Functional cloning and characterization of a UDP- glucuronic acid decarboxylase: the pathogenic fungus Cryptococcus neoformans elucidates UDP-xylose synthesis.

UDP-xylose is a sugar donor required for the synthesis of diverse and important glycan structures in animals, plants, fungi, and bacteria. Xylose-containing glycans are particularly abundant in plants and in the polysaccharide capsule that is the major virulence factor of the pathogenic fungus Cryptococcus neoformans. Biosynthesis of UDP-xylose is mediated by UDP-glucuronic acid decarboxylase, which converts UDP-glucuronic acid to UDP-xylose. Although this enzymatic activity was described over 40 years ago it has never been fully purified, and the gene encoding it has not been identified. We used homology to a bacterial gene, hypothesized to encode a related function, to identify a cryptococcal sequence as putatively encoding a UDP-glucuronic acid decarboxylase. A soluble 47-kDa protein derived from bacteria expressing the C. neoformans gene catalyzed conversion of UDP-glucuronic acid to UDP-xylose, as confirmed by NMR analysis. NADH, UDP, and UDP-xylose inhibit the activity. Close homologs of the cryptococcal gene, which we termed UXS1, appear in genome sequence data from organisms ranging from bacteria to humans.

Spatial and temporal sequence of capsule construction in Cryptococcus neoformans.

The pathogenic yeast Cryptococcus neoformans is distinguished by an extensive polysaccharide capsule, which impedes host defences and is absolutely required for fungal virulence. Despite the biological importance of the capsule, nothing is known about how it is assembled. Substantial capsule growth occurs in two distinct situations relevant to cryptococcal pathogenesis: formation of new buds and induction of capsule on mature cells. We developed pulse-chase protocols to examine these events in a dynamic way using a variety of microscopy techniques. We show that the capsule overlying buds is newly synthesized and differs physically from the corresponding parental material. New capsule formed by mature cells upon induction of synthesis is added at the inner aspect of the existing structure, displacing pre-existing material outwards. Surprisingly, new polysaccharide material is also deposited throughout the capsule, yielding a progressively denser structure. These results yield the first model of capsule synthesis and open new lines of investigation into the underlying mechanisms.

Detection of glycophospholipid anchors on proteins.

Many eukaryotic proteins are tethered to the plasma membrane by glycosyl phosphatidylinositol (GPI) membrane anchors. This unit provides a general approach for detecting GPI-anchored proteins. First, the detergent-partitioning behavior of a protein of interest is examined for characteristics of GPI-linked species. The partitioning of total cellular and isolated proteins with Triton X-114 is described in this unit, and precondensation of Triton X-114, which is necessary to remove hydrophilic contaminants before partitioning, is outlined in a Support Protocol 1. The protein may also be subjected to specific enzymatic or chemical cleavages to release it from its GPI anchor. Phospholipase cleavage (starting with intact cells or membranes, or with isolated protein) is detailed, and chemical cleavage with nitrous acid is also described. If GPI-anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. A protocol for separation of lipid moieties released from proteins is provided and base hydrolysis of proteins is also presented.

Detection of glycophospholipid anchors on proteins.

Many eukaryotic proteins are tethered to the plasma membrane by glycosyl phosphatidylinositol (GPI) membrane anchors. This unit provides a general approach for detecting GPI-anchored proteins. First, the detergent-partitioning behavior of a protein of interest is examined for characteristics of GPI-linked species. The protein may also be subjected to specific enzymatic or chemical cleavages to release the protein from its GPI anchor. Protocols for phospholipase cleavage and chemical cleavage with nitrous acid are provided for this purpose. If GPI-anchored proteins are radiolabeled with fatty acids, it facilitates the detection of the GPI protein products following the cleavage reactions. Separation of lipid moieties and base hydrolysis of proteins are detailed herein.

How does Cryptococcus get its coat?

During the past few decades, increasing attention has focused on pathogenic fungi both as fascinating research subjects and as the agents of serious illness in diverse patient populations. In particular, opportunistic fungi such as Cryptococcus neoformans command notice as the ranks of their immunocompromised victims grow. C. neoformans is unique among fungal pathogens for its major virulence factor, a complex polysaccharide capsule. In this article, our current understanding of the structure and biosynthesis of the capsule is reviewed, as are the many questions that remain to be answered about how Cryptococcus gets its coat.

A unique alpha-1,3 mannosyltransferase of the pathogenic fungus Cryptococcus neoformans.

The major virulence factor of the pathogenic fungus Cryptococcus neoformans is an extensive polysaccharide capsule which surrounds the cell. Almost 90% of the capsule is composed of a partially acetylated linear alpha-1,3-linked mannan substituted with D-xylose and D-glucuronic acid. A novel mannosyltransferase with specificity appropriate for a role in the synthesis of this glucuronoxylomannan is active in cryptococcal membranes. This membrane-associated activity transfers mannose in vitro from GDP-mannose to an alpha-1, 3-dimannoside acceptor, forming a second alpha-1,3 linkage. Product formation by the transferase is dependent on protein, time, temperature, divalent cations, and each substrate. It is not affected by amphomycin or tunicamycin but is inhibited by GDP and mannose-1-phosphate. The described activity is not detectable in the model yeast Saccharomyces cerevisiae, consistent with the absence of a similar polysaccharide structure in that organism. A second mannosyltransferase from C. neoformans membranes adds mannose in alpha-1,2 linkage to the same dimannoside acceptor. The two activities differ in pH optimum and cation preference. While the alpha-1,2 transferase does not have specificity appropriate for a role in glucuronoxylomannan synthesis, it may participate in production of mannoprotein components of the capsule. This study suggests two new targets for antifungal drug discovery.

Melanin as a potential cryptococcal defence against microbicidal proteins.

Cryptococcus neoformans is an important fungal pathogen that synthesizes melanin when grown in the presence of phenolic substrates. The ability of C. neoformans to produce melanin is associated with virulence, but the specific role of melanin in the pathogenesis of infection is not clear. In this study the ability of C. neoformans melanin to bind proteins and protect against microbicidal peptides was investigated. Melanin was shown to bind a variety of proteins of fungal and mammalian origin. Melanin-protein interactions were dependent on the pH of the solution and on the amount of protein and melanin present. Melanized cells were less susceptible to killing by three microbicidal peptides: a defensin, a protegrin, and a magainin. Incubation of the microbicidal peptides with melanin particles, followed by removal of the melanin, reduced or abolished fungicidal activity, demonstrating interactions between peptides and melanin. The ability of melanin to bind proteins and to protect against microbicidal peptides suggests a protective function for melanin, whereby it sequesters microbicidal peptides and abrogates their activity.

Antibody response to Cryptococcus neoformans proteins in rodents and humans.

The prevalence and specificity of serum antibodies to Cryptococcus neoformans proteins was studied in mice and rats with experimental infection, in individuals with or without a history of potential laboratory exposure to C. neoformans, human immunodeficiency virus (HIV)-positive individuals who developed cryptococcosis, in matched samples from HIV-positive individuals who did not develop cryptococcosis, and in HIV-negative individuals. Rodents had little or no serum antibody reactive with C. neoformans proteins prior to infection. The intensity and specificity of the rodent antibody response were dependent on the species, the mouse strain, and the viability of the inoculum. All humans had serum antibodies reactive with C. neoformans proteins regardless of the potential exposure, the HIV infection status, or the subsequent development of cryptococcosis. Our results indicate (i) a high prevalence of antibodies reactive with C. neoformans proteins in the sera of rodents after cryptococcal infection and in humans with or without HIV infection; (ii) qualitative and quantitative differences in the antibody profiles of HIV-positive individuals; and (iii) similarities and differences between humans, mice, and rats with respect to the specificity of the antibodies reactive with C. neoformans proteins. The results are consistent with the view that C. neoformans infections are common in human populations, and the results have implications for the development of vaccination strategies against cryptococcosis.

Inositol acylation of glycosylphosphatidylinositols in the pathogenic fungus Cryptococcus neoformans and the model yeast Saccharomyces cerevisiae.

Cryptococcus neoformans, an opportunistic fungus responsible for life-threatening infection in immunocompromised patients, is able to synthesize glycosylphosphatidylinositol (GPI) structures. Radiolabelling experiments in vitro with the use of a cryptococcal cell-free system showed that the pathway begins as in other eukaryotes, with the addition of N-acetylglucosamine to phosphatidylinositol, followed by deacetylation of the sugar residue. The third step, acylation of the inositol ring, seemed to involve a fatty acid other than palmitate, in contrast with previous findings in Saccharomyces cerevisiae and mammalian GPI pathways. A systematic study of inositol acylation in C. neoformans and S. cerevisiae showed that both organisms used a variety of fatty acids in this step; these were transferred directly from acyl-CoA to inositol without modification. However, the specificity of fatty acid utilization was quite distinct in the two fungi, with the pathogen being substantially more restrictive. In mammalian cells fatty acids added exogenously as acyl-CoAs are not transferred directly to inositol. These results suggest significant differences in the GPI biosynthetic pathway between mammalian and C. neoformans cells that could represent targets for anti-cryptococcal therapy.

Glycosyl-phosphatidylinositol anchor attachment in a yeast in vitro system.

The yeast mating pheromone precursor prepro-alpha factor was fused to C-terminal signals for glycosyl-phosphatidylinositol (GPI) anchor attachment, based on the sequence of the Saccharomyces cerevisiae protein Gas1p. Maturation of fusion proteins expressed in vivo required the presence of both a functional GPI attachment site and the synthesis of GPI precursors. Constructs were translated in vitro for use in cell-free studies of glycolipid attachment. The radiolabelled polypeptides were post-translationally translocated into yeast microsomes, where at least one third of the molecules received a GPI anchor. This approach offers distinct advantages over anchor attachment reactions that require co-translational translocation of secretory peptide substrates.

Specific requirements for the ER to Golgi transport of GPI-anchored proteins in yeast.

GPI-anchored proteins are attached to the membrane via a glycosylphosphatidylinositol-(GPI) anchor whose carbohydrate core is conserved in all eukaryotes. Apart from membrane attachment, the precise role of the GPI-anchor is not known, but it has been proposed to play a role in protein sorting. We have investigated the transport of the yeast GPI-anchored protein Gas1p. We identified two mutant strains involved in very different cellular processes that are blocked selectively in the transport of GPI-anchored proteins before arrival to the Golgi. The end8-1/lcb1-100 mutant is defective in ceramide synthesis. In vitro data suggest a requirement for ceramides after the exit from the ER. We therefore propose that ceramides might function in the fusion of a GPI-containing vesicle with the Golgi, but we cannot exclude a role in the ER. The second mutant that blocks the transport of GPI-anchored proteins to the Golgi is ret1-1, a mutant in the alpha-subunit of coatomer. In both mutants, GPI-anchor attachment is normal and in ret1-1 cells, the GPI-anchors are remodeled with ceramide to the same extent as in wild-type cells.

GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles.

Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur.

Toxicity of myristic acid analogs toward African trypanosomes.

New drugs are needed for treatment of diseases caused by African trypanosomes. One possible target for chemotherapy is the biosynthesis of the glycosyl phosphatidyl-inositol (GPI) of this parasite's variant surface glycoprotein (VSG). Unlike mammalian GPIs, the diacylglycerol moiety of the VSG anchor contains only myristate (tetradecanoate), added in unique remodeling reactions. We previously found that 11-oxatetradecanoic acid [i.e., 10-(propoxy)decanoic acid] is selectively toxic to trypanosomes. We have now assayed 244 different fatty acid analogs, most with chain lengths comparable to that of myristate, for trypanocidal effects. In these assays we surveyed the effects on toxicity of systematic alterations in the analogs' steric, conformational, and hydrophobic properties. We also used three 3H-labeled oxatetradecanoic acids to explore the mechanism of analog action. Their incorporation into VSG correlated roughly with toxicity, although they also were incorporated into phospholipids and other proteins. Myristate analogs are useful for studying the mechanism of GPI myristolyation, and they are candidates for antitrypanosomal chemotherapy.

The fatty acids in unremodelled trypanosome glycosyl-phosphatidylinositols.

Glycolipid A, the precursor of the glycosyl-phosphatidylinositol (GPI) anchor of the trypanosome variant surface glycoprotein, is constructed in two phases. First, the glycan is assembled on phosphatidylinositol (PI), yielding a glycolipid termed A'. Second, glycolipid A' undergoes fatty acid remodelling, by deacylation and reacylation, to become the dimyristoyl species glycolipid A. In this paper, we examine the fatty acid content of glycolipid A' and its cellular progenitors. A' contains exclusively stearate at the sn-1 position and a complex mixture of fatty acids (including 18:0, 18:1, 18:2, 20:4 and 22:6) at sn-2. Presumably these fatty acids derive from stearate-containing PI species which initially enter the biosynthetic pathway. We compared the diacylglycerol species from glycolipid A' with those from phosphatidylinositol to determine whether a subset of stearate-containing PIs is utilized for GPI biosynthesis. We found that the spectrum of stearate-containing diacylglycerols in PI is similar to that in A', although the proportions of each compound differ. Total PI in general was highly enriched in stearate-containing species. Differences in composition between glycosylated PI and total cellular PI may be due to the substrate specificity of the sugar transferase which initiates the GPI biosynthetic pathway. Alternatively, the species of PI present at the endoplasmic reticulum site of GPI biosynthesis may differ from those in total PI.

Trypanosome metabolism of myristate, the fatty acid required for the variant surface glycoprotein membrane anchor.

The trypanosome variant surface glycoprotein (VSG) is anchored to the outer leaflet of the parasite plasma membrane by a glycosyl phosphatidylinositol (GPI). The VSG anchor is unique among GPIs in containing exclusively dimyristoylglycerol as its lipid moiety. Myristate is incorporated into the anchor precursor by sequential deacylation and specific reacylation with myristate. Although myristate is required for the VSG anchor, trypanosomes cannot synthesize this fatty acid and must import their entire supply from the host bloodstream, where it exists in low abundance. Chemical analysis of these parasites reveals that most of their myristate is in VSG protein, with no major lipid storage form. Unexpectedly, when these cells are radiolabeled with [3H]myristate in culture, most of the label is incorporated into phospholipids, with little into VSG. This apparent contradiction is explained by the fact that trypanosomes in culture medium elongate much of the [3H]myristate into palmitate and stearate, probably because the medium (with only 5% serum) contains limiting amounts of these fatty acids. In contrast, trypanosomes radiolabeled in whole blood (with higher concentrations of palmitate and stearate) do not modify most of the [3H]myristate, and instead utilize the major portion of it for GPI synthesis. Our studies suggest that bloodstream trypanosomes have evolved highly efficient means of directing myristate into the GPI biosynthetic pathway.

An analog of myristic acid with selective toxicity for African trypanosomes.

Trypanosoma brucei, the protozoan parasite responsible for African sleeping sickness, evades the host immune response through the process of antigenic variation. The variant antigen, known as the variant surface glycoprotein (VSG), is anchored to the cell surface by a glycosyl phosphatidylinositol (GPI) structure that contains myristate (n-tetradecanoate) as its only fatty acid component. The utilization of heteroatom-containing analogs of myristate was studied both in a cell-free system and in vivo. Results indicated that the specificity of fatty acid incorporation depends on chain length rather than on hydrophobicity. One analog, 10-(propoxy)decanoic acid, was highly toxic to trypanosomes in culture although it is nontoxic to mammalian cells.

Fatty acid remodeling: a novel reaction sequence in the biosynthesis of trypanosome glycosyl phosphatidylinositol membrane anchors.

The trypanosome variant surface glycoprotein (VSG) is anchored to the plasma membrane via a glycosyl phosphatidylinositol (GPI). The GPI is synthesized as a precursor, glycolipid A, that is subsequently linked to the VSG polypeptide. The VSG anchor is unusual, compared with anchors in other cell types, in that its fatty acid moieties are exclusively myristic acid. To investigate the mechanism for myristate specificity we used a cell-free system for GPI biosynthesis. One product of this system, glycolipid A', is indistinguishable from glycolipid A except that its fatty acids are more hydrophobic than myristate. Glycolipid A' is converted to glycolipid A through highly specific fatty acid remodeling reactions involving deacylation and subsequent reacylation with myristate. Therefore, myristoylation occurs in the final phase of trypanosome GPI biosynthesis.