Phenotypic switching in Cryptococcus neoformans

Cryptococcus neoformans strains exhibit considerable phenotype variability with regards to the capsular polysaccharide, sterol composition of the cell wall, and cell and colony morphology. Phenotypic changes can occur spontaneously during in vitro passage of strains or during chronic infection in vivo and may be associated with differences in virulence. Studies from our laboratory have demonstrated that phenotype variability can be the result of phenotypic switching. Phenotypic switching is defined as a reversible change of an observable colony phenotype that occurs at a frequency above the expected frequency for somatic mutations. This implies that phenotypic switching represents controlled and programmed changes in this pathogenic yeast rather than random mutations. We have shown that a phenotypic switch from a smooth colony phenotype to a mucoid colony phenotype occurs in vitro and in vivo during chronic infection of mice. More importantly we have now demonstrated that the switch is associated with an increase in virulence and a change in the host immune response. Implications of these findings for the pathogenesis of cryptococcosis are discussed.     

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.     

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.     

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.     

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.     

Capsule structural heterogeneity and antigenic variation in Cryptococcus neoformans

Cryptococcus neoformans is a human pathogenic fungus with a capsule composed primarily of glucuronoxylomannan (GXM) that is important for virulence. Current views of GXM structure postulate a polymer composed of repeating mannose trisaccharide motifs bearing a single beta(1,2) glucuronic acid with variable xylose and O-acetyl substitutions to form six triads. GXM from different strains is notoriously variable in triad composition, but it is not known if the polymer consists of one or more motif-repeating units. We investigated the polymeric organization of GXM by using mass spectrometry to determine if its compositional motif arrangement was similar to that of bacterial capsular polysaccharides, namely, a polymer of a single repeating unit. The results were consistent with, and confirmatory for, the current view that the basic unit of GXM is a repeating mannose trisaccharide motif, but we also found evidence for the copolymerization of different GXM repeating units in one polysaccharide molecule. Analysis of GXM from isogenic phenotypic switch variants suggested structural differences caused by glucuronic acid positional effects, which implied flexibility in the synthetic pathway. Our results suggest that cryptococcal capsule synthesis is fundamentally different from that observed in prokaryotes and employs a unique eukaryotic approach, which theoretically could synthesize an infinite number of structural combinations. The biological significance of this capsule construction scheme is that it is likely to confer a powerful avoidance strategy for interactions with the immune system and phagocytic environmental predators. Consistent with this premise, the antigenic variation of a capsular epitope recognized by a nonprotective antibody was observed under different growth conditions.     

Phenotypic switching and its implications for the pathogenesis of Cryptococcus neoformans

Phenotypic switching has been described in several strains of Cryptococcus neoformans. It occurs in vivo during chronic infection and is associated with differential gene expression and changes in virulence. The switch involves changes in the polysaccharide capsule and cell wall that affect the yeast's ability to resist phagocytosis. In addition, the phenotypic switch variants elicit qualitatively different inflammatory responses in the host. The host's immune response ultimately affects selection of the switch variants in animal models of chronic cryptococcosis. The biological relevance of phenotypic switching is demonstrated in several murine infection models and further underlines the importance of phenotypic switching in the setting of human disease. This includes the association of switching and poor outcome in chronic infection, the ability of the mucoid variant of strain RC-2 (RC-2 MC) but not the smooth variant (RC-2 SM) to promote increased intracranial pressure in a rat model, and lastly the observation that antifungal interventions can promote the selection of more virulent switch variants during chronic murine infection.     

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.     

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.     

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.     

Paradoxical role of capsule in murine bronchoalveolar macrophage-mediated killing of Cryptococcus neoformans

Infections with the encapsulated fungus Cryptococcus neoformans are usually acquired via inhalation, and the presence of a capsule has been identified as a virulence factor. Therefore, we studied murine bronchoalveolar macrophage (BAM)-mediated killing and phagocytosis of encapsulated and acapsular strains of C. neoformans. After 2 h, BAM killed encapsulated strains CN52 and MP415 more readily than acapsular strains CN602 and CAP67 (54.9 and 36.2% vs 26.1 and 6.7%, respectively, p less than 0.001). Pre-incubating CN602 with purified capsular polysaccharide increased killing to 42.7% (p = 0.04). Significantly greater killing of the encapsulated strains also occurred in vivo. BAM-mediated killing of CN52 appeared to proceed by non-oxidative mechanisms, as BAM released minimal amounts of H2O2 after stimulation with CN52, and killing was not reduced by inhibitors or scavengers of the respiratory burst. The association between encapsulation and susceptibility to BAM fungicidal effects was not attributable to differences in yeast ingestion. Using the same low ratio of organisms to BAM as in the killing assay, greater than 95% of both CN52 and CN602 were phagocytosed. However, BAM phagocytosed significantly greater numbers of acapsular CN602 when incubated with a higher inoculum. Phagocytosis and killing of CN52 and CN602 required fresh serum as a source of C. Phagocytosis of CN52, but not CN602, was profoundly inhibited if BAM were plated on surfaces coated with mAb against the C3bR (CR1). mAb against the iC3b receptor (CR3) did not affect phagocytosis of either strain. These data demonstrate the innate ability of BAM to preferentially kill, by apparently non-oxidative mechanisms, an encapsulated as opposed to acapsular organism. Inasmuch as different receptors appear involved in phagocytosis of encapsulated versus acapsular C. neoformans, the disparity in killing may result from the greater ability of receptors mediating uptake of encapsulated organisms to trigger the antimicrobial armamentarium of the BAM.     

Isolation and characterization of capsule structure mutant strains of Cryptococcus neoformans

The capsule of Cryptococcus neoformans is the most obvious virulence factor of this pathogenic yeast. The main capsule constituents are glucuronoxylomannans (GXM). Although several studies have focused on GXM composition and structure, very little is known about their genetics. To elucidate the relationship between the capsule structure and the pathophysiology of the cryptococcosis, genetic screening for mutant strains producing a structurally modified capsule was set up. Using monoclonal antibodies specific for different capsule sugar epitopes, we isolated strains with different mutated capsule structures (Cas mutants). According to their reactivities with various monoclonal antibodies, the mutants were classified into six groups (Cas1 to Cas6). One Cas2 mutant was used to clone the corresponding gene by complementation. This gene (USX1) encodes the previously identified UDP-xylose synthase. We demonstrated that it is necessary for both capsule xylosylation and C. neoformans virulence.     

新型隐球酵母铜应答转录因子Cuf1与荚膜的生物合成的相关性研究

摘要：【目的】新型隐球酵母是人类条件致病真菌,主要感染免疫缺陷患者。该酵母最显著的特征是细胞外包被主要的致病因子-多糖荚膜,其调控机制复杂。本文研究旨在阐述编码铜依赖转录因子的CUF1基因对其荚膜生物合成的负调控作用。【方法】以野生型菌株为对照,对CUF1缺失的突变菌株进行菌落形态观察、荚膜墨汁染色的显微观察、细胞聚沉试验以及荚膜定量分析。【结果】与野生型菌株相比,Δcuf1突变株产生的菌落更粘,显微镜下亦可明显观察到荚膜更厚。同样数量的细胞,突变株聚沉平衡后体积更大。此外,荚膜粗提物定量称重分析也证明突     

新型隐球酵母铜应答转录因子Cuf1与荚膜的生物合成相关性

[目的]新型隐球酵母是人类条件致病真菌,主要感染免疫缺陷患者.该酵母最显著的特征是细胞外包被着多糖荚膜,这一重要致病因子的调控机制复杂.本文研究旨在阐述编码铜依赖转录因子的CUF1基因对其荚膜生物合成的负调控作用.[方法]以野生型菌株为对照,对CUF1缺失的突变菌株进行菌落形态观察、荚膜墨汁染色的显微观察、细胞聚沉试验以及荚膜定量分析.[结果]与野生型菌株相比,△cuf1突变株产生的菌落更粘,显微镜下亦可明显观察到荚膜更厚.同样数量的细胞,突变株聚沉平衡后体积更大.此外,荚膜粗提物定量称重分析也证明突变株产生了更多的荚膜.并且外源铁可以回复△cuf1突变株荚膜过量产生的表型.[结论]铜应答转录因子1(Cuf1)对荚膜的生物合成具有负调控作用.Cuf1可能通过铁的高亲和吸收途径调控铁吸收而实现该作用的.