Synthesis of cell envelope glycoproteins of Cryptococcus laurentii

Fungi of the genus Cryptococcus are encapsulated basidiomycetes that are ubiquitously found in the environment. These organisms infect both lower and higher animals. Human infections that are common in immune-compromised individuals have proven difficult to cure or even control with currently available antimycotics that are quite often toxic to the host. The virulence of Cryptococcus has been linked primarily to its polysaccharide capsule, but also to cell-bound glycoproteins. In this review, we show that Cryptococcus laurentii is an excellent model for studies of polysaccharide and glycoprotein synthesis in the more pathogenic relative C. neoformans. In particular, we will discuss the structure and biosynthesis of O-linked carbohydrates on cell envelope glycoproteins of C. laurentii. These O-linked structures are synthesized by at least four mannosyltransferases, two galactosyltransferases, and at least one xylosyltransferase that have been characterized. These glycosyltransferases have no known homologues in human tissues. Therefore, enzymes involved in the synthesis of cryptococcal glycoproteins, as well as related enzymes involved in capsule synthesis, are potential targets for the development of specific inhibitors for treatment of cryptococcal disease.     

Biosynthesis of Glycogen and Starch in Cryptococcus laurentii

Cells of Cryptococcus laurentii, when grown in liquid culture on 2% glucose close to neutral pH, showed glycogen granules throughout the cytoplasm. Glycogen levels of C. laurentii cells reached maximal levels just before onset of stationary phase. Concomitantly, a sharp rise in total and specific activity of glycogen synthetase was observed. Conversely, glycogen phosphorylase reached its highest specific activity approximately 3 hr after the glycogen peaked and remained high until most of the endogenous glycogen was utilized. Uridine diphosphoglucose pyrophosphorylase activity was always an order of magnitude higher than glycogen synthetase during log phase, but fell off rapidly after the cells reached stationary growth. Kinetic properties of the glycogen synthetase showed that the enzyme is always activated by glucose-6-phosphate, although the degree of activation by glucose-6-phosphate was found to be somewhat variable. The accelerated uptake of glucose commencing with the onset of stationary phase is explained by the rapid formation of extracellular acidic polysaccharide, which continues as long as there is glucose in the medium. In cells grown at pH 3.4, where no detectable extracellular acidic polysaccharide was formed, glucose uptake drastically declined when the cells reached stationary phase. These cells also contained glycogen-like granules in the cytoplasm. The evidence presented indicates that these granules are in fact glycogen, and that its structure does not resemble that of the starch excreted by cells grown at acidic pH.     

Phototoxicity of polyacetylenes to Cryptococcus laurentii

Isolates of Cryptococcus laurentii from leaves of Hawaiian species of Bidens were tested for photosensitivity to polyacetylenes from Bidens. Organisms from host plants with or without leaf acetylenes did not differ in their responses to the phototoxic compounds. Two methods were used to determine photosensitivity, a qualitative paper disc assay and a quantitative Titertek assay in which dose response curves for four polyacetylenes were prepared. The significance of pigmentation in phylloplane fungi is discussed.     

Biosynthesis of ubiquinone compounds with conjugated prenyl side chains

Enzymatic steps from two different biosynthetic pathways were combined in Escherichia coli, directing the synthesis of a new class of biomolecules--ubiquinones with prenyl side chains containing conjugated double bonds. This was achieved by the activity of a C(30) carotenoid desaturase, CrtN, from Staphylococcus aureus, which exhibited an inherent flexibility in substrate recognition compared to other carotenoid desaturases. By utilizing the known plasticity of E. coli's native ubiquinone biosynthesis pathway and the unusual activity of CrtN, modified ubiquinone structures with prenyl side chains containing conjugated double bonds were generated. The side chains of the new structures were confirmed to have different degrees of desaturation by mass spectrometry and nuclear magnetic resonance analysis. In vivo (14)C labeling and in vitro activity studies showed that CrtN desaturates octaprenyl diphosphates but not the ubiquinone compounds directly. Antioxidant properties of conjugated side chain ubiquinones were analyzed in an in vitro beta-carotene-linoleate model system and were found to be higher than the corresponding unmodified ubiquinones. These results demonstrate that by combining pathway steps from different branches of biosynthetic networks, classes of compounds not observed in nature can be synthesized and structural motifs that are functionally important can be combined or enhanced.     

Phylogenetic study of Cryptococcus laurentii mycocinogenic strains

Previous studies reported Cryptococcus laurentii strains to secrete mycocins of different inhibition range. These mycocinogenic strains were studied by molecular methods to verify their phylogenetic relationships. Based on the combined analysis of LSU and ITS sequence data, these strains were confirmed to belong to the species C. laurentii. Therefore, different strains of the same basidiomycetous yeast C. laurentii secrete a distinct mycocin. This trait has not been demonstrated for basidiomycetous yeasts before. Additionally, this study provides several DNA-barcodes (LSU, ITS and RPB1) and reports their variability for this species.     

An extracellular galactoxylomannan of acapsular Cryptococcus laurentii mutant

An extracellular galactoxylomannan (GalXM) composed of D-Gal (34.0%), D-Xyl (26.6%) and D-Man (31.0%), and a small amount of L-Ara (4.0%) and D-Glc (4.4%) has been isolated from culture medium of acapsulated mutant of Cryptococcus laurentii by ethanol precipitation and gel filtration. Phosphorylated polymer of Mw approximately 75,000 contained 90% carbohydrates, 3.9% phosphorus and 5.3% proteins. Results of chemical and spectroscopic studies showed a highly branched structure of GalXM with a 1,6-linked mannopyranosyl-galactopyranosyl backbone (approximately 44%) branched predominantly at C-2 and C-3 of mannosyl, and C-3 of galactosyl residues by side chains terminated mainly by xylosyl and mannosyl residues, and to a less extent by arabinosyl and glucosyl ones.     

Ultrastructure of the septal complex in hyphae of Cryptococcus laurentii.

Electron microscopy of hyphae produced by Cryptococcus laurentii revealed typical basidiomycetous dolipore septa between the cells. Parenthesomes were not observed.     

Repeated isolation of Cryptococcus laurentii from the oropharynx of an immunocompromized patient

Cryptococcus laurentii is one of the non-neoformans cryptococci that have rarely been isolated from humans. We report a case of repeated colonization of the oropharynx by Cr. laurentii in a patient with erythroleukaemia. The isolate was identified by phenotypic and genotypic tests and showed resistance to fluconazole. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biosynthesis of an unglycosylated envelope glycoprotein of Rous sarcoma virus in the presence of tunicamycin.

Cells stably infected with Rous sarcoma virus were treated with tunicamycin to prevent the glycosylation of the precursor (pr92gp) to the two viral envelope glycoproteins gp85 and gp35. Pretreatment of the cells for 4 h with the antibiotic resulted in a 90% reduction in [3H]mannose incorporation into total cellular glycoproteins, intracellular viral glycoproteins, and released virus particles. Protein synthesis and virus particle formation were not significantly affected by the treatment. A new polypeptide made in the presence of the drug was identified by immunoprecipitation of pulse-labeled cell lysates with monospecific anti-gp85 and anti-gp35 sera. This polypeptide, migrating on sodium dodecyl sulfate-polyacrylamide gels as a molecule of 62,000 daltons (pr62), contained no [3H]mannose, was labeled with [S35]methionine and [3H]arginine, could not be chased into the higher-molecular-weight glycosylated form, and contained the same [3H]arginine tryptic peptides as pr92gp. The unglycosylated pr62 was still detectable 2 h after the pulse labeling of the cells. The lack of glycosylation of pr62 did not seem to reduce its stability. No clear evidence for the incorporation of this molecule or its cleavage products into viral particles could be obtained. To code for an envelope polypeptide of 62,000 daltons, only about 1,500 nucleotides or 15% of the total coding capacity of the virus are needed.     

Synthesis and immunogenicity of polysaccharide-protein conjugate composed of galactoglucoxylomannan of Cryptococcus laurentii

Galactoglucoxylomannan (GalGXMan) antigen of Cryptococcus laurentii was conjugated to protein carrier by a simple one step reaction. The conjugate was immunogenic in rabbits and reinjection elicited booster response with significant increase of serum IgG (H+L) level. Induction of this Ig-isotype was confirmed in experiments with Protein A. Effectiveness of immune serum to inhibit the growth of C. laurentii was demonstrated. The results indicate that the prepared conjugate could be considered as effective immunogen with potential for incorporation in the vaccine.     

Salicylic Acid Enhances Biocontrol Efficacy of the Antagonist Cryptococcus laurentii in Apple Fruit

Biological control and induced resistance are two of the promising approaches to the control of postharvest diseases. This study was conducted to evaluate the efficacy of salicylic acid (SA) alone or in combination with an antagonistic yeast, Cryptococcus laurentii, in controlling the blue mold disease caused by Penicillium expansum on apple fruit wounds. SA alone significantly inhibited the spore germination of P. expansum in vitro when its concentration was increased to 1000 μg ml⁻¹, but it was not effective in controlling the disease in vivo. Simultaneous application of SA and C. laurentii to the wounds on the apple fruit surface showed that SA could improve the efficacy of C. laurentii against P. expansum in a concentration-dependent manner, being most effective at 10 μg ml⁻¹ but less effective at a higher or lower concentrations. Besides reducing the blue mold incidence in the local wound sites, the combination of C. laurentii with SA at 10 μg ml⁻¹ also had a synergistic effect on the induction of fruit resistance to the disease, which might be associated with a rapid increase in peroxidase, phenylalanineamonialyase and lipoxygenase activities. In addition, SA at 100 μg ml⁻¹ or above showed an adverse effect on the growth of C. laurentii in vitro and in vivo, whereas it had no effect when its concentration was decreased to 10 μg ml⁻¹ or lower. This suggested that SA could enhance the biological activity of C. laurentii in apple fruit by inducing resistance to pathogens based on the antagonistic activity of C. laurentii.