Tamara L. Doering

A novel pathway for glycan assembly: Biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein

The trypanosome variant surface glycoprotein (VSG), like many other eukaryotic cell surface proteins, is anchored to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) moiety. This glycolipid is assembled first as a precursor (glycolipid A) that is then covalently attached to the newly synthesized polypeptide. We have developed a trypanosome cell-free system capable of performing all of the steps in the biosynthesis of the glycan portion of glycolipid A. Using [3H]sugar nucleotides as substrates, several biosynthetic intermediates have been identified. From structural analyses of these intermediates, we propose a pathway for GPI biosynthesis. Based on comparisons between the VSG GPI anchor and similar structures in other cells, we believe that this same pathway will apply to the GPI anchors, and the related insulin-mediator compound, of higher eukaryotes.

A Yeast under Cover: the Capsule of Cryptococcus neoformans

Few fungi are pathogenic to humans. Of these, Cryptococcus neoformans has emerged as an important cause of mortality in immunocompromised patients, especially those with AIDS. As a result, extensive research efforts have addressed the pathogenesis and virulence of this organism. C. neoformans is a basidiomycetous fungus that is ubiquitous in the environment, where it is found in soil, in association with certain trees, and in bird guano (16). Because of its ubiquity, it has been suggested that most people are exposed to C. neoformans early in life (41). The fungus is heterothallic, with mating types MATa and MAT. Asexual reproduction takes place either by budding or, in the case of MAT cells, by haploid fruiting in response to nutrient deprivation or exposure to the mating pheromone a factor (106). Sexual reproduction occurs when cells of opposite mating types come together to form a heterokaryon that ultimately leads to the production of basidia and basidiospores (64). Desiccated cells and the spores formed by haploid fruiting or sexual reproduction have all been suggested to serve as infective particles, which must be less than 2 m in diameter to penetrate the lung parenchyma (44, 86). Infection occurs when the fungal particles are inhaled and enter the alveolar space. In most immunocompetent individuals, this infection is either cleared or remains dormant until an immune imbalance leads to further development. In the setting of compromised immune function, however, the fungus disseminates, with particular tropism for the central nervous system. In severe cases, cryptococcal infection progresses to a meningoencephalitis that is fatal if left untreated. C. neoformans virulence is mediated predominantly by a polysaccharide capsule that surrounds its cell wall and has multiple effects on the host immune system. This structure provides a physical barrier that interferes with normal phagocytosis and clearance by the immune system. Capsule components inhibit the production of proinflammatory cytokines, deplete complement components (by efficiently binding them), and reduce leukocyte migration to sites of inflammation (11). The capsule also constitutes the major diagnostic feature of cryptococcosis, because its components can be detected in the bloodstream and it can be visualized with light microscopy by using India ink staining. The capsule excludes the ink particles and forms characteristic halos (Fig. 1A) whose diameters are often several times that of the cell. The elaborate structure of the capsule may also be appreciated by electron microscopy (Fig. 1B and C). Given the importance of the capsule in cryptococcal disease, tremendous effort has been applied in recent years to understanding its biology. This review focuses on the resulting advances in our understanding of the structure and synthesis of the capsular components, the incorporation of these components into the existing capsular network, the association between the capsule and the cell wall, and the regulation of capsule growth.

Cell wall α‐1,3‐glucan is required to anchor the Cryptococcus neoformans capsule

Cryptococcus neoformans is an opportunistic pathogen responsible for serious disease in humans. Critical for virulence of this fungus is an elaborate polysaccharide capsule, which impedes the host immune response. We found that association of the capsule with the cell requires a specific component of the cell wall, α‐1,3‐glucan. Post‐transcriptional inhibition of α‐1,3‐glucan synthase expression, using double‐stranded RNA interference, yields cells that are unable to assemble a capsule although they generate its polysaccharide components. The resulting cryptococci are slow‐growing and acapsular. This finding demonstrates a novel mode of polysaccharide attachment and an important application of RNA interference in fungi. The elimination of the capsule by reducing the expression of a single gene suggests a potential avenue for antifungal chemotherapy.

How Sweet it is! Cell Wall Biogenesis and Polysaccharide Capsule Formation in Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus responsible for severe opportunistic infections. The most prominent feature of this yeast is its elaborate polysaccharide capsule, a complex structure that is required for virulence. The capsule is intimately associated with the cell wall, which underlies the capsule and offers the organism strength and flexibility in potentially hostile environments. Both structures are primarily composed of polysaccharides, offering a glimpse of the tremendous variation inherent in natural carbohydrate structures and their multiple biological functions. The steps in cell wall and capsule biosynthesis and assembly pose fascinating questions of metabolism, enzymology, cell biology, and regulation; the answers have potential application to treatment of a deadly infection. This article reviews current knowledge of cryptococcal cell wall and capsule biosynthesis and outstanding questions for the future.

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.

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.

Eukaryotic UDP-Galactopyranose Mutase (GLF Gene) in Microbial and Metazoal Pathogens

ABSTRACT Galactofuranose (Galf) is a novel sugar absent in mammals but present in a variety of pathogenic microbes, often within glycoconjugates that play critical roles in cell surface formation and the infectious cycle. In prokaryotes, Galf is synthesized as the nucleotide sugar UDP-Galf by UDP-galactopyranose mutase (UGM) (gene GLF). Here we used a combinatorial bioinformatics screen to identify a family of candidate eukaryotic GLFs that had previously escaped detection. GLFs from three pathogens, two protozoa (Leishmania major and Trypanosoma cruzi) and one fungus (Cryptococcus neoformans), had UGM activity when expressed in Escherichia coli and assayed in vivo and/or in vitro. Eukaryotic GLFs are closely related to each other but distantly related to prokaryotic GLFs, showing limited conservation of core residues around the substrate-binding site and flavin adenine dinucleotide binding domain. Several eukaryotes not previously investigated for Galf synthesis also showed strong GLF homologs with conservation of key residues. These included other fungi, the alga Chlamydomonas and the algal phleovirus Feldmannia irregularis, parasitic nematodes (Brugia, Onchocerca, and Strongyloides) and Caenorhabditis elegans, and the urochordates Halocynthia and Cionia. The C. elegans open reading frame was shown to encode UGM activity. The GLF phylogenetic distribution suggests that Galf synthesis may occur more broadly in eukaryotes than previously supposed. Overall, GLF/Galf synthesis in eukaryotes appears to occur with a disjunct distribution and often in pathogenic species, similar to what is seen in prokaryotes. Thus, UGM inhibition may provide an attractive drug target in those eukaryotes where Galf plays critical roles in cellular viability and virulence.

Loss of cell wall alpha(1‐3) glucan affects Cryptococcus neoformans from ultrastructure to virulence

Yeast cell walls are critical for maintaining cell integrity, particularly in the face of challenges such as growth in mammalian hosts. The pathogenic fungus Cryptococcus neoformans additionally anchors its polysaccharide capsule to the cell surface via α(1‐3) glucan in the wall. Cryptococcal cells disrupted in their alpha glucan synthase gene were sensitive to stresses, including temperature, and showed difficulty dividing. These cells lacked surface capsule, although they continued to shed capsule material into the environment. Electron microscopy showed that the alpha glucan that is usually localized to the outer portion of the cell wall was absent, the outer region of the wall was highly disorganized, and the inner region was hypertrophic. Analysis of cell wall composition demonstrated complete loss of alpha glucan accompanied by a compensatory increase in chitin/chitosan and a redistribution of beta glucan between cell wall fractions. The mutants were unable to grow in a mouse model of infection, but caused death in nematodes. These studies integrate morphological and biochemical investigations of the role of alpha glucan in the cryptococcal cell wall.

Tissue specific expression of p422 protein, a putative lipid carrier, in mouse adipocytes

The differentiation of 3T3-L1 preadipocytes leads to the expression of a new protein, p422, and its mRNA. This protein has 70% and 20-30% amino acid sequence homology to myelin P2 and the fatty acid binding proteins of liver and intestine, respectively. Investigation of the distribution in mouse tissues of p422 protein by immunoblotting and of p422 mRNA by cDNA hybridization indicates that they are expressed only in adipose tissue. Liver and intestinal fatty acid binding protein mRNAs were not detectable in mouse adipose tissue or in 3T3-L1 adipocytes. It is suggested that p422 functions as an adipocyte fatty acid binding protein.

Cryptococcus neoformans and Cryptococcus gattii, the Etiologic Agents of Cryptococcosis

Cryptococcus neoformans and Cryptococcus gattii are the two etiologic agents of cryptococcosis. They belong to the phylum Basidiomycota and can be readily distinguished from other pathogenic yeasts such as Candida by the presence of a polysaccharide capsule, formation of melanin, and urease activity, which all function as virulence determinants. Infection proceeds via inhalation and subsequent dissemination to the central nervous system to cause meningoencephalitis. The most common risk for cryptococcosis caused by C. neoformans is AIDS, whereas infections caused by C. gattii are more often reported in immunocompetent patients with undefined risk than in the immunocompromised. There have been many chapters, reviews, and books written on C. neoformans. The topics we focus on in this article include species description, pathogenesis, life cycle, capsule, and stress response, which serve to highlight the specializations in virulence that have occurred in this unique encapsulated melanin-forming yeast that causes global deaths estimated at more than 600,000 annually.