Cara L. Griffith

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.

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.

UDP-glucose Dehydrogenase Plays Multiple Roles in the Biology of the Pathogenic Fungus Cryptococcus neoformans

Cryptococcus neoformans is a pathogenic fungus surrounded by an elaborate polysaccharide capsule that is strictly required for its virulence in humans and other mammals. Nearly half of the sugar residues in the capsule are derived from UDP-glucuronic acid or its metabolites. To examine the role of these nucleotide sugars in C. neoformans, the gene encoding UDP-glucose dehydrogenase was disrupted. Mass spectrometry analysis of nucleotide sugar pools showed that the resulting mutant lacked both UDP-glucuronic acid and its downstream product, UDP-xylose, thus confirming the effect of the knockout and indicating that an alternate pathway for UDP-glucuronic acid production was not used. The mutant was dramatically affected by the lack of specific sugar donors, demonstrating altered cell integrity, temperature sensitivity, lack of growth in an animal model of cryptococcosis, and morphological defects. Additionally, the polysaccharide capsule could not be detected on the mutant cells, although the possibility remains that abbreviated forms of capsule components are made, possibly without proper surface display. The capsule defect is largely independent of the other observed changes, as cells that are acapsular because of mutations in other genes show lack of virulence but do not exhibit alterations in cell integrity, temperature sensitivity, or cellular morphology. All of the observed alterations were reversed by correction of the gene disruption.

An efficiently regulated promoter system for Cryptococcus neoformans utilizing the CTR4 promoter

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for serious meningitis. Although many useful molecular tools have been developed for its study, there are currently few inducible promoters available for general use. To address this need, we have constructed expression plasmids incorporating upstream elements of the C. neoformans copper transporter gene CTR4, and tested them in C. neoformans serotypes A and D. In response to copper deprivation, these plasmids mediate high‐level expression of a reporter protein. This expression can be completely repressed using physiologically low concentrations of copper. Notably, this new family of copper‐sensing promoters demonstrates excellent expression in serotype A, contrasting with other available promoters. These plasmids therefore offer efficient and regulated expression for both serotypes A and D, and should be valuable tools for the C. neoformans research community. Copyright

The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis

ABSTRACT Cryptococcus neoformans is a fungal pathogen that is responsible for life-threatening disease, particularly in the context of compromised immunity. This organism makes extensive use of mannose in constructing its cell wall, glycoproteins, and glycolipids. Mannose also comprises up to two-thirds of the main cryptococcal virulence factor, a polysaccharide capsule that surrounds the cell. The glycosyltransfer reactions that generate cellular carbohydrate structures usually require activated donors such as nucleotide sugars. GDP-mannose, the mannose donor, is produced in the cytosol by the sequential actions of phosphomannose isomerase, phosphomannomutase, and GDP-mannose pyrophosphorylase. However, most mannose-containing glycoconjugates are synthesized within intracellular organelles. This topological separation necessitates a specific transport mechanism to move this key precursor across biological membranes to the appropriate site for biosynthetic reactions. We have discovered two GDP-mannose transporters in C. neoformans, in contrast to the single such protein reported previously for other fungi. Biochemical studies of each protein expressed in Saccharomyces cerevisiae show that both are functional, with similar kinetics and substrate specificities. Microarray experiments indicate that the two proteins Gmt1 and Gmt2 are transcribed with distinct patterns of expression in response to variations in growth conditions. Additionally, deletion of the GMT1 gene yields cells with small capsules and a defect in capsule induction, while deletion of GMT2 does not alter the capsule. We suggest that C. neoformans produces two GDP-mannose transporters to satisfy its enormous need for mannose utilization in glycan synthesis. Furthermore, we propose that the two proteins have distinct biological roles. This is supported by the different expression patterns of GMT1 and GMT2 in response to environmental stimuli and the dissimilar phenotypes that result when each gene is deleted.

Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans

UDP-glucose dehydrogenase catalyses the conversion of UDP-glucose into UDP-GlcA, a critical precursor for glycan synthesis across evolution. We have cloned the gene encoding this important enzyme from the opportunistic pathogen Cryptococcus neoformans. In this fungus, UDP-GlcA is required for the synthesis of capsule polysaccharides, which in turn are essential for virulence. The gene was expressed in Escherichia coli and the 51.3-kDa recombinant protein from wild-type and five mutants was purified for analysis. The cryptococcal enzyme is strongly inhibited by UDP-xylose and NADH, has highest activity at pH 7.5 and demonstrates Km (app) values of 0.1 and 1.5 mM for NAD+ and UDP-glucose respectively. Its activity was significantly decreased by mutations in the putative sites of NAD+ and UDP-glucose binding. Unlike previously reported eukaryotic UDP-glucose dehydrogenases, which are hexamers, the cryptococcal enzyme is a dimer.

Delaying S-phase progression rescues cells from heat-induced S-phase hypertoxicity.

The mechanism by which a cell protects itself from the lethal effects of heat shock and other stress‐inducing agents is the subject of much research. We have investigated the relationship between heat‐induced damage to DNA replication machinery and the lethal effects of heat shock, in S‐phase cells, which are more sensitive to heat shock than either G1 or G2. We found that maintaining cells in aphidicolin, which prevents the passage of cells through S‐phase, can rescue S‐phase HeLa cells from the lethal effects of heat shock. When S‐phase, HeLa cells were held for 5–6 h in 3 μM aphidicolin the measured clonogenic survival was similar to that for exponentially growing cells. It is known, that heat shock induces denaturation or unfolding of proteins, rendering them less soluble and more likely to co‐isolate with the nuclear matrix. Here, we show that enhanced binding of proteins involved in DNA replication (PCNA, RPA, and cyclin A), with the nuclear matrix, correlates with lethality of S‐phase cells following heat shock under four different experimental conditions. Specifically, the amounts of RPA, PCNA, and cyclin A associated with the nuclear matrix when cells resumed progression through S‐phase correlated with cell killing. Heat‐induced enhanced binding of nuclear proteins involved with other aspects of DNA metabolism, (Mrell, PDI), do not show this correlation. These results support the hypothesis that heat‐induced changes in the binding of proteins associated with DNA replication factories are the potentially lethal lesions, which become fixed to lethal lesions by S‐phase progression but are repairable if S‐phase progression is delayed.

Evidence that protein disulfide isomerase (PDI) is involved in DNA-nuclear matrix anchoring.

DNA–nuclear matrix (NM) anchoring plays a critical role in the organization of DNA within the nucleus and in functional access to DNA for transcription, replication, and DNA repair. The cellular response to oxidative stress involves both gene expression and DNA repair. We, therefore, determined if changes in the oxidative–reductive environment can affect DNA–NM anchoring. The present study used two approaches to study the effect of the reducing agent DTT on DNA–NM anchoring. First, the relative stringency of the DNA–NM attachment was determined by measuring the ability of NM attached DNA loops to undergo supercoiling changes. Second, the effects of DTT on the association of nuclear proteins with DNA were determined by cisplatin crosslinking. When nucleoids (nuclear matrices with attached DNA loops) were prepared from HeLa cells with 1 mM dithiothreitol (DTT), supercoiled DNA loops unwound more efficiently compared with control in the presence of increasing propidium iodide (PI) concentrations. In addition, the rewinding of DNA supercoils in nucleoids treated with DTT was inhibited. Both effects on DNA supercoiling ability were reversed by diamide suggesting that they are dependent on the oxidation state of the protein thiols. When DTT treated nucleoids were isolated from γ‐irradiated cells, the inhibition of DNA supercoil rewinding was equal to the sum of the inhibition due to DTT and γ‐rays alone. Nucleoids isolated from heat‐shocked cells with DTT, showed no inhibition of DNA rewinding, except a small inhibition at high PI concentrations. Nuclear DNA in DTT‐treated nuclei was digested faster by DNase I than in untreated nuclei. These results suggest that DTT is altering DNA–NM anchoring by affecting the protein component(s) of the anchoring complex. Extracting NM with increasing concentrations of DTT did not solubilize any protein to a significant extent until measurable NM disintegration occurred. Therefore, we determined if 1 mM DTT affected the ability of 1 mM cisplatin to crosslink proteins to DNA. Isolated nuclei were treated with 1 mM DTT for 30 min or left untreated prior to crosslinking with 1 mM cisplatin for 2 h at 4°C. The ability of capsulation to crosslink DNA to proteins per se, did not appear to be affected by 1 mM DTT because relative amounts of at least four proteins, 69, 60, 40, and 35 kDa, were crosslinked to DNA to the same extent in DTT‐treated and untreated nuclei. However, protein disulfide isomerase (PDI) crosslinked to DNA in untreated nuclei, but did not crosslink DNA in nuclei that were treated with 1 mM DTT; 1 mM DTT did not affect the intranuclear localization of PDI. Thus, DTT appears to alter the conformation of PDI, as suggested by the DTT‐induced change in DNA association, but not its NM association. These results also imply that DNA–NM anchoring involves the redox state of protein sulfhydryl groups. J. Cell. Biochem. 85: 689–702, 2002.

Cryptococcus neoformans Dual GDP-Mannose Transporters and Their Role in Biology and Virulence

ABSTRACT Cryptococcus neoformans is an opportunistic yeast responsible for lethal meningoencephalitis in humans. This pathogen elaborates a polysaccharide capsule, which is its major virulence factor. Mannose constitutes over one-half of the capsule mass and is also extensively utilized in cell wall synthesis and in glycosylation of proteins and lipids. The activated mannose donor for most biosynthetic reactions, GDP-mannose, is made in the cytosol, although it is primarily consumed in secretory organelles. This compartmentalization necessitates specific transmembrane transporters to make the donor available for glycan synthesis. We previously identified two cryptococcal GDP-mannose transporters, Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar kinetics and substrate specificities in vitro. We have now examined these proteins in vivo and demonstrate that cells lacking Gmt1 show significant phenotypic differences from those lacking Gmt2 in terms of growth, colony morphology, protein glycosylation, and capsule phenotypes. Some of these observations may be explained by differential expression of the two genes, but others suggest that the two proteins play overlapping but nonidentical roles in cryptococcal biology. Furthermore, gmt1 gmt2 double mutant cells, which are unexpectedly viable, exhibit severe defects in capsule synthesis and protein glycosylation and are avirulent in mouse models of cryptococcosis.