Characterization of glycoinositolphosphoryl ceramide structure mutant strains of Cryptococcus neoformans

In fungi, glycoinositolphosphoryl ceramide (GIPC) biosynthetic pathway produces essential molecules for growth, viability, and virulence. In previous studies, we demonstrated that the opportunistic fungus Cryptococcus neoformans synthesizes a complex family of xylose-(Xyl) branched GIPCs, all of which have not been previously reported in fungi. As an effort to understand the biosynthesis of these sphingolipids, we have now characterized the structures of GIPCs from C. neoformans wild-type (KN99alpha) and mutant strains that lack UDP-Xyl, by disruption of either UDP-glucose dehydrogenase (NE321) or UDP-glucuronic acid decarboxylase (NE178). The structures of GIPCs were determined by a combination of nuclear magnetic resonance (NMR) spectroscopy, tandem mass spectrometry (MS), and gas chromatography-MS. The main and largest GIPC from wild-type strain was identified as an alpha-Manp(1 --> 6)alpha-Manp(1 --> 3)alpha-Manp[beta-Xylp(1 --> 2)]alpha-Manp(1 --> 4)beta-Galp(1 --> 6)alpha-Manp(1 --> 2) Ins-1-P-Ceramide, whereas the most abundant GIPC from both mutant strains was found to be an alpha-Manp(1 --> 3)alpha-Manp(1 --> 4)beta-Galp(1 --> 6)alpha-Manp(1 --> 2)Ins-1-P-Ceramide. The ceramide moieties of C. neoformans wild-type and mutant strains were composed of a C(18) phytosphingosine, which was N-acylated with 2-hydroxy tetra-, or hexacosanoic acid, and 2,3-dihydroxy-tetracosanoic acid. Our structural analysis results indicate that the C. neoformans mutant strains are unable to complete the assembly of the GIPC-oligosaccharide moiety due the absence of Xyl side chain.     

Genetic and phenotypic characterization of capsule mutants of Cryptococcus neoformans

Stable mutants with reduced capacity to produce capsules were isolated from suspensions of Cryptococcus neoformans after treatment of the wild type with a mutagen. The mutants could be assigned one of two phenotypes, hypocapsular or acapsular. Hypocapsular mutants were immunochemically and physicochemically indistinguishable from the wild type, whereas acapsular mutants lacked a major capsular antigen and a negatively charged exterior. In genetic analysis, the mutant trait segregated as a Mendelian gene (1:1) when random basidiospores from an outcross were studied, and analysis of products of single meiotic events from outcrossed mutants was likewise consistent with meiotic segregation. Two-factor crosses yielded the expected four classes of progeny, with recombinants equal to parentals. We concluded that chromosomal genes are responsible for synthesis of the cryptococcal capsule and that random basidiospore analysis represents a useful technique for genetic analysis in this species.     

Cryptococcus neoformans capsule biosynthesis and regulation

The capsule is certainly the most prominent virulence factor in Cryptococcus neoformans: acapsular strains are avirulent, and capsular polysaccharides have a deleterious effect on the immune system. Until very recently, very few genes involved in capsule biosynthesis had been identified - and this despite the existence of a detailed body of work concerning the capsule's composition, structure and their regulation by environmental factors. The tremendous development of experimental tools and techniques suited to the study of C. neoformans biology together with the sequencing of three complete genomes have, over the last three years, enabled the identification of a number of proteins which participate directly in biosynthesis of the capsule or which regulate its size. Even though this knowledge is still preliminary, it gives us a clearer picture of the various events needed for biosynthesis of this fascinating structure.     

Isolation and characterization of the Cryptococcus neoformans MATa pheromone gene

Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATalpha. The mating pathway of this fungus has a number of conserved genes, including a MATalpha-specific pheromone (MFalpha1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATalpha cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfalpha1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFalpha1 gene, which is found in MATalpha strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans.     

Molecular architecture of the Cryptococcus neoformans capsule

Many microbes are surrounded by phagocytosis-inhibiting capsules. We took advantage of the large size of the polysaccharide capsule of the pathogenic yeast Cryptococcus neoformans to examine capsular architecture and the relationship between molecular architecture and the interaction of the capsule with potentially opsonic serum proteins. Our experimental design used complementary approaches in which (i) assessment of permeability to macromolecules of different Stokes radii; (ii) determination of the binding of Fab fragments of anticapsular antibodies as a measure of matrix density; (iii) capsular deconstruction by treatment with dimethyl sulphoxide; and (iv) evaluation of capsule plasticity, were used to probe the molecular structure of the capsule. The results showed that the capsule is a matrix with a variable porosity that increases with distance from the cell wall. A high density of the matrix at the capsule interior prevents penetration of large macromolecules to sites near the cell wall. In contrast, the capsular edge that is the interface with phagocytes presents capsular polysaccharide in a very low density that exhibits considerable plasticity and permeability to macromolecules. Notably, the capsule of yeast cells harvested from infected tissue showed a greater matrix density than yeast cells grown in vitro under capsule induction conditions.     

Primary Isolation Medium for Cryptococcus neoformans

The isolation and identification of Cryptococcus neoformans is improved by use of a potato dextrose medium containing urea-antibiotic supplements and a pH of 3.5.     

The volume and hydration of the Cryptococcus neoformans polysaccharide capsule

We present a new method to measure capsule size in the human fungal pathogen Cryptococcus neoformans that avoids the limitations and biases inherent in India ink measurements. The method is based on the use of gamma-radiation, which efficiently releases the capsule from the cell. By comparing the volume of irradiated and non-irradiated cells, one can accurately estimate the relative size of the capsule per cell. This method was also used to obtain an estimate of the capsule weight and water content. The C. neoformans capsule is a highly hydrated structure in all the conditions measured. However, after capsule enlargement, the amount of capsular polysaccharide significantly increases, suggesting a that capsule growth has a high energy cost for the cell.     

Recombinational mapping of capsule mutations in Cryptococcus neoformans.

Seven capsule-negative mutants of Cryptococcus neoformans were isolated. All mutations were linked (maximum map distance, 38 U); two mutations were found to be allelic.     

Occurrence of diploid strains of Cryptococcus neoformans.

A mating between niacin and pantothenate auxotrophs of Cryptococcus neoformans gave a few prototrophic progeny that were self-fertile. These were uninuclear but contained twice as much DNA as the parental strains. Segregation of nutritional markers was observed upon sporulation. We conclude that these self-fertile strains are diploids.     

Characterization of pathogenic constituents of Cryptococcus neoformans strains

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 X 10(5) or 1 X 10(7) viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; a cell surface structure for responsible for resistance to intra- or extracellular killing and induction of the granulomatous lesion; a growth rate suitable for interacting with phagocytic elimination.     

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.     

Molecular characterization and evaluation of virulence factors of Cryptococcus laurentii and Cryptococcus neoformans strains isolated from external hospital areas

Cryptococcosis is a common opportunistic fungal infection that is mainly caused by the species Cryptococcus neoformans and Cryptococcus gattii, but there have recently been several reports of infection by non-neoformans Cryptococcus species. The aims of this study were to genetically characterize Cryptococcus spp. isolated from external hospital areas in Minas Gerais State, Brazil, and to evaluate their pathogenic potential, analyzing their phospholipase and melanin production and the capacity for capsule enlargement. Seventy-three different samples were collected: 62 from bird droppings and 11 from tree detritus. C.?neoformans alone was isolated from 43.8% of the samples, Cryptococcus laurentii alone from 23.3% and both fungi were found together in 10.9%. C. laurentii was exclusively isolated from 45% (5/11) of the tree samples (Anacardium occidentale, Guazuma ulmifolia, Mangifera indica and Ficus benjamina). Among the 51 C. neoformans isolates, 47 were classified as type VNI and four as type VNII. All of the C. neoformans isolates were of MATα type. Among the 21 isolates of C. laurentii genotyped using the URA5-RFLP technique, 16 amplified a 1.6kb amplicon which produced a specific restriction profile in 15 isolates. In C.?neoformans, 76.4% of the isolates were capable of capsule enlargement in the induction medium and 92.1% were phospholipase producers. In C. laurentii, 7.4% of the isolates were capable of capsule enlargement and 85.1% were phospholipase producers. Characterization of the genotypes and the pathogenic potential of the Cryptococcus spp. isolates studied may contribute towards better understanding of the epidemiology of cryptococcosis and the ecology of agents causing this disease in our region.     

Equatorial ring-like channels in the Cryptococcus neoformans polysaccharide capsule

Under certain conditions, India ink particles can penetrate the capsule of the opportunistic pathogen Cryptococcus neoformans. India ink penetration gave two distinct patterns, one as a ring in the middle of the capsule, and another as a double spot located at opposite poles of the cells. These spots were perpendicularly orientated to the bud. This pattern suggests the existence of a localized structure deep in the capsule that can accumulate large insoluble particles. Although the mechanisms responsible for the assemblage and maintenance of ring-like channels are not understood, their existence deep within the capsule implies a new level of complexity for this enigmatic structure.     

Phenotype characterization of environmental Cryptococcus neoformans isolates

Cryptococcosis is caused by the three varieties of C. neoformans with physiological and virulence differences, some of which have been studied to determine biological aspects of this microorganism. The phenotypical aspects of environmental isolates from varieties grubii and gattii were evaluated to establish differences associated with their life cycle and virulence. To this end, 28 and 31 strains of C. neoformans serotypes A (var. grubii) and C (var. gattii) were studied. The microscopic and macroscopic morphology on Sabouraud agar and soils, growth rate at 37 degrees C, production of 22 extracellular enzymes, haploid fructification, mating type, killer toxin sensitivity patterns and virulence in BALB/c mice were evaluated. No differences were observed between the two varieties regarding microscopic and macroscopic morphology or growth at 37 degrees C (p > 0.05). However, a decrease in the cellular and capsular sizes of yeast in soil, as compared to Sabouraud, was observed (p < 0.05). Additionally, higher enzimatic activity of proteases, phospholipases, phenoloxidase and beta-glucosidase was observed in var. grubii isolates as compared to var. gattii (p < 0.05). In both varieties, structures related with haploid fruitification were observed and all isolates were mating type alpha. Killer toxin sensitivity patterns of the isolates of var. grubii were I and II; in contrast, in var. gattii, seven different patterns were found: I, V, IX-XIII. In the animal model we found that 12 of 22 (54.5%) isolates of var. grubii caused the death of the mice during the observation period, while none of the 14 var. gattii isolates caused it. The decrease in capsular and cellular sizes of the yeast in soil and the frequency of mating type alpha with structures related to haploid fructification suggest an important mechanism of production of infectious particles in nature. Additionally, greater enzimatic activity of var. grubii can be associated with the virulence in the animal model.     

Preparation and characterization of Cryptococcus neoformans synchronous culture

We have developed a method for preparation of synchronous culture in Cryptococcus neoformans. The method is based on age fractionation of exponentially growing asynchronous culture through differential sedimentation in 10-20% (w/v) lactose gradient. C. neoformans capsule thickness should be reduced to a minimum to ensure most accurate age fractionation, which is necessary to obtain a higher degree of synchrony. The C. neoformans synchronous culture system has revealed important characteristics with respect to cellular morphology, DNA content and cell volume distribution. The method can be used for further cell cycle studies.