracoccidioides brasiliensis (18), and Sporotrix schenckii (19); and in other fungi (reviewed in refs. 20-22). The gene encoding for GCS (GCS1) has been isolated from P. pastoris, M. grisea, and C. albicans (12). The GCS1 gene has been deleted in the pathogenic fungus C. albicans, but its role in the pathogenicity of this fungus has yet to be elucidated.
Other studies have suggested a role for GlcCer in the regulation of fungal growth and pathogenesis. For instance, in C. neoformans GlcCer is mainly localized in the cell wall and mostly accumulates at the budding site of dividing cells. Interestingly, antibodies against C. neoformans GlcCer produced by patients affected with cryptococcosis inhibit budding and division of C. neoformans cells grown in vitro (10) as well as differentiation and germ-tube formation of Pseudallescheria boydii and C. albicans (23). Additionally, production of antibodies against fungal glycolipids has been demonstrated in patients with paracoccidioidomycosis (24), although in this disease the antibodies are mostly interacting with galactosylceramide instead of GlcCer. In other fungi, disruption of the GlcCer biosynthetic pathway altered spore germination, hyphal development, and fungal growth (16, 25). Monoclonal antibodies against fungal GlcCer have been produced (11), and, interestingly, treatment with anti-GlcCer antibody enhanced macrophage function against the fungus Fonsecaea pedrosoi (26). Together, these studies suggest an important role for GlcCer in fungal cell growth and differentiation and — because germ-tube formation and hyphal growth are provirulent factors — also suggest that GlcCer might be implicated in the regulation of fungal virulence. Thus we wondered whether GlcCer would have a role in the development and progression of cryptococcosis.
Here we have identified that GCS is a key regulator of pathogenicity of C. neoformans by ensuring cell cycle progression and growth of fungal cells in environments characterized by neutral/alkaline pH and physiological concentrations of CO2. Since these environments are characteristically found in the lung alveolar spaces, GCS regulates survival of C. neoformans upon inhalation through the respiratory tract, with important implications for the dissemination of fungal cells to the brain and the development of meningo-encephalitis.
Results
The C. neoformans GCS1 gene encodes for GCS activity.
By sequence homology to the human GCS cDNA, a candidate C. neoformans GCS1 cDNA was identified. To investigate the potential activity of the C. neoformans GCS1 gene product as a GCS, the effect of GCS1 expression on sphingolipid metabolism was studied in S. cerevisiae, which totally lacks endogenous GCS activity. The human GCS gene was also expressed in a pYES vector as a positive control, and S. cerevisiae carrying the pYES empty vector was used as a negative control. Yeast cells were labeled in vivo with tritiated dihydrosphingosine ([3H]-DHS; Figure 1A), a precursor of ceramide/phytoceramide (the substrate of GCS) in mammalian and yeast cells. The results showed that, when induced by galactose, C. neoformans Gcs1 produced a radiolabeled sphingolipid that migrated similarly to a soy GlcCer standard; this sphingolipid was absent when repressed by glucose. As expected, expression of human GCS1 showed the formation of the putative GlcCer under conditions of induction but not repression (Figure 1A). Cells carrying the empty vector lacked this sphingolipid under conditions of induction and repression. These results suggest that C. neoformans Gcs1 is functional when expressed in S. cerevisiae and that it may use endogenous ceramides as substrates.
Mammalian GCS efficiently recognizes fluorescently labeled short-chain ceramide analogs as substrates in vitro and in vivo. To study the substrate specificity of the C. neoformans Gcs1, 7-nitro-2-1,3-benzoxadiazol-4-yl–C6–ceramide (NBD-C6-ceramide) and NBD-C6-phytoceramide were tested as substrates in vitro, using cell protein extracts of S. cerevisiae expressing C. neoformans GCS1 or human GCS genes, under conditions of induction or repression. In contrast to what we observed for human GCS, C. neoformans Gcs1 did not recognize NBD-C6-ceramides as substrates (Figure 1B). The inability of C. neoformans Gcs1 to use short-chain fluorescent ceramide analogs was also confirmed by in vivo labeling using NBD-ceramides (data not shown). Together, these results show that C. neoformans Gcs1 does not share the same substrate specificity as human GCS.
C. neoformans Gcs1 produces GlcCer.
To examine whether the C. neoformans GCS1 gene encodes for GCS, the C. neoformans GCS1 gene was deleted in the C. neoformans var. grubii strain (WT), creating the gcs1 mutant, and was subsequently reconstituted, generating the gcs1 + GCS1 strain (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI27890DS1). To verify that the C. neoformans gcs1 mutant lacks the putative GlcCer, in vivo labeling using [3H]-DHS in WT, gcs1, and gcs1 + GCS1 strains was performed. As illustrated in Figure 2A, a radiolabeled compound that migrated onto a TLC similarly to the soy GlcCer standard was clearly lost in the gcs1 strain, whereas it was completely restored in the reconstituted strain. Use of NBD-ceramides in C. neoformans for in vitro or in vivo labeling did not result in the formation of the corresponding identifiable sphingolipid in the tested strains (data not shown), in agreement with the finding that C. neoformans Gcs1 expressed in S. cerevisiae was unable to recognize NBD-ceramide analogs as substrates.
To confirm that the missing sphingolipid in the gcs1 is GlcCer, we purified and concentrated the putative sphingolipid from C. neoformans strains, and the extracted lipids were analyzed by high-performance TLC (HPTLC), electrospray tandem mass spectrometry, and NMR. The analysis of the purified lipid extracts onto the HPTLC plate stained with orcinol clearly showed a band of a sugar-containing lipid migrating in tandem with the GlcCer standard from soy. This band was absent in the gcs1 strain and reconstituted in the gcs1 + GCS1 strain (Figure 2B).
The structure of the GlcCer was confirmed by syringe infusion of the glycosphingolipid into an API 3000 triple quadrupole mass spectrometer. Neutral loss scans of m/z 180 (loss of hexose headgroup), as well as precursor ion scans for the types of sphingoid bases commonly found in fungi, were performed. A major peak in C. neoformans WT was observed at m/z 756.7, which corresponded to a [M+H]+ charge for a monohexosylceramide with a lipid backbone composed of 37 carbons, 2 double bonds, and an additional hydroxyl (Figure 2C). Fragmentation of this ion yielded a product ion of m/z 276.4, which identified the lipid backbone as having an 18-carbon sphingadienine base with one additional methyl group (for a total of 19 carbons) plus an amide-linked hydroxy-C18:1 fatty acid. This is consistent with the backbone structure of a GlcCer that has been previously isolated from this organism (9-methyl-4,8-sphingadienine in amide linkage to 2-hydroxyoctadecanoic acid) (10). Precursor ion scans for m/z 276.4 also revealed lower-abundance species that probably reflect GlcCer with a non–hydroxy-C18:0 fatty acid (at m/z 740.7) and a hydroxy-C16:0 fatty acid (at m/z 728.7). Precursor scans in the gcs1 mutant revealed minor peaks also present in the WT spectrum, whereas none of these GlcCers were detected (Figure 2C). Importantly, the reconstituted strain displayed a strong m/z 756.7 ion for the major GlcCer of the WT cells; interestingly, the minor C18:0 and hydroxy-C16:0 species were not detected.
Since no standards are available for these compounds, liquid chromatography mobility could not definitely identify the sugar head group. Thus we performed 2-dimensional NMR spectroscopy to uniquely define the structure of the hexose moiety of the purified sphingolipid (WT), as shown in Figure 2, D and E. The NMR characterization of the hexose commenced with the assignment of the glycoside protons in the 1H-NMR spectrum of the monohexosylceramide (Supplemental Table 1). The unambiguous identification of the H1' anomeric proton was straightforward due to its unique chemical shift (1H, 4.17 ppm; 13C, 103.11 ppm) and doublet multiplicity (3JH1',H2', 7.7 Hz). A combination of 2-dimensional 1H-1H–double quantum filtered correlation spectroscopy and total correlated spectroscopy spectra clearly revealed the complete hexose proton spin system, which was supported by the 13C heteronuclear single quantum correlation data (Figure 2, D and E). The stereochemistry of the hexose could be unambiguously determined from the first 4 vicinal 3J-coupling constants. The large 3JHH-coupling constants observed (>7 Hz) indicated antiperiplanar orientations of the vicinal hexapyranose H1'/H2', H2'/H3' (3JH2',H3', 8.9 Hz), H3'/H4' (3JH3',H4', 8.2 Hz), and H4'/H5' (3JH4',H5', 9.1 Hz) ring protons (Supplemental Table 1). The analysis of 1D–nuclear Overhauser effect (1D-NOE) difference experiments corroborated the previous -glycopyranosyl assignment. NOEs were observed from H1' to H3' and H5'; saturation at H2' produced a strong NOE at H4'. Thus, based on our results and on previous NMR analysis of GlcCer species from other pathogenic fungi (27, 28), we conclude that glucose is attached to the ceramide backbone of the sphingolipid produced by the C. neoformans Gcs1 enzyme.
Role of Gcs1 in the pathogenicity of C. neoformans.
To determine the role of GCS1 in the pathogenicity of C. neoformans, we performed virulence studies using a murine animal model (CBA/J) of cryptococcosis. The average survival of mice infected intranasally with WT and gcs1 + GCS1 strains was 24.6 ± 3.9 and 27.3 ± 4.8 days, respectively (P < 0.0001; Figure 3A), whereas mice infected with C. neoformans gcs1 were all alive after 90 days of observation. At the ninetieth day, even though the 11 mice infected with C. neoformans gcs1 mutant showed no clinical signs of meningoencephalitis, they were sacrificed, and lungs and brains were removed and analyzed for CFUs and histology. Yeast cells were absent in 8 of 11 brains. The 3 yeast cell–positive brains showed approximately 6 x 105, 500, and 28,000 cells per organ. Among the 11 lungs, 6 lungs were used to determine fungal tissue burden, and an average of 5–10 x 103 yeast cells/lung were found, which corresponds to an approximately 100-fold reduction of the initial inoculum. These results were confirmed by a study aiming to determine the lung fungal burden up to 70 days after infection with WT or gcs1 strains (Supplemental Figure 2). These findings suggest that Gcs1 plays a key role in the pathogenicity of C. neoformans and growth of yeast cells in the lung.
To examine whether the lung environment plays a role in protecting the host against the dissemination of the gcs1 strain, we challenged CBA/J mice intravenously with WT, gcs1, and gcs1 + GCS1 strains and monitored their survival. Mice infected with the WT and gcs1 + GCS1 strains showed a similar average survival rate of 6.3 ± 0.5 days and 6.2 ± 0.4 days, respectively (Figure 3B). Mice infected with the gcs1 mutant were apparently healthy until 13–15 days of infection, when they rapidly showed severe clinical signs of meningoencephalitis such as intense tremor, rigidity, lack of balance, and inability to coordinate movements. On the fifteenth day, mice were sacrificed because they were unable to reach food or water. Brains, lungs, kidneys, spleens, and livers were removed and assayed for tissue burden culture and histology. Analysis of CFUs of brains showed an average number of 8.4 ± 0.06 (log10 CFU ± SD) yeast cells, which was significantly higher (P < 0.01) than CFU in other organs (5.5 ± 0.5 in lung, 5.6 ± 0.1 in kidney, 5.3 ± 0.2 in spleen, and 5.6 ± 0.1 in liver). These results clearly suggest that when CBA/J mice are challenged intravenously, they cannot efficiently prohibit gcs1 cells from disseminating throughout the body.
Inflammatory response against the C. neoformans gcs1 strain.
To examine the host inflammatory response against C. neoformans gcs1, 5 lungs and 5 brains from CBA/J mice challenged intranasally and 4 lungs and 4 brains of CBA/J mice challenged intravenously with C. neoformans gcs1 were analyzed for histology. Figure 4, A–D, and Supplemental Figure 3, A and C, show representative fields from 4 different lungs recovered from mice infected intranasally. Supplemental Figure 3, E and G, show a representative field of 2 lungs and Supplemental Figure 3, F and H, show a representative field of 2 brains recovered from mice infected intravenously.
The lungs of mice challenged intranasally showed a strong granulomatous response consisting of the formation of extensive spheric nodules, in which gcs1 cells were mainly trapped in the center with abundant necrotic tissue and numerous neutrophils (Figure 4). The necrotic tissue was surrounded by a ring of foamy macrophages and giant cells loaded with yeast cells and/or cellular debris or "ghosts" of yeast cells, which appeared to be degenerated (Figure 4C). The ring of macrophages was then surrounded by lymphocytes, fibroblasts, and fibrotic tissue (Figure 4A), in which a significant deposition of collagen was observed (Figure 4, C and D). These nodules ranged from 500 to 2,500 μm in diameter and were confined in the lung, with no invasion into the mediastinal area. In other cases, smaller nodules were observed — such as those shown in Supplemental Figure 3, A and C — in which few C. neoformans cells were engulfed in macrophages surrounded by other macrophages containing "ghosts" of C. neoformans cells (Supplemental Figure 3B). Few lymphocytic interstitial infiltrations were localized in the proximity of the nodules (Supplemental Figure 3). Other portions of the lung appeared to be normal. These structures were present only in lungs infected with C. neoformans gsc1, with different degrees of organization, whereas the nodules were not found in lungs of mice infected with the C. neoformans WT strain during the course of the infection (data not shown). All brains selected from mice infected intranasally with gsc1 and analyzed by histology showed no sign of yeast cells.
Histology analysis was also performed on lungs, brains, and other organs of mice infected intravenously. In the lungs, gcs1 cells were contained within small, spheric, nodule-like structures (Supplemental Figure 3, E and G) similar to those illustrated in Supplemental Figure 3, A and C. In the brain, C. neoformans cells were almost exclusively localized within brain abscesses of different size, which were not surrounded by any host inflammatory response (Supplemental Figure 3, F and H). Numerous abscesses containing gcs1 cells were also found in the kidney (data not shown). In the liver, the gcs1 cells were found in very small periportal lesions with no host cellular infiltrates, whereas in the spleen the gcs1 strain was surrounded by a granulomatous response invading the white pulp and characterized by the presence of yeast cells surrounded by macrophages, lymphocytes, neutrophils, and a few megakaryocytes (data not shown). These results suggest that when C. neoformans gsc1 cells are introduced intranasally, the host organizes a strong granulomatous response, in which yeast cells are confined by macrophages, lymphocytes, fibroblasts, fibrotic tissue, and collagen deposition, with almost total containment of the infection within the lung tissue. In contrast, when yeast cells are introduced intravenously, C. neoformans gsc1 cells do disseminate to the brain and other organs, eventually killing the animal.
Effect of Gcs1 on intracellular and extracellular growth of C. neoformans.
In the host, C. neoformans proliferates intracellularly, within the acidic environment of the phagolysosome, and extracellularly, an environment characterized by alkaline/neutral pH and high CO2 concentration. Thus we investigated the effect of the loss of GlcCer on the growth of C. neoformans intracellularly and in in vitro acidic and alkaline environments. For intracellular growth, C. neoformans WT, gcs1, and gcs1 + GCS1 strains were incubated with J774.16 macrophage-like J774.16 cells, and the number of buds of internalized cells per phagocytic index was measured. As shown in Supplemental Figure 4, we found no differences in intracellular growth among the tested strains, suggesting that GlcCer has no effect on fungal growth once C. neoformans is internalized within macrophages.
In order to evaluate in vitro cell culture growth, we performed growth curves of C. neoformans WT, gcs1, and gcs1 + GCS1 strains incubated in 5% CO2 at either a neutral/alkaline or an acidic pH. As shown in Figure 5A, the gcs1 mutant was unable to grow at a neutral/alkaline pH in the presence of 5% CO2 (Figure 5A), whereas growth was observed at an acidic pH (Figure 5B). This difference in growth was not observed in the presence of atmospheric CO2 concentration (0.04%) at pH 7.4 or 4.0 (data not shown). Importantly, the growth defect at 5% CO2 and neutral pH was reversible because when yeast cells were switched to fresh acidic medium, cell growth was completely restored (data not shown), suggesting that growth of gcs1 cells is arrested at alkaline pH and 5% CO2.
To determine which phases of the C. neoformans cell cycle are affected by lack of GlcCer, we performed flow cytometry analysis of C. neoformans cells incubated for 24 hours and 36 hours at alkaline pH and 5% CO2. We found that a significantly greater percentage of gcs1 cells were locked in S and G2/M phases compared with the WT and gcs1 + GCS1 cells at both 24 (Table 1) and 36 hours (data not shown), suggesting that GlcCer is important for exiting from the S and G2/M phases of the cell cycle.
Discussion
In the present study the role of C. neoformans GCS in the virulence of this pathogenic fungus was investigated. Our results show that C. neoformans GCS was an essential factor in determining the success of the fungal infection by regulating survival of C. neoformans during the initial colonization of the lung. In addition, we found that GCS guaranteed survival of C. neoformans in the lung extracellular space by ensuring cell-cycle progression at neutral/alkaline pH and physiologic CO2 concentration (Table 2).
The dramatic protection against pathogenicity imparted by loss of GCS underlines the importance of the extracellular component of fungal cells during C. neoformans infection. C. neoformans infects the host through inhalation, and upon its arrival in the alveoli it encounters 2 environments in which normally grows: the extracellular space, characterized by a neutral/alkaline pH and a physiologic CO2 concentration of approximately 5.0% (compared to 0.04% present in the air, ref. 29); and the intracellular environment of the phagolysosome of alveolar macrophages, characterized by an acidic pH. Our results suggest that GlcCer is critical for C. neoformans to grow in the extracellular (alkaline) but not in the intracellular (acidic) environment of the lung. Since during the infection most C. neoformans cells are found in the host extracellular space (30), the overall effect of the loss of GlcCer on C. neoformans pathogenicity is significant. Indeed, when the gcs1 strain was introduced intranasally, the overall growth of yeast cells in the alveoli was arrested (Supplemental Figure 2); even if the mutant grew intracellularly (Supplemental Figure 4), the host responded with the formation of well-organized granulomas (Figure 4 and Supplemental Figure 3), which efficiently contained gcs1 cells in the lung. This allowed the host to survive the cryptococcal infection without any evident clinical sign of disease (Figure 3A).
When the gcs1 strain was introduced intravenously, mice survived longer than those infected with the WT or reconstituted strains (Figure 3B). In the blood, gcs1 also finds a hostile environment because the pH is approximately 7.4 and the CO2 concentration is approximately 5% (partial pressure of CO2 [pCO2], 40 mmHg). Thus growth of gcs1 cells in the bloodstream was arrested until yeast cells were either internalized by phagocytic cells or they left the bloodstream and invaded the organs, in which they formed abscesses such as those found in the brain (Supplemental Figure 3, F and H) and the kidney (data not shown). In both the intracellular environment and in abscesses, the pH was characteristically acidic, and growth of gcs1 could resume. Indeed, it was interesting to note that in the brain upon intravenous challenge of gcs1, yeast cells were almost exclusively contained within abscesses (Supplemental Figure 3, F and H), whereas in the WT strain, in addition to being present in abscesses, cells were also present in the surrounding tissue (data not shown). Thus we hypothesize that once the acidic abscess is formed, gcs1 cells promptly restore growth, and the consequent destruction of brain tissue will eventually manifest abruptly with clinical sign of meningoencephalitis. The animal infected intravenously with gcs1 would eventually die, although it would take longer than mice infected with the control strains due to the transient growth arrest that yeast cells undergo in the bloodstream upon injection.
We believe the results from these studies identify GlcCer as a novel virulence factor for C. neoformans because loss of this glycosphingolipid does not affect other fungal characteristics, such as capsule production, melanin formation, or growth at ambient CO2 and 37°C (Supplemental Figure 5), known to be required to cause disease (1). Interestingly, a greater sensitivity of gcs1 mutant to SDS was observed (Supplemental Figure 5), suggesting an alteration of cell wall integrity. This hypothesis is supported by previous observations of the corrleation of GlcCer density at the cell surface with the cell wall thickness of C. neoformans (10). However, the difference in cell growth of gcs1 in the presence of SDS compared with control strains was not so dramatic to account for the significant loss of virulence of the mutant. Instead, the growth of gcs1 mutant was arrested in environments characterized by alkaline pH and 5% CO2.
In budding yeasts such as C. neoformans, the S and G2 phases of the cell cycle are characterized by bud formation and maturation (Figure 5C). Interestingly, previous studies have shown that GlcCer is localized at the budding site of C. neoformans, and addition of human antibodies against C. neoformans GlcCer to in vitro C. neoformans culture inhibits cell budding and fungal cell growth (10). Our studies showed that loss of GlcCer significantly prolonged the S and the G2/M phases in alkaline environments, therefore suggesting a mechanism whereby GlcCer regulates fungal cell growth by allowing yeast cells to complete the cell cycle.
The role of GCS in alkaline tolerance is not restricted to C. neoformans, as recent findings showed that a Kluyveromyces lactis mutant defective in GlcCer cannot grow at pH 8.5 (31). On the other hand, the molecular mechanism by which GlcCer regulates alkaline tolerance at high but not low CO2 is not known. Studies have proposed that fungal cells sense different concentrations of CO2 and, in conditions in which CO2 is elevated (e.g., 5%), fungi respond by upregulating virulence factors. For instance, CO2 stimulates capsule formation in C. neoformans (32) and the transformation of yeast cells to filamentous forms in C. albicans through adenylyl cyclase (33). In other pathogenic fungi, such as Coccidioides immitis, CO2 promotes the formation of endosporulating spherules (34), which is a process required for virulence. These studies suggest the existence of a conserved signaling pathway that regulates fungal virulence through the sensing of CO2. C. neoformans senses CO2 through 2 -class carbonic anhydrases (Can1 and Can2), which have been recently identified (35, 36), but whether GlcCer regulates signaling events mediated by CO2-carbonic anhydrases at different pHs awaits further investigation.
In conclusion, these studies showed that GlcCer plays a key role in the pathogenicity of C. neoformans through a mechanism that ensures the transition through the cell cycle of yeast cells in alkaline environments with a physiological concentration of CO2. Since in cases of cryptococcosis extracellular organisms predominate, the function of GlcCer becomes clinically relevant. Interestingly, Gcs1 and GlcCer are found in a variety of pathogenic fungi, and because a significant biochemical difference between human and C. neoformans Gcs1 exists, compounds that would specifically inhibit the fungal enzyme might represent a novel class of antifungal agents against fungal infections (16, 25). Furthermore, since antibodies against the fungal GlcCer inhibit C. neoformans growth in vitro and do not interact with the human GlcCer (11), they may represent a new therapeutic approach to control infections by fungal microorganisms producing GlcCer.
Methods
Strains, plasmids, and growing conditions.
The strains used in this study were the C. neoformans var. grubii serotype A strain H99 (WT), the gcs1 mutant derived from H99, and the gcs1 + GCS1 reconstituted strain derived from gcs1; as well as the S. cerevisiae strains JK9-3d (MAT-, trp1, leu2-3, his4, ura3, ade2, rme1) and JK9-3d containing the pYES2 vector, the pYES2 vector expressing human GCS1 (pYES2-HuGCS1), and the pYES2 vector expressing C. neoformans GCS1 (pYES2-CnGCS1). Escherichia coli strains DH5- (Invitrogen Corp.) and TOP10 (Invitrogen Corp.) were used in this study. Plasmids pBluescript SK (Stratagene) and pCR2.1-TOPO (Invitrogen Corp.) were used for cloning, and pYES2 (Invitrogen Corp.) was used for expression of GCS genes in S. cerevisiae. C. neoformans strains were routinely grown in yeast extract/peptone/2% dextrose–rich (YPD-rich) medium. S. cerevisiae strain JK9-3d transformed with the pYES2, pYES2-HuGCS1, or pYES2-CnGCS1 vector was routinely grown in synthetic medium containing 6.7 g/l yeast nitrogen base (YNB) without amino acids, 1.2 g/l amino acid mixture lacking uracil, and 2% glucose or 1% glucose and 1% galactose for 16 hours at 30°C to induce either human or cryptococcal GCS1 gene expression. A full-length cDNA clone of the human GCS1 gene was cloned into pYES2 (Invitrogen Corp.) vector as described previously (37), generating the pYES2-HuGCS1 plasmid. Bacterial strains for plasmid amplification were routinely grown at 37°C in Luria-Bertani medium containing 75 mg/l of ampicillin (Sigma-Aldrich). DMEM (#11995; BD Biosciences) buffered with HEPES at pH 7.4 or pH 4.0 was used for growing the C. neoformans strain at 37°C in presence of 5% CO2.
Isolation and cloning of the C. neoformans GCS1 gene.
The GCS1 gene from C. neoformans was identified by a BLAST search of the human GCS sequence (GenBank D50840) in the genome database of C. neoformans var. grubii serotype A strain H99 from Duke Center for Genome Technology (http://cneo.genetics.duke.edu/blast.html). One putative sequence, with an E value of 9e-51 corresponding to chr4Bpiece18, was found. The GCS1 cDNA was then isolated by RT-PCR using the PRGCS-5' forward (5'-ATGAGCGATTCCGGCACATTGTCC-3') and reverse (5'-TCAATTTCTATCACCCAAACGAATTG-3') primers, and total RNA was extracted from C. neoformans strain H99. A 1,353-bp fragment was obtained, cloned into the pCR2.1-TOPO vector generating plasmid pCR-CnGCS1, and sequenced. Amino acid alignment of C. neoformans Gcs1 with other Gcs1 proteins from different organisms revealed a homology (identity + similarity) of 47%, 16%, 43%, 46%, 43%, and 35% to human, Arabidopsis thaliana, C. albicans, P. pastoris, M. grisea, and Aspergillus nidulans, respectively. The C. neoformans Gcs1 contains the motifs [D1, D2, D3 (R/QXXRW)] common to other GCSs required for GCS activity (38) and the characteristic N terminus hydrophobic transmembrane domain, which contains a signal-anchor sequence to the Golgi outer membrane (37). The C. neoformans GCS1 cDNA sequence was deposited in the GenBank database (AY956317). The GCS1 gene was deleted and reconstituted in the WT C. neoformans var. grubii strain, generating C. neoformans gcs1 and gcs1 + GCS1 strains (see Supplemental Methods).
In vitro growth studies.
From overnight YPD broth cultures of C. neoformans WT, gcs1, and gcs1 + GCS1 strains, cells were washed twice in sterile distilled water (SDW), resuspended, and diluted into 40 ml fresh YPD broth to a final density of 104 cells/ml and incubated in a shaker incubator at 30°C or 37°C. Aliquots were taken at the time points indicated in Supplemental Figure 5A, and serial dilutions were plated onto YPD plates for assessment of CFU. For in vitro growth studies on DMEM at pH 7.4, 7.0, or 4.0, 104 cells/ml were incubated at 37°C in the presence of 5% CO2, aliquots were taken at the time points indicated in Figure 5A, and serial dilutions were plated onto YPD plates for assessment of CFU. Capsule formation and melanin production were examined using media as previously described (1). For cell wall stability, cells were spotted in serial dilutions on YPD plates containing 0.05% SDS. For osmotic stress, cells were spotted in serial dilutions on YPD plates containing 1M NaCl. For oxidative or nitrosative stresses, cells were spotted in serial dilutions on YNB plates containing amino acids adjusted to pH 4.0 and supplemented with H2O2 to a final concentration of 5 mM or nitrite to a final concentration of 1 mM. After incubation at 30°C or 37°C for 72 hours, cell growth was examined and recorded by photography. Results show plates incubated at 37°C.
For intracellular growth studies, J774.16 macrophage-like cells were used, and fungal growth of C. neoformans WT, gcs1, and gcs1 + GCS1 strains was determined as previously described (2, 5-9).
For cell-cycle studies, yeast cells were incubated in DMEM, pH 7.4, at 37°C in the presence of 5% CO2 for 24 and 36 hours. Cells were then washed twice with ice-cold water, sonicated, and fixed in 7 ml of 95% ethanol for 16 hours. After fixing, the cells were pelleted, washed with 5 ml of 50 mM sodium citrate, resuspended in 1 ml of 50 mM sodium citrate containing 0.25 mg/ml RNase A, and incubated at 50°C for 1 hour. Next, proteinase K was added at a final concentration of 20 mg/ml, and cells were kept at 50°C for an additional hour. Finally, 1 ml of 50 mM sodium citrate containing 50 μg/ml propidium iodine was added. Cells were kept for 24 hours in the dark at 4°C and were analyzed using a BD fluorescence-activated cell analyzer. Data were modeled using the program ModFit LT (version 3.1; Verity Software).
In vitro activity assay of GCS.
To express C. neoformans GCS1 gene in S. cerevisiae, the cDNA fragment was digested with EcoRI from pCR-CnGCS1 and subcloned into EcoRI-restricted pYES2 plasmid, generating pYES2-CnGCS1. The S. cerevisiae JK9-3d strain was chemically transformed with pYES2-CnGCS1 plasmid. Transformants were selected on YNB ura– agar plates containing 2% glucose, randomly chosen, purified, and stored. For gene induction, cell cultures were pelleted and washed twice with SDW. Cells were resuspended, inoculated into YNB ura– broth containing 1% galactose plus 1% glucose or 2% glucose, and incubated in a 30°C shaker for 16 hours. For in vitro GCS activity in C. neoformans strains, yeast cells were grown on YPD media in a shaker incubator for 24 hours at 30°C, harvested by centrifugation, and washed with SDW, and pellets were stored at –80°C if not used immediately. Once cells were collected, proteins were extracted as previously described (9) and quantitated by the method of Bradford (39). The in vitro GCS activity assay was performed by using the fluorescent ceramide analog NBD-C6-ceramide (Avanti Polar Lipids Inc.) as a substrate and monitoring the formation of NBD-C6-GlcCer as described previously (37), with some modifications. Briefly, 200 μg of yeast proteins were incubated for 30 minutes at 30°C in 50 mM bis-Tris-HCl buffer (pH 6.5) containing 12.5 mM KCl, 250 μM EDTA, 50 μM phosphatidylcholine, 500 μM UDP-glucose, 5 mM Triton X-100, and 20 μM NBD-C6-ceramide in a final reaction volume of 100 μl. The reaction was terminated by the addition of 0.3 ml CHCl3/MeOH (1:1 ratio). The samples were mixed, and the phases were separated by centrifugation at 2,000 g for 5 minutes. The lower phase was dried down using the SPD 2010 Speedvac (Thermo Electron Corp.). The chloroform-soluble product was analyzed by analytical TLC on silica gel 60 plates (EM Science) using the solvent system chloroform/methanol/water (65:25:4). Purified GlcCer from soy (Avanti Polar Lipids Inc.) was also loaded onto the TLC plate and used as a standard (40). NBD-GlcCer was identified as a fluorescent band migrating in tandem with soy GlcCer and quantitated by direct fluorescence using a phosphorimager Molecular Dynamics Inc. 840 STORM unit and by ImageQuant analysis (GE Healthcare).
In vivo activity assay of GCS.
Approximately 5 hours before cell collection, yeast cells were counted, and 5 x 107 cells were resuspended in the same growing media and incubated with 2 μM/ml NBD-C6-ceramide. Similar experiments were performed using NBD-C6-phytoceramide, NBD-C12-ceramide, and NBD-C12-phytoceramide. Yeast cells were incubated for 5 hours, shaken at 30°C. Cells were harvested by centrifugation for 10 minutes at 2,000 g and washed with SDW, and the total lipid was extracted using the method of Mandala et al. (41) followed by base hydrolysis. The extracted lipids were loaded onto a TLC using the solvent system chloroform/methanol/water (65:25:4 ratio). Formation of NBD-GlcCer was analyzed as described above.
For in vivo labeling using [3H]-DHS, approximately 2 hours prior to cell collection, 5 x 107 yeast cells/ml were incubated with 2 μCi/ml of [3H]-DHS for 2 hours at 30°C in the appropriate growing media in a shaker incubator. The cells were harvested by centrifugation (10 minutes at 2,500 g), washed with SDW, and stored at –80°C if not used immediately. Lipids were extracted as described by Mandala et al. (41) and after base hydrolysis were loaded onto a TLC plate and analyzed using a phosphoimager and quantitation of radioactive GlcCer performed by ImageQuant analysis. As a control, 10 μl of 20 mM soy GlcCer (Avanti Polar Lipids Inc.) was loaded onto a TLC plate and visualized using iodine stain.
Mass spectrometry analysis.
Purified GlcCer was obtained as described in Supplemental Methods and used for mass spectrometry (MS) analysis. The MS and MS/MS scans of GlcCer were conducted using an API 3000 triple quadrupole mass spectrometer with electrospray ionization as described previously by Merrill et al. (42). The analyses used syringe infusion and included neutral loss scans (with m/z 180 for loss of the hexose headgroup) and precursor ion scans for the characteristic product ion m/z 276.4.
NMR analysis.
NMR experiments were recorded in 500 μl dimethyl-sulfoxide-d6 (Cambridge Isotope Laboratories Inc.) using 160 μg putative monohexosylceramide obtained from HPTLC purification; subsequently, the sample was dissolved in dimethyl-sulfoxide-d6 and deuterium oxide (98:2, vol/vol). NMR spectra were recorded on a Varian Inova 600 MHz spectrometer equipped with an actively shielded z-gradient triple-resonance (1H, 13C, 31P) probe at 25°C. All spectra were processed and analyzed using FELIX 2000 Insights II version 2000 (MSI) and NMRPipe version 2.3 (43) program packages. 1H chemical shifts were internally referenced to 3-(trimethylsilyl)-1-propane sulfonic acid. 13C chemical shifts were referenced against the center line of the dimethyl-sulfoxide septet ( = 39.52 ppm). The decoupling in the presence of scalar interactions-2 (DISPI-2) mixing time was 62.6 ms (44). NOE difference experiments were measured with saturation delays of 6 seconds (45).
Virulence studies and histology analysis in a murine mouse model of cryptococcosis.
Four- to 6-week-old CBA/J (NCI/Frederick Laboratories) mice were used. Mice were anesthetized with an intraperitoneal injection of 60 μl xylazine/ketamine mixture containing 95 mg ketamine and 5 mg xylazine per kilogram of body weight. C. neoformans WT, gcs1, and gcs1 + GCS1 strains were prepared by growing cells for 24 hours at 30°C in YPD medium. The cells were pelleted, washed twice, and resuspended in PBS, pH 7.4, at a concentration of 2.5 x 107 cells/ml. Ten CBA/J mice were infected with 5 x 105 cells of WT, gcs1, or gcs1 + GCS1 strains in a volume of 20 μl through nasal inhalation. Eight CBA/J mice were infected with 106 cells of WT, gcs1, or gcs1 + GCS1 strains in a volume of 100 μl through tail vein injection. The mice were fed ad libitum and followed with twice-daily inspections. Mice that appeared moribund or in pain or with clinical signs of meningoencephalitis were sacrificed using CO2 inhalation followed by cervical dislocation. All animal procedures were approved by the Medical University of South Carolina Institutional Animal Care and Use Committee and followed the guidelines of the American Veterinary Medical Association. Histology analysis was performed as described in Supplemental Methods.
Statistics.
Survival data from the mouse experiments were analyzed by Kluskal-Wallis test. For other statistical analysis, a 2-tailed Student’s t test was used. A P value of less than 0.05 was considered to be significant.
Acknowledgments
We are grateful to Helio Takahashi, Yusuf Hannun, Lina Obeid, Deepak Bastia, Edward Balish, Lincoln Scott, and Carolina Westwater for discussion. Special thanks to Margaret Romano and to Russell Harley for invaluable help and discussion with the histology staining and to Stefka Spassieva for helping in the purification of GlcCer. We also thank all members of the M. Del Poeta and C. Luberto laboratories for guidance. We gratefully acknowledge John Oatis for helping with NMR analysis. This work was supported in part by the Burroughs Welcome Fund; NIH grant AI56168 (to M. Del Poeta); grants RR17677 Project 2 (to M. Del Poeta) and Project 6 (to C. Luberto) from the Centers of Biomedical Research Excellence Program of the National Center for Research Resources; National Science Foundation/EPSCoR grant #EPS-0132573 (to C. Luberto); NIH grant C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources; and, for the sphingolipid analysis by mass spectrometry, NIH grant GM069338 (LIPID MAPS, to A.H. Merrill). M. Del Poeta is a Burroughs Wellcome New Investigator in Pathogenesis of Infectious Diseases.
Footnotes
Philipp C. Rittershaus and Talar B. Kechichian contributed equally to this work.
Nonstandard abbreviations used: GCS, GlcCer synthase; GlcCer, glucosylceramide; [3H]-DHS, tritiated dihydrosphingosine; HPTLC, high-performance TLC; NBD, 7-nitro-2-1,3-benzoxadiazol-4-yl; NOE, nuclear Overhauser effect; pYES2-CnGCS1, pYES2 vector expressing C. neoformans GCS1; pYES2-HuGCS1, pYES2 vector expressing human GCS1; SDW, sterile distilled water; YNB, yeast nitrogen base; YPD, yeast extract/peptone/2% dextrose.
Conflict of interest: The authors have declared that no conflict of interest exists.
References
Casadevall, A., and Perfect, J.R. 1998. Cryptococcus neoformans. ASM Press. Washington, DC, USA. 351–380..
Feldmesser, M., Tucker, S., and Casadevall, A. 2001. . Intracellular parasitism of macrophages by Cryptococcus neoformans. Trends Microbiol. 9::273-278.
Levitz, S.M. et al. 1999. . Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages. Infect. Immun. 67::885-890.
Chretien, F. et al. 2002. . Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia. J. Infect. Dis. 186::522-530.
Feldmesser, M., Kress, Y., Novikoff, P., and Casadevall, A. 2000. . Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect. Immun. 68::4225-4237.
Lee, S.C., Kress, Y., Zhao, M.L., Dickson, D.W., and Casadevall, A. 1995. . Cryptococcus neoformans survive and replicate in human microglia. Lab. Invest. 73::871-879.
Tucker, S.C., and Casadevall, A. 2002. . Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm. Proc. Natl. Acad. Sci. U. S. A. 99::3165-3170.
Steenbergen, J.N., Shuman, H.A., and Casadevall, A. 2001. . Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc. Natl. Acad. Sci. U. S. A. 98::15245-15250.
Luberto, C. et al. 2001. . Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans. Genes Dev. 15::201-212.
Rodrigues, M.L. et al. 2000. . Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth. Infect. Immun. 68::7049-7060.
Toledo, M.S., Suzuki, E., Levery, S.B., Straus, A.H., and Takahashi, H.K. 2001. . Characterization of monoclonal antibody MEST-2 specific to glucosylceramide of fungi and plants. Glycobiology. 11::105-112.
Leipelt, M. et al. 2001. . Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi. J. Biol. Chem. 276::33621-33629.
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.
Leipelt, M., Warnecke, D.C., Hube, B., Zahringer, U., and Heinz, E. 2000. . Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms. Biochem. Soc. Trans. 28::751-752.
Boas, M.H., Egge, H., Pohlentz, G., Hartmann, R., and Bergter, E.B. 1994. . Structural determination of N-2'-hydroxyoctadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine from species of Aspergillus. Chem. Phys. Lipids. 70::11-19.
Levery, S.B. et al. 2002. . Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth. FEBS Lett. 525::59-64.
Toledo, M.S., Levery, S.B., Suzuki, E., Straus, A.H., and Takahashi, H.K. 2001. . 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. Glycobiology. 11::113-124.
Takahashi, H.K. et al. 1996. . Isolation and possible composition of glucosylceramides from Paracoccidioides brasiliensis. Braz. J. Med. Biol. Res. 29::1441-1444.
Toledo, M.S., Levery, S.B., Glushka, J., Straus, A.H., and Takahashi, H.K. 2001. . Structure elucidation of sphingolipids from the mycopathogen Sporothrix schenckii: identification of novel glycosylinositol phosphorylceramides with core manalpha1-->6Ins linkage. Biochem. Biophys. Res. Commun. 280::19-24.
Warnecke, D., and Heinz, E. 2003. . Recently discovered functions of glucosylceramides in plants and fungi. Cell. Mol. Life Sci. 60::919-941.
Hanada, K. 2005. . Sphingolipids in infectious diseases. Jpn. J. Infect. Dis. 58::131-148.
Heung, L.J., Luberto, C., and Del Poeta, M. 2006. . Role of sphingolipids in microbial pathogenesis. Infect. Immun. 74::28-39.
Pinto, M.R. et al. 2002. . Characterization of glucosylceramides in Pseudallescheria boydii and their involvement in fungal differentiation. Glycobiology. 12::251-260.
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.
Thevissen, K. et al. 2004. . Defensins from insects and plants interact with fungal glucosylceramides. J. Biol. Chem. 279::3900-3905.
Nimrichter, L. et al. 2004. . A monoclonal antibody to glucosylceramide inhibits the growth of Fonsecaea pedrosoi and enhances the antifungal action of mouse macrophages. Microbes Infect. 6::657-665.
Toledo, M.S. et al. 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.
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.
Villee, C.A. 1957. Biology. Saunders. Philadelphia, Pennsylvania, USA. 615 pp..
Levitz, S.M. 2001. . Cryptococcus neoformans: intracellular or extracellular Trends Microbiol. 9::417-418.
Saito, K., Takakuwa, N., Ohnishi, M., and Oda, Y. 2006. . Presence of glucosylceramide in yeast and its relation to alkali tolerance of yeast. Appl. Microbiol. Biotechnol. 69::1-7.
Granger, D.L., Perfect, J.R., and Durack, D.T. 1985. . Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide. J. Clin. Invest. 76::508-516.
Klengel, T. et al. 2005. . Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence. Curr. Biol. 15::2021-2026.
Klotz, S.A., Drutz, D.J., Huppert, M., Sun, S.H., and DeMarsh, P.L. 1984. . The critical role of CO2 in the morphogenesis of Coccidioides immitis in cell-free subcutaneous chambers. J. Infect. Dis. 150::127-134.
Mogensen, E.G. et al. 2006. . Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1. Eukaryot. Cell. 5::103-111.
Bahn, Y.S., Cox, G.M., Perfect, J.R., and Heitman, J. 2005. . Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence. Curr. Biol. 15::2013-2020.
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. Proc. Natl. Acad. Sci. U. S. A. 93::4638-4643.
Marks, D.L., Dominguez, M., Wu, K., and Pagano, R.E. 2001. . Identification of active site residues in glucosylceramide synthase. A nucleotide-binding catalytic motif conserved with processive beta-glycosyltransferases. J. Biol. Chem. 276::26492-26498.
Bradford, M.M. 1976. . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72::248-254.
Sullards, M.C., Lynch, D.V., Merrill, A.H. Jr., and Adams, J. 2000. . Structure determination of soybean and wheat glucosylceramides by tandem mass spectrometry. J. Mass. Spectrom. 35::347-353.
Mandala, S.M. et al. 1995. . The discovery of australifungin, a novel inhibitor of sphinganine N-acyltransferase from Sporormiella australis. Producing organism, fermentation, isolation, and biological activity. J. Antibiot. (Tokyo). 48::349-356.
Merrill, A.H. Jr., Sullards, M.C., Allegood, J.C., Kelly, S., and Wang, E. 2005. . Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry. Methods. 36::207-224.
Delaglio, F. et al. 1995. . NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR. 6::277-293.
Shaka, A.J., Lee, C.J., and Pines, A. 1988. . Iterative schemes for bilinear operators; application to spin decoupling. J. Magn. Reson. 77::274-293.
Mersh, J.D., and Sanders, J.K.M. 1982. . The suppression of Bloch-Siegert shifts and subtraction artifacts in double-resonance difference spectroscopy. J. Magn. Reson. 50::289-298.(Philipp C. Rittershaus, T)
2School of Biology, Georgia Institute of Technology, Atlanta, Georgia, USA.
3Department of Molecular Biology, Scripps Research Institute, La Jolla, California, USA.
4Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina, USA.
Abstract
The pathogenic fungus Cryptococcus neoformans infects humans upon inhalation and causes the most common fungal meningoencephalitis in immunocompromised subjects worldwide. In the host, C. neoformans is found both intracellularly and extracellularly, but how these two components contribute to the development of the disease is largely unknown. Here we show that the glycosphingolipid glucosylceramide (GlcCer), which is present in C. neoformans, was essential for fungal growth in host extracellular environments, such as in alveolar spaces and in the bloodstream, which are characterized by a neutral/alkaline pH, but not in the host intracellular environment, such as in the phagolysosome of macrophages, which is characteristically acidic. Indeed, a C. neoformans mutant strain lacking GlcCer did not grow in vitro at a neutral/alkaline pH, yet it had no growth defect at an acidic pH. The mechanism by which GlcCer regulates alkali tolerance was by allowing the transition of C. neoformans through the cell cycle. This study establishes C. neoformans GlcCer as a key virulence factor of cryptococcal pathogenicity, with important implications for future development of new antifungal strategies.
See the related Commentary beginning on page 1481
Introduction
Cryptococcus neoformans is an environmental yeast distributed worldwide and is the most common cause of fungal meningoencephalitis in immunocompromised hosts (1). Infection is initiated upon inhalation of spores or desiccated yeast. In the lung, the fungus proliferates in the alveolar space, and in immunocompetent subjects the infection is normally contained in this organ. However, in immunocompromised subjects, dissemination of the yeast cells from the lung to the brain can occur intracellularly (within host cells) and extracellularly (in the bloodstream), leading to the development of a life-threatening disease (2-4). Since C. neoformans is a facultative intracellular pathogen, many studies have addressed how the fungus survives and grows within host cells (2, 5-9), but how fungal cells survive and proliferate extracellularly is not well understood.
One of the cell surface molecules of this fungus is glucosylceramide (GlcCer), an antigenic glycosphingolipid that elicits an antibody response in patients affected with cryptococcosis (10) and in mice (11). GlcCer is synthesized by uridine diphosphate–glucose:ceramide (UDP-glucose:ceramide) glucosyltransferases encoded by GlcCer synthase (GCS) genes, which catalyze the first step in glycosphingolipid biosynthesis leading to the formation of the membrane lipid GlcCer. Very little is known about the role and function of GCS in fungal cells. The synthesis of GlcCer is absent in the yeast Saccharomyces cerevisiae, but it has been demonstrated in other fungi, such as Pichia pastoris; in fungi pathogenic to plants, such as Magnaporthe grisea (12); in fungi pathogenic to humans, such as C. neoformans (10), Candida albicans (12-14), Aspergillus fumigatus (15, 16), Histoplasma capsulatum (17), Paracoccidioides brasiliensis (18), and Sporotrix schenckii (19); and in other fungi (reviewed in refs. 20-22). The gene encoding for GCS (GCS1) has been isolated from P. pastoris, M. grisea, and C. albicans (12). The GCS1 gene has been deleted in the pathogenic fungus C. albicans, but its role in the pathogenicity of this fungus has yet to be elucidated.
Other studies have suggested a role for GlcCer in the regulation of fungal growth and pathogenesis. For instance, in C. neoformans GlcCer is mainly localized in the cell wall and mostly accumulates at the budding site of dividing cells. Interestingly, antibodies against C. neoformans GlcCer produced by patients affected with cryptococcosis inhibit budding and division of C. neoformans cells grown in vitro (10) as well as differentiation and germ-tube formation of Pseudallescheria boydii and C. albicans (23). Additionally, production of antibodies against fungal glycolipids has been demonstrated in patients with paracoccidioidomycosis (24), although in this disease the antibodies are mostly interacting with galactosylceramide instead of GlcCer. In other fungi, disruption of the GlcCer biosynthetic pathway altered spore germination, hyphal development, and fungal growth (16, 25). Monoclonal antibodies against fungal GlcCer have been produced (11), and, interestingly, treatment with anti-GlcCer antibody enhanced macrophage function against the fungus Fonsecaea pedrosoi (26). Together, these studies suggest an important role for GlcCer in fungal cell growth and differentiation and — because germ-tube formation and hyphal growth are provirulent factors — also suggest that GlcCer might be implicated in the regulation of fungal virulence. Thus we wondered whether GlcCer would have a role in the development and progression of cryptococcosis.
Here we have identified that GCS is a key regulator of pathogenicity of C. neoformans by ensuring cell cycle progression and growth of fungal cells in environments characterized by neutral/alkaline pH and physiological concentrations of CO2. Since these environments are characteristically found in the lung alveolar spaces, GCS regulates survival of C. neoformans upon inhalation through the respiratory tract, with important implications for the dissemination of fungal cells to the brain and the development of meningo-encephalitis.
Results
The C. neoformans GCS1 gene encodes for GCS activity.
By sequence homology to the human GCS cDNA, a candidate C. neoformans GCS1 cDNA was identified. To investigate the potential activity of the C. neoformans GCS1 gene product as a GCS, the effect of GCS1 expression on sphingolipid metabolism was studied in S. cerevisiae, which totally lacks endogenous GCS activity. The human GCS gene was also expressed in a pYES vector as a positive control, and S. cerevisiae carrying the pYES empty vector was used as a negative control. Yeast cells were labeled in vivo with tritiated dihydrosphingosine ([3H]-DHS; Figure 1A), a precursor of ceramide/phytoceramide (the substrate of GCS) in mammalian and yeast cells. The results showed that, when induced by galactose, C. neoformans Gcs1 produced a radiolabeled sphingolipid that migrated similarly to a soy GlcCer standard; this sphingolipid was absent when repressed by glucose. As expected, expression of human GCS1 showed the formation of the putative GlcCer under conditions of induction but not repression (Figure 1A). Cells carrying the empty vector lacked this sphingolipid under conditions of induction and repression. These results suggest that C. neoformans Gcs1 is functional when expressed in S. cerevisiae and that it may use endogenous ceramides as substrates.
Mammalian GCS efficiently recognizes fluorescently labeled short-chain ceramide analogs as substrates in vitro and in vivo. To study the substrate specificity of the C. neoformans Gcs1, 7-nitro-2-1,3-benzoxadiazol-4-yl–C6–ceramide (NBD-C6-ceramide) and NBD-C6-phytoceramide were tested as substrates in vitro, using cell protein extracts of S. cerevisiae expressing C. neoformans GCS1 or human GCS genes, under conditions of induction or repression. In contrast to what we observed for human GCS, C. neoformans Gcs1 did not recognize NBD-C6-ceramides as substrates (Figure 1B). The inability of C. neoformans Gcs1 to use short-chain fluorescent ceramide analogs was also confirmed by in vivo labeling using NBD-ceramides (data not shown). Together, these results show that C. neoformans Gcs1 does not share the same substrate specificity as human GCS.
C. neoformans Gcs1 produces GlcCer.
To examine whether the C. neoformans GCS1 gene encodes for GCS, the C. neoformans GCS1 gene was deleted in the C. neoformans var. grubii strain (WT), creating the gcs1 mutant, and was subsequently reconstituted, generating the gcs1 + GCS1 strain (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI27890DS1). To verify that the C. neoformans gcs1 mutant lacks the putative GlcCer, in vivo labeling using [3H]-DHS in WT, gcs1, and gcs1 + GCS1 strains was performed. As illustrated in Figure 2A, a radiolabeled compound that migrated onto a TLC similarly to the soy GlcCer standard was clearly lost in the gcs1 strain, whereas it was completely restored in the reconstituted strain. Use of NBD-ceramides in C. neoformans for in vitro or in vivo labeling did not result in the formation of the corresponding identifiable sphingolipid in the tested strains (data not shown), in agreement with the finding that C. neoformans Gcs1 expressed in S. cerevisiae was unable to recognize NBD-ceramide analogs as substrates.
To confirm that the missing sphingolipid in the gcs1 is GlcCer, we purified and concentrated the putative sphingolipid from C. neoformans strains, and the extracted lipids were analyzed by high-performance TLC (HPTLC), electrospray tandem mass spectrometry, and NMR. The analysis of the purified lipid extracts onto the HPTLC plate stained with orcinol clearly showed a band of a sugar-containing lipid migrating in tandem with the GlcCer standard from soy. This band was absent in the gcs1 strain and reconstituted in the gcs1 + GCS1 strain (Figure 2B).
The structure of the GlcCer was confirmed by syringe infusion of the glycosphingolipid into an API 3000 triple quadrupole mass spectrometer. Neutral loss scans of m/z 180 (loss of hexose headgroup), as well as precursor ion scans for the types of sphingoid bases commonly found in fungi, were performed. A major peak in C. neoformans WT was observed at m/z 756.7, which corresponded to a [M+H]+ charge for a monohexosylceramide with a lipid backbone composed of 37 carbons, 2 double bonds, and an additional hydroxyl (Figure 2C). Fragmentation of this ion yielded a product ion of m/z 276.4, which identified the lipid backbone as having an 18-carbon sphingadienine base with one additional methyl group (for a total of 19 carbons) plus an amide-linked hydroxy-C18:1 fatty acid. This is consistent with the backbone structure of a GlcCer that has been previously isolated from this organism (9-methyl-4,8-sphingadienine in amide linkage to 2-hydroxyoctadecanoic acid) (10). Precursor ion scans for m/z 276.4 also revealed lower-abundance species that probably reflect GlcCer with a non–hydroxy-C18:0 fatty acid (at m/z 740.7) and a hydroxy-C16:0 fatty acid (at m/z 728.7). Precursor scans in the gcs1 mutant revealed minor peaks also present in the WT spectrum, whereas none of these GlcCers were detected (Figure 2C). Importantly, the reconstituted strain displayed a strong m/z 756.7 ion for the major GlcCer of the WT cells; interestingly, the minor C18:0 and hydroxy-C16:0 species were not detected.
Since no standards are available for these compounds, liquid chromatography mobility could not definitely identify the sugar head group. Thus we performed 2-dimensional NMR spectroscopy to uniquely define the structure of the hexose moiety of the purified sphingolipid (WT), as shown in Figure 2, D and E. The NMR characterization of the hexose commenced with the assignment of the glycoside protons in the 1H-NMR spectrum of the monohexosylceramide (Supplemental Table 1). The unambiguous identification of the H1' anomeric proton was straightforward due to its unique chemical shift (1H, 4.17 ppm; 13C, 103.11 ppm) and doublet multiplicity (3JH1',H2', 7.7 Hz). A combination of 2-dimensional 1H-1H–double quantum filtered correlation spectroscopy and total correlated spectroscopy spectra clearly revealed the complete hexose proton spin system, which was supported by the 13C heteronuclear single quantum correlation data (Figure 2, D and E). The stereochemistry of the hexose could be unambiguously determined from the first 4 vicinal 3J-coupling constants. The large 3JHH-coupling constants observed (>7 Hz) indicated antiperiplanar orientations of the vicinal hexapyranose H1'/H2', H2'/H3' (3JH2',H3', 8.9 Hz), H3'/H4' (3JH3',H4', 8.2 Hz), and H4'/H5' (3JH4',H5', 9.1 Hz) ring protons (Supplemental Table 1). The analysis of 1D–nuclear Overhauser effect (1D-NOE) difference experiments corroborated the previous -glycopyranosyl assignment. NOEs were observed from H1' to H3' and H5'; saturation at H2' produced a strong NOE at H4'. Thus, based on our results and on previous NMR analysis of GlcCer species from other pathogenic fungi (27, 28), we conclude that glucose is attached to the ceramide backbone of the sphingolipid produced by the C. neoformans Gcs1 enzyme.
Role of Gcs1 in the pathogenicity of C. neoformans.
To determine the role of GCS1 in the pathogenicity of C. neoformans, we performed virulence studies using a murine animal model (CBA/J) of cryptococcosis. The average survival of mice infected intranasally with WT and gcs1 + GCS1 strains was 24.6 ± 3.9 and 27.3 ± 4.8 days, respectively (P < 0.0001; Figure 3A), whereas mice infected with C. neoformans gcs1 were all alive after 90 days of observation. At the ninetieth day, even though the 11 mice infected with C. neoformans gcs1 mutant showed no clinical signs of meningoencephalitis, they were sacrificed, and lungs and brains were removed and analyzed for CFUs and histology. Yeast cells were absent in 8 of 11 brains. The 3 yeast cell–positive brains showed approximately 6 x 105, 500, and 28,000 cells per organ. Among the 11 lungs, 6 lungs were used to determine fungal tissue burden, and an average of 5–10 x 103 yeast cells/lung were found, which corresponds to an approximately 100-fold reduction of the initial inoculum. These results were confirmed by a study aiming to determine the lung fungal burden up to 70 days after infection with WT or gcs1 strains (Supplemental Figure 2). These findings suggest that Gcs1 plays a key role in the pathogenicity of C. neoformans and growth of yeast cells in the lung.
To examine whether the lung environment plays a role in protecting the host against the dissemination of the gcs1 strain, we challenged CBA/J mice intravenously with WT, gcs1, and gcs1 + GCS1 strains and monitored their survival. Mice infected with the WT and gcs1 + GCS1 strains showed a similar average survival rate of 6.3 ± 0.5 days and 6.2 ± 0.4 days, respectively (Figure 3B). Mice infected with the gcs1 mutant were apparently healthy until 13–15 days of infection, when they rapidly showed severe clinical signs of meningoencephalitis such as intense tremor, rigidity, lack of balance, and inability to coordinate movements. On the fifteenth day, mice were sacrificed because they were unable to reach food or water. Brains, lungs, kidneys, spleens, and livers were removed and assayed for tissue burden culture and histology. Analysis of CFUs of brains showed an average number of 8.4 ± 0.06 (log10 CFU ± SD) yeast cells, which was significantly higher (P < 0.01) than CFU in other organs (5.5 ± 0.5 in lung, 5.6 ± 0.1 in kidney, 5.3 ± 0.2 in spleen, and 5.6 ± 0.1 in liver). These results clearly suggest that when CBA/J mice are challenged intravenously, they cannot efficiently prohibit gcs1 cells from disseminating throughout the body.
Inflammatory response against the C. neoformans gcs1 strain.
To examine the host inflammatory response against C. neoformans gcs1, 5 lungs and 5 brains from CBA/J mice challenged intranasally and 4 lungs and 4 brains of CBA/J mice challenged intravenously with C. neoformans gcs1 were analyzed for histology. Figure 4, A–D, and Supplemental Figure 3, A and C, show representative fields from 4 different lungs recovered from mice infected intranasally. Supplemental Figure 3, E and G, show a representative field of 2 lungs and Supplemental Figure 3, F and H, show a representative field of 2 brains recovered from mice infected intravenously.
The lungs of mice challenged intranasally showed a strong granulomatous response consisting of the formation of extensive spheric nodules, in which gcs1 cells were mainly trapped in the center with abundant necrotic tissue and numerous neutrophils (Figure 4). The necrotic tissue was surrounded by a ring of foamy macrophages and giant cells loaded with yeast cells and/or cellular debris or "ghosts" of yeast cells, which appeared to be degenerated (Figure 4C). The ring of macrophages was then surrounded by lymphocytes, fibroblasts, and fibrotic tissue (Figure 4A), in which a significant deposition of collagen was observed (Figure 4, C and D). These nodules ranged from 500 to 2,500 μm in diameter and were confined in the lung, with no invasion into the mediastinal area. In other cases, smaller nodules were observed — such as those shown in Supplemental Figure 3, A and C — in which few C. neoformans cells were engulfed in macrophages surrounded by other macrophages containing "ghosts" of C. neoformans cells (Supplemental Figure 3B). Few lymphocytic interstitial infiltrations were localized in the proximity of the nodules (Supplemental Figure 3). Other portions of the lung appeared to be normal. These structures were present only in lungs infected with C. neoformans gsc1, with different degrees of organization, whereas the nodules were not found in lungs of mice infected with the C. neoformans WT strain during the course of the infection (data not shown). All brains selected from mice infected intranasally with gsc1 and analyzed by histology showed no sign of yeast cells.
Histology analysis was also performed on lungs, brains, and other organs of mice infected intravenously. In the lungs, gcs1 cells were contained within small, spheric, nodule-like structures (Supplemental Figure 3, E and G) similar to those illustrated in Supplemental Figure 3, A and C. In the brain, C. neoformans cells were almost exclusively localized within brain abscesses of different size, which were not surrounded by any host inflammatory response (Supplemental Figure 3, F and H). Numerous abscesses containing gcs1 cells were also found in the kidney (data not shown). In the liver, the gcs1 cells were found in very small periportal lesions with no host cellular infiltrates, whereas in the spleen the gcs1 strain was surrounded by a granulomatous response invading the white pulp and characterized by the presence of yeast cells surrounded by macrophages, lymphocytes, neutrophils, and a few megakaryocytes (data not shown). These results suggest that when C. neoformans gsc1 cells are introduced intranasally, the host organizes a strong granulomatous response, in which yeast cells are confined by macrophages, lymphocytes, fibroblasts, fibrotic tissue, and collagen deposition, with almost total containment of the infection within the lung tissue. In contrast, when yeast cells are introduced intravenously, C. neoformans gsc1 cells do disseminate to the brain and other organs, eventually killing the animal.
Effect of Gcs1 on intracellular and extracellular growth of C. neoformans.
In the host, C. neoformans proliferates intracellularly, within the acidic environment of the phagolysosome, and extracellularly, an environment characterized by alkaline/neutral pH and high CO2 concentration. Thus we investigated the effect of the loss of GlcCer on the growth of C. neoformans intracellularly and in in vitro acidic and alkaline environments. For intracellular growth, C. neoformans WT, gcs1, and gcs1 + GCS1 strains were incubated with J774.16 macrophage-like J774.16 cells, and the number of buds of internalized cells per phagocytic index was measured. As shown in Supplemental Figure 4, we found no differences in intracellular growth among the tested strains, suggesting that GlcCer has no effect on fungal growth once C. neoformans is internalized within macrophages.
In order to evaluate in vitro cell culture growth, we performed growth curves of C. neoformans WT, gcs1, and gcs1 + GCS1 strains incubated in 5% CO2 at either a neutral/alkaline or an acidic pH. As shown in Figure 5A, the gcs1 mutant was unable to grow at a neutral/alkaline pH in the presence of 5% CO2 (Figure 5A), whereas growth was observed at an acidic pH (Figure 5B). This difference in growth was not observed in the presence of atmospheric CO2 concentration (0.04%) at pH 7.4 or 4.0 (data not shown). Importantly, the growth defect at 5% CO2 and neutral pH was reversible because when yeast cells were switched to fresh acidic medium, cell growth was completely restored (data not shown), suggesting that growth of gcs1 cells is arrested at alkaline pH and 5% CO2.
To determine which phases of the C. neoformans cell cycle are affected by lack of GlcCer, we performed flow cytometry analysis of C. neoformans cells incubated for 24 hours and 36 hours at alkaline pH and 5% CO2. We found that a significantly greater percentage of gcs1 cells were locked in S and G2/M phases compared with the WT and gcs1 + GCS1 cells at both 24 (Table 1) and 36 hours (data not shown), suggesting that GlcCer is important for exiting from the S and G2/M phases of the cell cycle.
Discussion
In the present study the role of C. neoformans GCS in the virulence of this pathogenic fungus was investigated. Our results show that C. neoformans GCS was an essential factor in determining the success of the fungal infection by regulating survival of C. neoformans during the initial colonization of the lung. In addition, we found that GCS guaranteed survival of C. neoformans in the lung extracellular space by ensuring cell-cycle progression at neutral/alkaline pH and physiologic CO2 concentration (Table 2).
The dramatic protection against pathogenicity imparted by loss of GCS underlines the importance of the extracellular component of fungal cells during C. neoformans infection. C. neoformans infects the host through inhalation, and upon its arrival in the alveoli it encounters 2 environments in which normally grows: the extracellular space, characterized by a neutral/alkaline pH and a physiologic CO2 concentration of approximately 5.0% (compared to 0.04% present in the air, ref. 29); and the intracellular environment of the phagolysosome of alveolar macrophages, characterized by an acidic pH. Our results suggest that GlcCer is critical for C. neoformans to grow in the extracellular (alkaline) but not in the intracellular (acidic) environment of the lung. Since during the infection most C. neoformans cells are found in the host extracellular space (30), the overall effect of the loss of GlcCer on C. neoformans pathogenicity is significant. Indeed, when the gcs1 strain was introduced intranasally, the overall growth of yeast cells in the alveoli was arrested (Supplemental Figure 2); even if the mutant grew intracellularly (Supplemental Figure 4), the host responded with the formation of well-organized granulomas (Figure 4 and Supplemental Figure 3), which efficiently contained gcs1 cells in the lung. This allowed the host to survive the cryptococcal infection without any evident clinical sign of disease (Figure 3A).
When the gcs1 strain was introduced intravenously, mice survived longer than those infected with the WT or reconstituted strains (Figure 3B). In the blood, gcs1 also finds a hostile environment because the pH is approximately 7.4 and the CO2 concentration is approximately 5% (partial pressure of CO2 [pCO2], 40 mmHg). Thus growth of gcs1 cells in the bloodstream was arrested until yeast cells were either internalized by phagocytic cells or they left the bloodstream and invaded the organs, in which they formed abscesses such as those found in the brain (Supplemental Figure 3, F and H) and the kidney (data not shown). In both the intracellular environment and in abscesses, the pH was characteristically acidic, and growth of gcs1 could resume. Indeed, it was interesting to note that in the brain upon intravenous challenge of gcs1, yeast cells were almost exclusively contained within abscesses (Supplemental Figure 3, F and H), whereas in the WT strain, in addition to being present in abscesses, cells were also present in the surrounding tissue (data not shown). Thus we hypothesize that once the acidic abscess is formed, gcs1 cells promptly restore growth, and the consequent destruction of brain tissue will eventually manifest abruptly with clinical sign of meningoencephalitis. The animal infected intravenously with gcs1 would eventually die, although it would take longer than mice infected with the control strains due to the transient growth arrest that yeast cells undergo in the bloodstream upon injection.
We believe the results from these studies identify GlcCer as a novel virulence factor for C. neoformans because loss of this glycosphingolipid does not affect other fungal characteristics, such as capsule production, melanin formation, or growth at ambient CO2 and 37°C (Supplemental Figure 5), known to be required to cause disease (1). Interestingly, a greater sensitivity of gcs1 mutant to SDS was observed (Supplemental Figure 5), suggesting an alteration of cell wall integrity. This hypothesis is supported by previous observations of the corrleation of GlcCer density at the cell surface with the cell wall thickness of C. neoformans (10). However, the difference in cell growth of gcs1 in the presence of SDS compared with control strains was not so dramatic to account for the significant loss of virulence of the mutant. Instead, the growth of gcs1 mutant was arrested in environments characterized by alkaline pH and 5% CO2.
In budding yeasts such as C. neoformans, the S and G2 phases of the cell cycle are characterized by bud formation and maturation (Figure 5C). Interestingly, previous studies have shown that GlcCer is localized at the budding site of C. neoformans, and addition of human antibodies against C. neoformans GlcCer to in vitro C. neoformans culture inhibits cell budding and fungal cell growth (10). Our studies showed that loss of GlcCer significantly prolonged the S and the G2/M phases in alkaline environments, therefore suggesting a mechanism whereby GlcCer regulates fungal cell growth by allowing yeast cells to complete the cell cycle.
The role of GCS in alkaline tolerance is not restricted to C. neoformans, as recent findings showed that a Kluyveromyces lactis mutant defective in GlcCer cannot grow at pH 8.5 (31). On the other hand, the molecular mechanism by which GlcCer regulates alkaline tolerance at high but not low CO2 is not known. Studies have proposed that fungal cells sense different concentrations of CO2 and, in conditions in which CO2 is elevated (e.g., 5%), fungi respond by upregulating virulence factors. For instance, CO2 stimulates capsule formation in C. neoformans (32) and the transformation of yeast cells to filamentous forms in C. albicans through adenylyl cyclase (33). In other pathogenic fungi, such as Coccidioides immitis, CO2 promotes the formation of endosporulating spherules (34), which is a process required for virulence. These studies suggest the existence of a conserved signaling pathway that regulates fungal virulence through the sensing of CO2. C. neoformans senses CO2 through 2 -class carbonic anhydrases (Can1 and Can2), which have been recently identified (35, 36), but whether GlcCer regulates signaling events mediated by CO2-carbonic anhydrases at different pHs awaits further investigation.
In conclusion, these studies showed that GlcCer plays a key role in the pathogenicity of C. neoformans through a mechanism that ensures the transition through the cell cycle of yeast cells in alkaline environments with a physiological concentration of CO2. Since in cases of cryptococcosis extracellular organisms predominate, the function of GlcCer becomes clinically relevant. Interestingly, Gcs1 and GlcCer are found in a variety of pathogenic fungi, and because a significant biochemical difference between human and C. neoformans Gcs1 exists, compounds that would specifically inhibit the fungal enzyme might represent a novel class of antifungal agents against fungal infections (16, 25). Furthermore, since antibodies against the fungal GlcCer inhibit C. neoformans growth in vitro and do not interact with the human GlcCer (11), they may represent a new therapeutic approach to control infections by fungal microorganisms producing GlcCer.
Methods
Strains, plasmids, and growing conditions.
The strains used in this study were the C. neoformans var. grubii serotype A strain H99 (WT), the gcs1 mutant derived from H99, and the gcs1 + GCS1 reconstituted strain derived from gcs1; as well as the S. cerevisiae strains JK9-3d (MAT-, trp1, leu2-3, his4, ura3, ade2, rme1) and JK9-3d containing the pYES2 vector, the pYES2 vector expressing human GCS1 (pYES2-HuGCS1), and the pYES2 vector expressing C. neoformans GCS1 (pYES2-CnGCS1). Escherichia coli strains DH5- (Invitrogen Corp.) and TOP10 (Invitrogen Corp.) were used in this study. Plasmids pBluescript SK (Stratagene) and pCR2.1-TOPO (Invitrogen Corp.) were used for cloning, and pYES2 (Invitrogen Corp.) was used for expression of GCS genes in S. cerevisiae. C. neoformans strains were routinely grown in yeast extract/peptone/2% dextrose–rich (YPD-rich) medium. S. cerevisiae strain JK9-3d transformed with the pYES2, pYES2-HuGCS1, or pYES2-CnGCS1 vector was routinely grown in synthetic medium containing 6.7 g/l yeast nitrogen base (YNB) without amino acids, 1.2 g/l amino acid mixture lacking uracil, and 2% glucose or 1% glucose and 1% galactose for 16 hours at 30°C to induce either human or cryptococcal GCS1 gene expression. A full-length cDNA clone of the human GCS1 gene was cloned into pYES2 (Invitrogen Corp.) vector as described previously (37), generating the pYES2-HuGCS1 plasmid. Bacterial strains for plasmid amplification were routinely grown at 37°C in Luria-Bertani medium containing 75 mg/l of ampicillin (Sigma-Aldrich). DMEM (#11995; BD Biosciences) buffered with HEPES at pH 7.4 or pH 4.0 was used for growing the C. neoformans strain at 37°C in presence of 5% CO2.
Isolation and cloning of the C. neoformans GCS1 gene.
The GCS1 gene from C. neoformans was identified by a BLAST search of the human GCS sequence (GenBank D50840) in the genome database of C. neoformans var. grubii serotype A strain H99 from Duke Center for Genome Technology (http://cneo.genetics.duke.edu/blast.html). One putative sequence, with an E value of 9e-51 corresponding to chr4Bpiece18, was found. The GCS1 cDNA was then isolated by RT-PCR using the PRGCS-5' forward (5'-ATGAGCGATTCCGGCACATTGTCC-3') and reverse (5'-TCAATTTCTATCACCCAAACGAATTG-3') primers, and total RNA was extracted from C. neoformans strain H99. A 1,353-bp fragment was obtained, cloned into the pCR2.1-TOPO vector generating plasmid pCR-CnGCS1, and sequenced. Amino acid alignment of C. neoformans Gcs1 with other Gcs1 proteins from different organisms revealed a homology (identity + similarity) of 47%, 16%, 43%, 46%, 43%, and 35% to human, Arabidopsis thaliana, C. albicans, P. pastoris, M. grisea, and Aspergillus nidulans, respectively. The C. neoformans Gcs1 contains the motifs [D1, D2, D3 (R/QXXRW)] common to other GCSs required for GCS activity (38) and the characteristic N terminus hydrophobic transmembrane domain, which contains a signal-anchor sequence to the Golgi outer membrane (37). The C. neoformans GCS1 cDNA sequence was deposited in the GenBank database (AY956317). The GCS1 gene was deleted and reconstituted in the WT C. neoformans var. grubii strain, generating C. neoformans gcs1 and gcs1 + GCS1 strains (see Supplemental Methods).
In vitro growth studies.
From overnight YPD broth cultures of C. neoformans WT, gcs1, and gcs1 + GCS1 strains, cells were washed twice in sterile distilled water (SDW), resuspended, and diluted into 40 ml fresh YPD broth to a final density of 104 cells/ml and incubated in a shaker incubator at 30°C or 37°C. Aliquots were taken at the time points indicated in Supplemental Figure 5A, and serial dilutions were plated onto YPD plates for assessment of CFU. For in vitro growth studies on DMEM at pH 7.4, 7.0, or 4.0, 104 cells/ml were incubated at 37°C in the presence of 5% CO2, aliquots were taken at the time points indicated in Figure 5A, and serial dilutions were plated onto YPD plates for assessment of CFU. Capsule formation and melanin production were examined using media as previously described (1). For cell wall stability, cells were spotted in serial dilutions on YPD plates containing 0.05% SDS. For osmotic stress, cells were spotted in serial dilutions on YPD plates containing 1M NaCl. For oxidative or nitrosative stresses, cells were spotted in serial dilutions on YNB plates containing amino acids adjusted to pH 4.0 and supplemented with H2O2 to a final concentration of 5 mM or nitrite to a final concentration of 1 mM. After incubation at 30°C or 37°C for 72 hours, cell growth was examined and recorded by photography. Results show plates incubated at 37°C.
For intracellular growth studies, J774.16 macrophage-like cells were used, and fungal growth of C. neoformans WT, gcs1, and gcs1 + GCS1 strains was determined as previously described (2, 5-9).
For cell-cycle studies, yeast cells were incubated in DMEM, pH 7.4, at 37°C in the presence of 5% CO2 for 24 and 36 hours. Cells were then washed twice with ice-cold water, sonicated, and fixed in 7 ml of 95% ethanol for 16 hours. After fixing, the cells were pelleted, washed with 5 ml of 50 mM sodium citrate, resuspended in 1 ml of 50 mM sodium citrate containing 0.25 mg/ml RNase A, and incubated at 50°C for 1 hour. Next, proteinase K was added at a final concentration of 20 mg/ml, and cells were kept at 50°C for an additional hour. Finally, 1 ml of 50 mM sodium citrate containing 50 μg/ml propidium iodine was added. Cells were kept for 24 hours in the dark at 4°C and were analyzed using a BD fluorescence-activated cell analyzer. Data were modeled using the program ModFit LT (version 3.1; Verity Software).
In vitro activity assay of GCS.
To express C. neoformans GCS1 gene in S. cerevisiae, the cDNA fragment was digested with EcoRI from pCR-CnGCS1 and subcloned into EcoRI-restricted pYES2 plasmid, generating pYES2-CnGCS1. The S. cerevisiae JK9-3d strain was chemically transformed with pYES2-CnGCS1 plasmid. Transformants were selected on YNB ura– agar plates containing 2% glucose, randomly chosen, purified, and stored. For gene induction, cell cultures were pelleted and washed twice with SDW. Cells were resuspended, inoculated into YNB ura– broth containing 1% galactose plus 1% glucose or 2% glucose, and incubated in a 30°C shaker for 16 hours. For in vitro GCS activity in C. neoformans strains, yeast cells were grown on YPD media in a shaker incubator for 24 hours at 30°C, harvested by centrifugation, and washed with SDW, and pellets were stored at –80°C if not used immediately. Once cells were collected, proteins were extracted as previously described (9) and quantitated by the method of Bradford (39). The in vitro GCS activity assay was performed by using the fluorescent ceramide analog NBD-C6-ceramide (Avanti Polar Lipids Inc.) as a substrate and monitoring the formation of NBD-C6-GlcCer as described previously (37), with some modifications. Briefly, 200 μg of yeast proteins were incubated for 30 minutes at 30°C in 50 mM bis-Tris-HCl buffer (pH 6.5) containing 12.5 mM KCl, 250 μM EDTA, 50 μM phosphatidylcholine, 500 μM UDP-glucose, 5 mM Triton X-100, and 20 μM NBD-C6-ceramide in a final reaction volume of 100 μl. The reaction was terminated by the addition of 0.3 ml CHCl3/MeOH (1:1 ratio). The samples were mixed, and the phases were separated by centrifugation at 2,000 g for 5 minutes. The lower phase was dried down using the SPD 2010 Speedvac (Thermo Electron Corp.). The chloroform-soluble product was analyzed by analytical TLC on silica gel 60 plates (EM Science) using the solvent system chloroform/methanol/water (65:25:4). Purified GlcCer from soy (Avanti Polar Lipids Inc.) was also loaded onto the TLC plate and used as a standard (40). NBD-GlcCer was identified as a fluorescent band migrating in tandem with soy GlcCer and quantitated by direct fluorescence using a phosphorimager Molecular Dynamics Inc. 840 STORM unit and by ImageQuant analysis (GE Healthcare).
In vivo activity assay of GCS.
Approximately 5 hours before cell collection, yeast cells were counted, and 5 x 107 cells were resuspended in the same growing media and incubated with 2 μM/ml NBD-C6-ceramide. Similar experiments were performed using NBD-C6-phytoceramide, NBD-C12-ceramide, and NBD-C12-phytoceramide. Yeast cells were incubated for 5 hours, shaken at 30°C. Cells were harvested by centrifugation for 10 minutes at 2,000 g and washed with SDW, and the total lipid was extracted using the method of Mandala et al. (41) followed by base hydrolysis. The extracted lipids were loaded onto a TLC using the solvent system chloroform/methanol/water (65:25:4 ratio). Formation of NBD-GlcCer was analyzed as described above.
For in vivo labeling using [3H]-DHS, approximately 2 hours prior to cell collection, 5 x 107 yeast cells/ml were incubated with 2 μCi/ml of [3H]-DHS for 2 hours at 30°C in the appropriate growing media in a shaker incubator. The cells were harvested by centrifugation (10 minutes at 2,500 g), washed with SDW, and stored at –80°C if not used immediately. Lipids were extracted as described by Mandala et al. (41) and after base hydrolysis were loaded onto a TLC plate and analyzed using a phosphoimager and quantitation of radioactive GlcCer performed by ImageQuant analysis. As a control, 10 μl of 20 mM soy GlcCer (Avanti Polar Lipids Inc.) was loaded onto a TLC plate and visualized using iodine stain.
Mass spectrometry analysis.
Purified GlcCer was obtained as described in Supplemental Methods and used for mass spectrometry (MS) analysis. The MS and MS/MS scans of GlcCer were conducted using an API 3000 triple quadrupole mass spectrometer with electrospray ionization as described previously by Merrill et al. (42). The analyses used syringe infusion and included neutral loss scans (with m/z 180 for loss of the hexose headgroup) and precursor ion scans for the characteristic product ion m/z 276.4.
NMR analysis.
NMR experiments were recorded in 500 μl dimethyl-sulfoxide-d6 (Cambridge Isotope Laboratories Inc.) using 160 μg putative monohexosylceramide obtained from HPTLC purification; subsequently, the sample was dissolved in dimethyl-sulfoxide-d6 and deuterium oxide (98:2, vol/vol). NMR spectra were recorded on a Varian Inova 600 MHz spectrometer equipped with an actively shielded z-gradient triple-resonance (1H, 13C, 31P) probe at 25°C. All spectra were processed and analyzed using FELIX 2000 Insights II version 2000 (MSI) and NMRPipe version 2.3 (43) program packages. 1H chemical shifts were internally referenced to 3-(trimethylsilyl)-1-propane sulfonic acid. 13C chemical shifts were referenced against the center line of the dimethyl-sulfoxide septet ( = 39.52 ppm). The decoupling in the presence of scalar interactions-2 (DISPI-2) mixing time was 62.6 ms (44). NOE difference experiments were measured with saturation delays of 6 seconds (45).
Virulence studies and histology analysis in a murine mouse model of cryptococcosis.
Four- to 6-week-old CBA/J (NCI/Frederick Laboratories) mice were used. Mice were anesthetized with an intraperitoneal injection of 60 μl xylazine/ketamine mixture containing 95 mg ketamine and 5 mg xylazine per kilogram of body weight. C. neoformans WT, gcs1, and gcs1 + GCS1 strains were prepared by growing cells for 24 hours at 30°C in YPD medium. The cells were pelleted, washed twice, and resuspended in PBS, pH 7.4, at a concentration of 2.5 x 107 cells/ml. Ten CBA/J mice were infected with 5 x 105 cells of WT, gcs1, or gcs1 + GCS1 strains in a volume of 20 μl through nasal inhalation. Eight CBA/J mice were infected with 106 cells of WT, gcs1, or gcs1 + GCS1 strains in a volume of 100 μl through tail vein injection. The mice were fed ad libitum and followed with twice-daily inspections. Mice that appeared moribund or in pain or with clinical signs of meningoencephalitis were sacrificed using CO2 inhalation followed by cervical dislocation. All animal procedures were approved by the Medical University of South Carolina Institutional Animal Care and Use Committee and followed the guidelines of the American Veterinary Medical Association. Histology analysis was performed as described in Supplemental Methods.
Statistics.
Survival data from the mouse experiments were analyzed by Kluskal-Wallis test. For other statistical analysis, a 2-tailed Student’s t test was used. A P value of less than 0.05 was considered to be significant.
Acknowledgments
We are grateful to Helio Takahashi, Yusuf Hannun, Lina Obeid, Deepak Bastia, Edward Balish, Lincoln Scott, and Carolina Westwater for discussion. Special thanks to Margaret Romano and to Russell Harley for invaluable help and discussion with the histology staining and to Stefka Spassieva for helping in the purification of GlcCer. We also thank all members of the M. Del Poeta and C. Luberto laboratories for guidance. We gratefully acknowledge John Oatis for helping with NMR analysis. This work was supported in part by the Burroughs Welcome Fund; NIH grant AI56168 (to M. Del Poeta); grants RR17677 Project 2 (to M. Del Poeta) and Project 6 (to C. Luberto) from the Centers of Biomedical Research Excellence Program of the National Center for Research Resources; National Science Foundation/EPSCoR grant #EPS-0132573 (to C. Luberto); NIH grant C06 RR015455 from the Extramural Research Facilities Program of the National Center for Research Resources; and, for the sphingolipid analysis by mass spectrometry, NIH grant GM069338 (LIPID MAPS, to A.H. Merrill). M. Del Poeta is a Burroughs Wellcome New Investigator in Pathogenesis of Infectious Diseases.
Footnotes
Philipp C. Rittershaus and Talar B. Kechichian contributed equally to this work.
Nonstandard abbreviations used: GCS, GlcCer synthase; GlcCer, glucosylceramide; [3H]-DHS, tritiated dihydrosphingosine; HPTLC, high-performance TLC; NBD, 7-nitro-2-1,3-benzoxadiazol-4-yl; NOE, nuclear Overhauser effect; pYES2-CnGCS1, pYES2 vector expressing C. neoformans GCS1; pYES2-HuGCS1, pYES2 vector expressing human GCS1; SDW, sterile distilled water; YNB, yeast nitrogen base; YPD, yeast extract/peptone/2% dextrose.
Conflict of interest: The authors have declared that no conflict of interest exists.
References
Casadevall, A., and Perfect, J.R. 1998. Cryptococcus neoformans. ASM Press. Washington, DC, USA. 351–380..
Feldmesser, M., Tucker, S., and Casadevall, A. 2001. . Intracellular parasitism of macrophages by Cryptococcus neoformans. Trends Microbiol. 9::273-278.
Levitz, S.M. et al. 1999. . Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages. Infect. Immun. 67::885-890.
Chretien, F. et al. 2002. . Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia. J. Infect. Dis. 186::522-530.
Feldmesser, M., Kress, Y., Novikoff, P., and Casadevall, A. 2000. . Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect. Immun. 68::4225-4237.
Lee, S.C., Kress, Y., Zhao, M.L., Dickson, D.W., and Casadevall, A. 1995. . Cryptococcus neoformans survive and replicate in human microglia. Lab. Invest. 73::871-879.
Tucker, S.C., and Casadevall, A. 2002. . Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm. Proc. Natl. Acad. Sci. U. S. A. 99::3165-3170.
Steenbergen, J.N., Shuman, H.A., and Casadevall, A. 2001. . Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc. Natl. Acad. Sci. U. S. A. 98::15245-15250.
Luberto, C. et al. 2001. . Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans. Genes Dev. 15::201-212.
Rodrigues, M.L. et al. 2000. . Human antibodies against a purified glucosylceramide from Cryptococcus neoformans inhibit cell budding and fungal growth. Infect. Immun. 68::7049-7060.
Toledo, M.S., Suzuki, E., Levery, S.B., Straus, A.H., and Takahashi, H.K. 2001. . Characterization of monoclonal antibody MEST-2 specific to glucosylceramide of fungi and plants. Glycobiology. 11::105-112.
Leipelt, M. et al. 2001. . Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi. J. Biol. Chem. 276::33621-33629.
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.
Leipelt, M., Warnecke, D.C., Hube, B., Zahringer, U., and Heinz, E. 2000. . Characterization of UDP-glucose:ceramide glucosyltransferases from different organisms. Biochem. Soc. Trans. 28::751-752.
Boas, M.H., Egge, H., Pohlentz, G., Hartmann, R., and Bergter, E.B. 1994. . Structural determination of N-2'-hydroxyoctadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine from species of Aspergillus. Chem. Phys. Lipids. 70::11-19.
Levery, S.B. et al. 2002. . Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc:ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth. FEBS Lett. 525::59-64.
Toledo, M.S., Levery, S.B., Suzuki, E., Straus, A.H., and Takahashi, H.K. 2001. . 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. Glycobiology. 11::113-124.
Takahashi, H.K. et al. 1996. . Isolation and possible composition of glucosylceramides from Paracoccidioides brasiliensis. Braz. J. Med. Biol. Res. 29::1441-1444.
Toledo, M.S., Levery, S.B., Glushka, J., Straus, A.H., and Takahashi, H.K. 2001. . Structure elucidation of sphingolipids from the mycopathogen Sporothrix schenckii: identification of novel glycosylinositol phosphorylceramides with core manalpha1-->6Ins linkage. Biochem. Biophys. Res. Commun. 280::19-24.
Warnecke, D., and Heinz, E. 2003. . Recently discovered functions of glucosylceramides in plants and fungi. Cell. Mol. Life Sci. 60::919-941.
Hanada, K. 2005. . Sphingolipids in infectious diseases. Jpn. J. Infect. Dis. 58::131-148.
Heung, L.J., Luberto, C., and Del Poeta, M. 2006. . Role of sphingolipids in microbial pathogenesis. Infect. Immun. 74::28-39.
Pinto, M.R. et al. 2002. . Characterization of glucosylceramides in Pseudallescheria boydii and their involvement in fungal differentiation. Glycobiology. 12::251-260.
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.
Thevissen, K. et al. 2004. . Defensins from insects and plants interact with fungal glucosylceramides. J. Biol. Chem. 279::3900-3905.
Nimrichter, L. et al. 2004. . A monoclonal antibody to glucosylceramide inhibits the growth of Fonsecaea pedrosoi and enhances the antifungal action of mouse macrophages. Microbes Infect. 6::657-665.
Toledo, M.S. et al. 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.
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.
Villee, C.A. 1957. Biology. Saunders. Philadelphia, Pennsylvania, USA. 615 pp..
Levitz, S.M. 2001. . Cryptococcus neoformans: intracellular or extracellular Trends Microbiol. 9::417-418.
Saito, K., Takakuwa, N., Ohnishi, M., and Oda, Y. 2006. . Presence of glucosylceramide in yeast and its relation to alkali tolerance of yeast. Appl. Microbiol. Biotechnol. 69::1-7.
Granger, D.L., Perfect, J.R., and Durack, D.T. 1985. . Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide. J. Clin. Invest. 76::508-516.
Klengel, T. et al. 2005. . Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence. Curr. Biol. 15::2021-2026.
Klotz, S.A., Drutz, D.J., Huppert, M., Sun, S.H., and DeMarsh, P.L. 1984. . The critical role of CO2 in the morphogenesis of Coccidioides immitis in cell-free subcutaneous chambers. J. Infect. Dis. 150::127-134.
Mogensen, E.G. et al. 2006. . Cryptococcus neoformans senses CO2 through the carbonic anhydrase Can2 and the adenylyl cyclase Cac1. Eukaryot. Cell. 5::103-111.
Bahn, Y.S., Cox, G.M., Perfect, J.R., and Heitman, J. 2005. . Carbonic anhydrase and CO2 sensing during Cryptococcus neoformans growth, differentiation, and virulence. Curr. Biol. 15::2013-2020.
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. Proc. Natl. Acad. Sci. U. S. A. 93::4638-4643.
Marks, D.L., Dominguez, M., Wu, K., and Pagano, R.E. 2001. . Identification of active site residues in glucosylceramide synthase. A nucleotide-binding catalytic motif conserved with processive beta-glycosyltransferases. J. Biol. Chem. 276::26492-26498.
Bradford, M.M. 1976. . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72::248-254.
Sullards, M.C., Lynch, D.V., Merrill, A.H. Jr., and Adams, J. 2000. . Structure determination of soybean and wheat glucosylceramides by tandem mass spectrometry. J. Mass. Spectrom. 35::347-353.
Mandala, S.M. et al. 1995. . The discovery of australifungin, a novel inhibitor of sphinganine N-acyltransferase from Sporormiella australis. Producing organism, fermentation, isolation, and biological activity. J. Antibiot. (Tokyo). 48::349-356.
Merrill, A.H. Jr., Sullards, M.C., Allegood, J.C., Kelly, S., and Wang, E. 2005. . Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry. Methods. 36::207-224.
Delaglio, F. et al. 1995. . NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR. 6::277-293.
Shaka, A.J., Lee, C.J., and Pines, A. 1988. . Iterative schemes for bilinear operators; application to spin decoupling. J. Magn. Reson. 77::274-293.
Mersh, J.D., and Sanders, J.K.M. 1982. . The suppression of Bloch-Siegert shifts and subtraction artifacts in double-resonance difference spectroscopy. J. Magn. Reson. 50::289-298.(Philipp C. Rittershaus, T)