Melanogenesis in Cryptococcus neoformans

Melanogenesis in Cryptococcus neoformans begins with the oxidation of dihydroxyphenylalanine by the enzyme phenol oxidase. The succeeding steps are very rapid. Two intermediates, dopachrome and 5,6-dihydroxyindole, have been isolated and characterized by high performance liquid chromatography. A pathway of melanin formation in C. neoformans is proposed, based on the presence of these intermediates.     

Anti-Chemotactic Activity of Capsular Polysaccharide of Cryptococcus neoformans In Vitro

In this study, we demonstrated the anti-chemotaetic activity of the capsular polysaccharides (CPSs) isolated from each of the heavily (H)- and weakly (W)-encapsulated strains of Cryptococcus neoformans in vitro. The capacity for activation of the alternative complement pathway (ACP) of cells of the two C. neoformans strains in fresh human sera was comparable to that of zymosan (insoluble control), whereas the capacity for generation of the chemotactic factor (CF) of the cells of the two strains in fresh murine sera was markedly lower in the order H- < W-strain than that of zymosan. Conversely, the capacities for ACP activation and CF generation of the CPSs were extremely lower than those of lipopolysaccharide (LPS, soluble control). When zymosan-activated murine serum was incubated with CPS, both CPSs inhibited CF activity dose dependently. When zymosan-activated serum was incubated with heat-killed cells of each strain of C. neoformans, H and W, the CF activity of the treated sera decreased significantly, suggesting that CPS per se did not affect the neutrophils directly, but CPS absorbed CF. On the other hand, both CPSs were shown to possess the O-acetyl groups in their molecules by ¹H-nuclear magnetic resonance spectroscopy. The de-O-acetylation of both CPSs increased the capacity for ACP activation to a level similar to that of LPS, and the de-O-acetylated CPS of both strains exhibited a lower ability to inhibit CF than did native CPS. Collectively, these results suggest that the anti-chemotactic activity of CPS accounts for its ability to absorb the CF which was mostly generated at the sites around the cell wall of whole cells via the ACP, thus suppressing the inflammatory response by preventing dispersal of CF to the extracellular space; and also that the O-acetyl group is partly, if any, involved in the mechanism for incompetence in ACP activation as well as the inhibition of CF.     

Production of Diagnostic Pigment by Phenoloxidase Activity of Cryptococcus neoformans

Cryptococcus neoformans produces brown pigmented colonies when grown on agar media made from an extract of potatoes and carrots, broad beans (Vicia faba), or Guizotia abyssinica seeds. Since other yeasts do not produce the pigment, these media are useful as differential isolation media for C. neoformans. Similar specific pigment was produced by C. neoformans on chemically defined agar media which contained six different substrates of phenoloxidase (o-diphenol: oxygen oxidoreductase EC 1.10.3.1) an enzyme which catalyses the oxidation of o-diphenols to melanin. Substrates were incorporated singly into the media and included L-3, 4-dihydroxyphenylalanine (L-DOPA), chlorogenic acid, protocatechuic acid, catechol, norepinephrine, and 3-hydroxytyramine hydrochloride (dopamine). No pigment was produced on media without substrate. Phenoloxidase activity in (NH(4))(2)SO(4) precipitates of C. neoformans cell-free extract was assayed by measuring increases in absorbance at 480 nm produced in solutions of L-DOPA. This reaction showed oxygen uptake and was effectively inhibited by copper chelators, but not by catalase. The enzyme also oxidized the five other substrates which induced pigment formation. Electron micrographs of cells incubated in L-DOPA showed deposition of the pigment in the cell wall.     

Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.     

Identification of Novel Hybrids Between Cryptococcus neoformans var. grubii VNI and Cryptococcus gattii VGII

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C. neoformans molecular type VNI and C. gattii molecular type VGII.     

Phospholipase activity in Cryptococcus neoformans

Phospholipases have only been detected in a few fungi and yeasts, in particular in Candida albicans. Secreted phospholipases are considered by some researchers to be a potential factor of virulence and pathogenicity in C. albicans. Twenty-three Cryptococcus neoformans strains were tested in order to observe phospholipase production. Twenty-two of the 23 strains tested were able to produce phospholipases, and the ratio diameter of the colony to total diameter of the colony plus zone of precipitation (Pz) ranged between 0.271 and 0.949. C. neoformans, just like C. albicans, can be divided on the basis of the Pz into different strains according to their virulence and pathogenicity. There also appeared to be a correlation between the phospholipase production and the size of the capsule in the strains isolated from AIDS patients. For this reason, further studies on C. neoformans phospholipase activity would be useful in evaluating the virulence of different strains.     

Lipid metabolism in Cryptococcus neoformans

In recent years, lipids have been shown to act as signalling molecules not only in mammalian cells but also in many other eukaryotes. Whereas in mammalian cells lipids regulate cellular functions that play crucial roles in the regulation of pathobiological processes, such as cancer, cardiovascular and neurodegenerative disorders, and inflammation, in the fungus Cryptococcus neoformans lipids play key roles in the regulation of pathogenic traits required for the development of cryptococcosis, an infectious disease particularly frequent in immunocompromised individuals. In this minireview we discuss recent advances in the understanding of lipid metabolism in this important human pathogen, highlighting the potential of fungal lipid enzymatic pathways as promising new drug targets.     

Melanin Biosynthesis in Cryptococcus neoformans

Pigment production by Cryptococcus neoformans is virulence associated. Dopamine- and 3,4-dihydroxyphenylalanine–melanin products were identified after acidic permanganate oxidation, alkaline hydrogen peroxide oxidation, or hydrolysis with hydriodic acid. These data provide direct chemical evidence for the formation of eumelanin polymers by catecholamine oxidation by laccase alone followed by oxidative coupling of dihydroxyindole.     

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.     

Micromorphology of Cryptococcus neoformans

Fine details of the internal and external morphology of Cryptococcus neoformans as seen in ultrathin sections are described and illustrated with electron micrographs. The capsule characteristic of this species contained microfibrils (30 to 40 A in diameter) that appeared to radiate from the cell wall and to coil and intertwine in various directions. These thin, uniformly structured, electron-dense filaments are believed to represent complex polysaccharide molecules. The internal morphology of C. neoformans was in many ways similar to that of yeasts studied by other authors. The cell was uninucleate with a single nucleolus. The nuclear envelope, a pair of unit membranes interrupted by pores, was typical of that found in eucaryotic organisms. Smooth endoplasmic reticulum, mitochondria, vacuoles, storage granules, and ribosomes were consistent features of the cytoplasm. In addition, C. neoformans presented membranous organelles derived from the plasma membrane and comparable to bacterial mesosomes and mitochondria of an annulate type.     

Epidemiology of Cryptococcus neoformans

The concept of the epidemiology of Cryptococcus neoformans as the causative agent of cryptococcosis and as a basidiomycetous yeast is based on the fact that bird manure has been until now its only known habitat but not plant material which likewise harbours various non-pathogenic Cryptococcus species.It could be shown that the possible influence of nutritional factors on the morphology and morphogenesis earns attention not only in view of the epidemiology of C. neoformans but of its perfect states, too.     

Creatinine metabolism in Cryptococcus neoformans and Cryptococcus bacillisporus.

The pathogenic species of Cryptococcus, C. neoformans and C. bacillisporus, utilized creatinine as a source of nitrogen but not of carbon. Chromatographic and autoradiographic studies suggest that creatinine metabolism in both species involves a single step resulting in the production of methylhydantoin and ammonia. The enzyme responsible for this step, creatinine deiminase, was produced by the cells only in the presence of creatinine in both species. The synthesis of creatinine deiminase was repressed by ammonia in C. neoformans, but not in C. bacillisporus. A possible explanation for this variation, based on the ecological differences between the two species, is discussed. A novel method for measuring creatinine deiminase activity is also described.     

Ptilomycalin A inhibits laccase and melanization in Cryptococcus neoformans

The antifungal spirocyclic guanidine alkaloid, ptilomycalin A, from marine sponge Monanchora arbuscula, inhibits melanogenesis of Cryptococcus neoformans in vitro through inhibition of biosynthesis of laccase in the melanin biosynthetic pathway with an IC(50) of 7.3 μM.     

A genetic linkage map of Cryptococcus neoformans variety neoformans serotype D (Filobasidiella neoformans)

To construct a genetic linkage map of the heterothallic yeast, Cryptococcus neoformans (Filobasidiella neoformans), we crossed two mating-compatible strains and analyzed 94 progeny for the segregation of 301 polymorphic markers, consisting of 228 restriction site polymorphisms, 63 microsatellites, two indels, and eight mating-type (MAT)-associated markers. All but six markers showed no significant (P < 0.05) segregation distortion. At a minimum LOD score of 6.0 and a maximum recombination frequency of 0.30, 20 linkage groups were resolved, resulting in a map length of approximately 1500 cM. Average marker density is 5.4 cM (range 1-28.7 cM). Hybridization of selected markers to blots of electrophoretic karyotypes unambiguously assigned all linkage groups to chromosomes and led us to conclude that the C. neoformans genome is approximately 20.2 Mb, comprising 14 chromosomes ranging in size from 0.8 to 2.3 Mb, with a ratio of approximately 13.2 kb/cM averaged across the genome. However, only 2 of 12 ungrouped markers hybridized to chromosome 10. The hybridizations revealed at least one possible reciprocal translocation involving chromosomes 8, 9, and 12. This map has been critical to genome sequence assembly and will be essential for future studies of quantitative trait inheritance.     

新生隐球菌嗜中枢性感染机制的研究进展

新生隐球菌是临床上最重要的侵袭性病原真菌之一,可感染免疫抑制和免疫正常人群引发具有致命威胁的隐球菌性脑膜脑炎.近年来,隐球菌嗜中枢神经系统感染的机制研究取得了长足的进展,隐球菌参与侵袭中枢神经系统的相关毒力因子及多条宿主细胞应答信号通路相继被发现.