Sexual compatibility between serotypes ofFilobasidiella neoformans (Cryptococcus neoformans)

Twenty-five of 37Cryptococcus neoformans strains of known serotype produced the basidiomycetousFilobasidiella state either alone or when paired with a strain of compatible mating-type. Sixteen strains were mating-type, four strains were a mating-type, and five strains were self-fertile.F. neoformans serotypes A and D were interfertile with compatible mating-types ofF. bacillispora serotypes B and C.C. neoformans var.gattii was interfertile with compatible mating-types ofF. neoformans andF. bacillispora. F. bacillispora strains, which utilized creatinine andl-malic acid, were interfertile with compatible mating-types ofF. neoformans, which did not utilize creatinine andl-malic acid. The interfertility between serotypes and biotypes eliminates the need for recognizing the names ofC. neoformans var.gattii, C. bacillisporus, andF. bacillispora. It is proposed thatC. neoformans var.gattii andC. bacillisporus be regarded as later, facultative synonyms ofC. neoformans and thatF. bacillispora be regarded as a later, facultative synonym ofF. neoformans.     

Diversity of laccase among Cryptococcus neoformans serotypes

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.     

Biochemical variation of Cryptococcus neoformans

Ninety-seven strains of Cryptococcus neoformans and C. bacillisporus were examined for 44 biochemical characters and the results were analyzed numerically. One phenon emerged at the 86% level of similarity when strains were clustered according to their M-similarity values. All strains grew in ten carbon sources (D-glucose, D-galactose, arbutin, maltose, sucrose, D-melezitose, D-xylose, D-mannitol, D-glucitol, and meso-inositol), and also grew at 37 ° C and produced urease and phenoloxidase. None of them grew in melibiose, lactose, nor valine, and none reduced nitrate to nitrite. Comparison of selected biochemical characters, creatinine utilization, and serotypes of 49 aberrant strains is presented. Forty-eight of the 97 strains produced the Filobasidiella state either alone or when paired with a strain of compatible mating-type. Filobasidiella neoformans serotypes A and D were interfertile with compatible mating-types of F. bacillispora serotypes B and C. The 44 biochemical characters and 4 serotypes did not predict barriers to mating competence. The present study further substantiates that Filobasidiella neoformans and F. bacillispora are one species.     

Mating types and serotypes of Cryptococcus neoformans isolated in Japan

Thirty-two isolates of Cryptococcus neoformans from patients and one from the wild obtained in Japan were characterized for their serotype, self-fertility, and mating behaviour by crossing them with two mating types of Filobasidiella neoformans var. neoformans and F. neoformans var. bacillispora. Of the 32 isolates from patients, 31 were of serotype A and the remaining one was of serotype D. Although these 32 isolates were all self-sterile, 23 serotype A and one serotype D isolates produced a complete sexual state when mixed with the alpha mating type of F. neoformans var. neoformans. The one natural isolate was of serotype A-D and self-fertile. The Japanese clinical isolates of C. neoformans appear to be predominantly serotype A and alpha mating type of F. neoformans var. neoformans as is the case in the U.S.A.     

Varietes and serotypes of Cryptococcus neoformans clinical isolates in Colombia

The aim of this study was to contribute to the knowledge of the geographic distribution of Cryptococcus neoformans varieties, in Colombian patients, and to determine the mating types of such varieties. A total of 370 clinical isolates were studied. C. neoformans var. neoformans was identified in 95.2% of them. C. neoformans var. gattii in 4.8%, 4.5% being serotype B and 0.3%, serotype C. Fifty-five of the 74 (74%) isolates studied were mating type "alpha", all of them C. neoformans var. neoformans. Serotype C had been previously reported only once in South American countries.     

Biochemical studies of phenoloxidase and utilization of catecholamines in Cryptococcus neoformans

Protoplasts of Cryptococcus neoformans contain phenoloxidase as a membrane-bound enzyme. The enzyme appeared to be attached on the inner side of cytoplasmic membranes. Synthesis of the enzyme was derepressed by low levels of glucose but was not affected by the level of ammonium. Copper chelators which inhibited the phenoloxidase of other organisms did not affect cryptococcal enzymes. However, cyanide- or iron-chelating agents such as hydroximide derivates or 8-hydroxyquinoline were effective inhibitors, suggesting that cryptococcal phenoloxidase is an iron-containing enzyme. Phenoloxidase of C. neoformans catalyzed the oxidation of various diphenols via dopachrome and labile intermediates to melanin polymers. The kinetic constants (Km) of the phenoloxidase and the permease for dopamine and norepinephrine were low. The correlation between phenoloxidase and the preferential growth of C. neoformans in the host brain is discussed.     

Cryptococcus neoformans: Comparisons of in vitro antifungal susceptibilities of serotypes AD and BC

Thirty-nine isolates of Cryptococcus neoformans, nineteen serotype AD and twenty serotype BC, were assayed for susceptibility to eight antifungal agents using an in vitro agar dilution assay. Media employed were Kimmig agar and yeast nitrogen base supplemented with 10% glucose. The antifungal agents used were ketoconazole, amphotericin B, 5-fluorocytosine, nystatin, miconazole, BAY N 7133, ICI 153,066, and itraconazole. No clinically significant differences in in vitro minimum inhibitory concentrations were detected between serotypes AD and BC against any of the compounds tested. An adverse medium effect was observed in two of the assays, but the outcome of the AD/BC comparison was not affected. This is the first report in which the in vitro antifungal susceptibilities of Cryptococcus neoformans serotypes are analyzed.     

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.     

Unique hybrids between the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii

Cryptococcus neoformans and Cryptococcus gattii are yeasts that cause meningoencephalitis, but that differ in host range and geographical distribution. Cryptococcus neoformans occurs world-wide and mostly infects immunocompromised patients, whereas C. gattii occurs mainly in (sub)tropical regions and infects healthy individuals. Anomalous C. neoformans strains were isolated from patients. These strains were found to be monokaryotic, and diploid or aneuploid. Amplified Fragment Length Polymorphism (AFLP) and sequence analyses indicated that AFLP genotypes 2 (C. neoformans) and 4 (C. gattii) were present. The strains were serologically BD. Mating- and serotype-specific PCR reactions showed that the strains were MATa-serotype D/MATalpha-serotype B. This study is the first to describe naturally occurring hybrids between C. neoformans and C. gattii.     

Genetic analyses of a hybrid cross between serotypes A and D strains of the human pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans has two varieties, var. grubii and var. neoformans, that correspond to serotypes A and D, respectively. Molecular phylogenetic analyses suggest that these two varieties have diverged from each other for approximately 18 million years. The discovery of pathogenic serotype AD hybrid strains in nature indicates that intervariety mating in C. neoformans occurs in the natural environment. However, little is known about the genetic consequences of hybridization in C. neoformans. Here, we analyzed a hybrid population of 163 progeny from a cross between strains of serotypes A (CDC15) and D (JEC20), using 114 codominant nuclear PCR-RFLP markers and 1 direct PCR marker. These markers were distributed on all 14 chromosomes of the sequenced strain JEC21 that was isogenic to one of the parents (JEC20) in our cross. Our analyses identified that of the 163 progeny, 5 were heterozygous at all 115 loci, 1 was completely homozygous and identical to one of the parents (CDC15), and the remaining 157 each contained at least 1 heterozygous locus. Because all 163 progeny inherited mitochondria from the MATa parent JEC20, none of the progeny had a genotype identical to either of the two parents or to a composite of the two parents. All 115 nuclear loci showed three different genotypes in the progeny population, consistent with Mendelian segregation during meiosis. While the linkage analysis showed independent reassortment among loci on different linkage groups, there were significant differences in recombination frequencies among chromosomes and among regions within certain chromosomes. Overall, the linkage-map length from this hybrid cross was much shorter and the recombination frequency much lower than those constructed using serotype D strains, consistent with suppressed recombination in the intervariety cross between strains of serotypes A and D. We discuss the implications of our results in our understanding of the speciation and evolution of the C. neoformans species complex.     

Recognition of cytoplasmic yeast antigens of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii by immune human sera

The humoral immune response of patients infected with Cryptococcus neoformans var. neoformans and C. neoformans var. gattii to cytoplasmic (non-capsular) antigens from the two varieties of Cryptococcus has been investigated. Cytoplasmic antigens from C. neoformans (one clinical isolate and one acapsular mutant of var. neoformans and two clinical isolates from var. gattii) were subject to isoelectric focusing, SDS-PAGE and Western blotting; patients sera was then used in the immunoenzyme development of the Western blots. The humoral response from the 20 patients (all HIV+) infected with var. neoformans against the var. neoformans antigens was predominantly IgG based, with a large number of bands recognised; the most commonly recognised bands were at 26, 52, 74, 100, 115 and 144 kDa. The IgM response was less pronounced and the IgA response was practically non-existent. The humoral response of the sera from the 15 patients (all but one HIV-) infected with var. gattii against var. gattii antigens was also predominantly IgG based with bands at 37, 55, 65, 74, 94 and 115 kDa being most commonly recognised. Periodate treatment of cytoplasmic antigens reduced the intensity of antigen recognition, though it did not absolutely destroy reactivity to any individual antigen. Comparison of immunodevelopment of cytoplasmic antigens from both varieties grown at 25°C and 37°C revealed that culture temperature made no differences in the number of bands recognised although there were differences in the intensity of recognition. This is the first report on the pattern of serological recognition of the non-capsular antigens from the two varieties of Cryptococcus and it identifies a number of major antigenic components.     

Cryptococcus neoformans I. Nonencapsulated Mutants

Seven nonencapsulated mutants of Cryptococcus neoformans were isolated from an encapsulated strain of human origin. Initially, the mutants were avirulent for mice. After several months of subculturing, six of the seven isolates reverted to the encapsulated state and possessed varying degrees of virulence. The results of these experiments suggest that a strong correlation exists between the presence of a capsule and the virulence of C. neoformans.     

Genetic complementation in Cryptococcus neoformans.

A complementation test was devised for the fungus Cryptococcus neoformans. Complementation was signalled by the growth of prototrophic heterokaryons generated in crosses of the type aB X Ab, where a and b represent any two of the genetic markers ilv1, cys1, cys2, and cys3. The cloned complementing heterokaryons formed characteristic hyphal colonies that contained both hyphae and yeast cells. The heterokaryon-derived yeasts were of three kinds: parental haploids, recombinant haploids, and diploids.     

Cryptococcus neoformans III. Inhibition of Phagocytosis

Isolated nonhydrolyzed cryptococcal polysaccharide is a rather specific potent inhibitor of the phagocytosis of Cryptococcus neoformans by human leukocytes in vitro. When an encapsulated strain of C. neoformans was cultured in the nonencapsulated state, the rate of phagocytosis was three times greater than when the encapsulated form was used. Our theory that capsular material plays a role in the pathogenesis of cryptococcosis requires (i) that C. neoformans exist in soil in a nonencapsulated state and (ii) that human phagocytes be capable of killing the organisms.     

Localization of mannoprotein in Cryptococcus neoformans.

Cell wall mannoprotein of nonpathogenic yeasts is surface exposed, since the cells are agglutinated by concanavalin A and antimannoprotein antibodies. However, nonencapsulated cells of Cryptococcus neoformans were agglutinated neither by concanavalin A nor by antimannoprotein antibodies. Immunogold electron microscopy located most mannoprotein in the inner cell wall. Chemical analysis of purified cell walls showed the lack of mannose, xylose, and galactose residues. These data indicate that cryptococcal mannoprotein recovered from the cultural supernatant is a nonstructural element of the cell wall.     

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.