Regulation of mannitol biosynthesis and degradation by Cryptococcus neoformans.

Cryptococcus neoformans, an encapsulated yeast that is an opportunistic pathogen of AIDS patients, produced and secreted mannitol when incubated with an appropriate carbon source. Glucose, fructose, and mannose were good growth substrates and were converted to mannitol. Maltose and xylose were good growth substrates but were not converted to mannitol. Cells of C. neoformans that were grown on a non-mannitol-generating carbon source, such as peptone or xylose, were able to convert glucose to mannitol only after a prolonged lag period in the presence of glucose. It was concluded that the enzymes of the mannitol biosynthetic pathway were not constitutively expressed but were induced in response to glucose or to a glucose metabolite. Enzymes required to catabolize mannitol, however, were constitutively expressed. The production of mannitol was inhibited by anaerobiosis, by the respiratory poison rotenone, and by polyethylenesulfonate, a specific inhibitor of fungal NADP-dependent dehydrogenases. When cells were incubated with deuterated glucose, the deuterium content of the mannitol produced was much lower than that of the glucose precursor, indicating that the glucose was diluted by an intracellular pool of an intermediate. We had previously shown that C. neoformans contains a large intracellular pool of glucose 6-phosphate, and we now conclude that this pool of glucose 6-phosphate is metabolically active.     

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.     

Regulation of Cryptococcus neoformans capsule size is mediated at the polymer level

The fungal pathogen Cryptococcus neoformans regulates its polysaccharide capsule depending on environmental stimuli. To investigate whether capsule polymers change under different growth conditions, we analyzed shed capsules at physiological concentrations without physical perturbation. Our results indicate that regulation of capsule size is mediated at the level of individual polysaccharide molecules.     

舍曲林抗新生隐球菌的体外及动物实验研究

目的评估舍曲林抗新生隐球菌的效果。方法实验分为6组,分别为空白对照组、10 mg/mL氟康唑、10 mg/mL舍曲林、20 mg/mL舍曲林、10 mg/mL氟康唑联用10 mg/mL舍曲林以及10 mg/mL氟康唑联用20 mg/mL舍曲林组。通过体外药敏试验及BALBc小鼠新生隐球菌动物探讨各组间抗隐球菌效果的差异。结果体外药敏试验发现舍曲林可有效降低新生隐球菌菌落数,当与氟康唑联用时抑菌效果更显著。动物实验发现2种浓度的舍曲林都可明显降低感染小鼠实验早期脑、肺组织的新生隐球菌菌落数,但在实验后期,低浓度的舍曲林对感染小鼠脑组织失去抑菌作用。脑、肺组织中,舍曲林治疗对新生隐球菌的抗菌效果均不如氟康唑。舍曲林与氟康唑联合用药对新生隐球菌模型小鼠肺组织的抗菌效果强于单用舍曲林或氟康唑。结论舍曲林具有抗新生隐球菌的作用,当与氟康唑联合用药时可起到协同作用。     

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.     

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.     

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.     

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.     

Complex selection on intron size in Cryptococcus neoformans

We conducted a genome-wide analysis of the roles of mutation and selection in sculpting intron size in the fungal pathogen Cryptococcus neoformans. We find that deletion rate is positively associated with intron length and that insertion rate exhibits a weak negative association with intron length. These patterns suggest that long introns as well as extremely short introns in this unusually intron-rich fungal genome are in mutation-selection disequilibrium and that the proportion of constrained functional sequence in introns does not scale linearly with size. We find that untranslated region introns are longer than coding-region introns and that first introns are substantially longer than subsequent introns, suggesting heterogeneous distribution of constrained functional sequence and/or selective pressures on intron size within genes. In contrast to Drosophila, we find a positive correlation between d(N) and first intron or last intron length and a negative correlation between d(N) and internal intron length. This contrasting pattern may indicate that terminal introns and internal introns are differentially subject to hypothesized selection pressures modulating intron size and provides further evidence of widespread selective constraints on noncoding sequences.     

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.     

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.     

Particle size of airborn Cryptococcus neoformans in a tower

Nearly 10(6) cells of Cryptococcus neoformans were cultured per g of pigeon droppings in a vacant tower. The air in the tower contained an average of 45 viable cells of C. neoformans per 100 liters: 60% of the cells were less than 4.7 micron in diameter. It is estimated that a human exposed to this atmosphere for 1 h would have 41 cells of c. neoformans deposited in the lungs. Sweeping resulted in the aerosolization of large numbers of cells of C. neoformans from 4.7 to 11 micron in diameter, the number of cells less than 4.7 micron remained relatively constant. One minute after sweeping, 4.4% of viable airborne cells of C. neoformans were less than 1.1 micron in diameter. We believe that this is the first report of isolating such small cells of C. neoformans from a natural site.     

In vivo and in vitro studies with an atypical,rhinotropic isolate of Cryptococcus neoformans

An atypical isolate of Cryptococcus neoformans was investigated because of its consistent and reproducible production of gross nasal pathology following i.v. injection in Swiss albino mice. Dose response to graded concentrations ranging from 1×l0²–1×l0⁷ cells/mouse yielded an LD₅₀ of 1.4×10³ cells/mouse for the atypical rhinotropic strain H140 which was significantly less virulent (p<0.01) than our reference strain of Cryptococcus neoformans. There was no significant difference in mortality following the injection of in vitro vs. in vivo passed inoculum. As early as two weeks after inoculation, this strain produced gross nasal enlargement to approximately 2–3 × normal dimensions with granulomatous and ulcerated lesions. The LD₆₀ resulted in the greatest percentage of nasal involvement (85%). C. neoformans was demonstrated by culture and histopathology in the noses, brains, lungs, livers and kidneys. A temperature selection was indicated by findings of a lower temperature minimum for subcultures isolated from the noses relative to those isolated from the brain, and by the fact that the most densely populated organs following intraperitoneal injection were the testes. This route of inoculation resulted in cutaneous nasal involvement in a manner analogous to that following i.v. injection. The atypical isolate was unable to assimilate trehalose or raffinose but otherwise was entirely consistent with identification as C. neoformans and produced characteristic CNS and general organ system disease in addition to the rhinotropic cutaneous manifestations. The model characterized here in normal mice may be of value in studies of fungal dermotropism.     

Phenotypic heterogeneity in expression of epitopes in the Cryptococcus neoformans capsule

The opportunistic yeast Cryptococcus neoformans is surrounded by a polysaccharide capsule comprised primarily of glucuronoxylomannan (GXM). GXM is a key component of the antigenic character of the capsule. Expression of the epitope that allows for binding of mAbs that require O -acetylation of GXM for mAb recognition was greatly influenced by cell age, growth conditions and serotype. Yeast cells of serotype A grown in vitro under capsule induction conditions showed considerable cell-to-cell variability in binding of two O -acetyl-dependent mAbs, and such mAbs uniformly failed to bind to GXM that covers yeast buds. Expression of the O -acetyl-dependent epitope increased with cell age. In contrast, all serotype A cells harvested from brain tissue bound the same O -acetyl-dependent mAbs. The ability of the cryptococcal capsule to activate the complement cascade and bind C3 occurred uniformly over the surface of all yeast cells, including the bud. Finally, the cell-to-cell variability in binding of O -acetyl-dependent mAbs with strains of serotype A was not found with strains of serotype D; almost all cells of serotype D showed homogeneous binding of O -acetyl-dependent mAbs. These results indicate that variability in expression of antigenic epitopes by GXM should be considered in selection of mAbs used for immunodiagnosis or immunotherapy.     

An unusual organelle in Cryptococcus neoformans links luminal pH and capsule biosynthesis

Cryptococcus neoformans is a basidiomycete that causes deadly infections in the immunocompromised. We previously generated a secretion mutant in this fungus by introducing a mutation in the SAV1 gene, which encodes a homolog of the Sec4/Rab8 subfamily GTPases. Under restrictive conditions there are two notable morphological changes in the sav1 mutant: accumulation of post-Golgi vesicles and the appearance of an unusual organelle, which we term the sav1 body (SB). The SB is an electron-transparent structure 0.2–1 μm in diameter, with vesicles or other membranous structures associated with the perimeter. Surprisingly, the SB was heavily labeled with anti-glucuronoxylomannan (GXM) antibodies, suggesting that it contains a secreted capsule component, GXM. A structure similar to the SB, also labeled by anti-GXM antibodies, was induced in wild type cells treated with the vacuolar-ATPase inhibitor, bafilomycin A₁. Bafilomycin A₁ and other agents that increase intraluminal pH also inhibited capsule polysaccharide shedding and capsule growth. These studies highlight an unusual organelle observed in C. neoformans with a potential role in polysaccharide synthesis, and a link between luminal pH and GXM biosynthesis.     

In vivo interaction between alveolar macrophages and Cryptococcus neoformans

In vivo interactions of rabbit alveolar macrophages (AM) and Cryptococcus neoformans, a yeast pathogenic for humans, were studied. As a control, inert silica particles of a similar diameter (5–6 μm) were used. Of 16 rabbits, 6 were instilled intratracheally with fluorescein-labelled heat-killed C. neoformans, 6 with fluorescein-labelled silica particles and 4 with saline only. After 24 h, the AM were collected by lung lavage, and phagocytosis, oxidative metabolism, phagolysosomal pH and morphology were studied. The accumulated number of yeasts attached to the AM was almost the same for C. neoformans as for the silica particles. The ingested fraction of C. neoformans was even higher than that of the silica particles. Quantitative NBT reduction by the AM, reflecting their oxidative metabolism, was markedly increased by exposure to C. neoformans for 24 h. The phagolysosomal pH was on the average lower in phagolysosomes with C. neoformans than with the silica particles, although approximately 2% of the phagolysosomes with C. neoformans had neutral pH. Phagolysosomes with neutral pH was not observed for silica particles. Electron microscopy showed presence of C. neoformans in phagolysosomes of AM. The conclusion of this study is that the phagocytic activity, oxidative metabolism and phagolysosomal pH AM against C. neoformans are significant 24 h after the exposure. This revised version was published online in June 2006 with corrections to the Cover Date.