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Cryptococcus neoformans var. neoformans was repeatedly isolated from decaying wood forming hollows in living trees growing in urban areas of Rio de Janeiro, Brazil. A new natural habitat for C. neoformans var. neoformans has been found that is not associated with specific trees.
The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in Cryptococcus neoformans. Since isolations of Cryptococcus species other than C. neoformans from clinical specimens have been increasing, we examined the laccase activities of C. albidus, C. laurentii, C. curvatus, and C. humicola. Incubation of cells with epinephrine produced adrenochrome color in C. albidus, C. laurentii, and C. curvatus but not in C. humicola. Activity was always less than in C. neoformans. Laccase was detected in the soluble fractions of disrupted C. albidus, C. laurentii, and C. curvatus cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from C. albidus, C. laurentii, and C. curvatus migrated more slowly than that from C. neoformans. One strain of C. curvatus exhibited two melanin bands. Thus, several clinically emerging Cryptococcus species express laccase and can synthesize melanin.
Yeasts of Cryptococcus neoformans in cerebrospinal fluid (CSF) were studied using polarized light and fluorescence microscopy. Ultraviolet-induced fluorescence of wet-fixed, Papanicolaou-stained smears and polarization of air-dried, Diff-Quik or Leishman-stained preparations are complementary methods that allow for rapid screening for and immediate identification of Cryptococcus in cytologic preparations of CSF.
Cryptococcus neoformans with a rare morphology of hand-mirror appearance was demonstrated by direct microscopic preparation of both cerebrospinal fluid (CSF) and sputum of a patient with AIDS. In addition, one to six blastoconidia were seen at the tip of a germ-tube like structure. Cr. neoformans was isolated in pure culture and the identification was confirmed by biochemical and serological tests as well as by animal pathogenicity.
The interaction between the fungal pathogen Cryptococcus neoformans and human fibronectin (HFN) was examined in this study. Polypeptides from cryptococcal whole homogenates and cell wall with molecular masses of 25 and 35 kDa, respectively reacted with HFN. The relevance of the occurrence of these proteins in intact cells was uncertain, since yeast cells from different strains and serotypes of C. neoformans did not significantly adhere to soluble or solid-phased HFN. In contrast, an exocellular proteolytic activity that cleaves HFN was suggested. Degradation of HFN by culture supernatant fluids was demonstrated by Western blotting using a monoclonal anti-HFN antibody. Several fragments of lower molecular weights were observed which reacted with the antibody. Proteolysis was mediated by a serine protease activity, since HFN cleavage was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), aprotinin, and N-tosyl-L-phenylalanyl chloromethylketone (TPCK), but not by inhibitors of metalo, cysteine, or aspartyl proteases. Similar results were obtained when the fluorogenic peptide carbobenzoxy-phenylalanyl-arginyl-7-amido-4-methylcoumarin (CBZ-Phe-Arg-NHmet-C) was used as substrate. The cryptococcal supernatant also cleaved laminin and type IV collagen, as demonstrated by polyacrylamide gel electrophoresis with co-polymerized proteins. The hydrolysis of these proteins was mediated by a single cryptococcal protease with a molecular mass of 75 kDa. The cleavage of key host components of the basement membrane and extracellular matrix by C. neoformans may be a relevant factor in the process of fungal invasion.
After preliminary results had shown the occurrence of Cryptococcus neoformans in canary droppings in southern Italy, the increasing epidemiological interest in cryptococcosis and the habitats of the agent led to more extensive studies. 180 samples of canary droppings were collected from pet shops and private households in two towns (Messina and Reggio Calabria) of southern Italy and culturally examined for C. neoformans. The examination was carried out with the help of the brown colour effect (BCE) specific of the C. neoformans colony on Guizotia abyssinica creatinine agar (Staib agar). 48 samples (26.6%) were positive; of these, 32/108 (29.6%) were from pet shops and 16/72 (22.2%) from private households. The colony growth was not mucous, i.e. microscopically the blastospores were found to be little or not encapsulated. All C. neoformans strains tested by the auxanographic method showed a weak assimilation of creatinine. The variety status of the isolates was tested with canavanine-glycine-bromothymol-blue (CGB) agar; all strains tested (n = 420) were found to be C. neoformans var. neoformans. In the light of extensive studies on C. neoformans and the canary in the pre-AIDS era in Germany, the present observations in Italy ask for further basic research, epidemiological studies, and education of the public.
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.
Reports of Cryptococcus neoformans isolations from patients in Panama have appeared since 1978, but to our knowledge, thIs is the first report of isolation of this organism from soil samples in the Republic. Of 25 soil samples analyzed, two (25%) yielded the organism on birdseed agar medium. Canavanine-glycine-bromthymol blue medium served to identify the isolates as belonging to C. neorformans var neoformans, and not C. Neoformans var. gattii. Initially, isolates did not have capsules, but upon passage through newborn mice, wide capsules developed. This finding has important public health implications, since non-encapsulated environmental organisms, once in the host, can develop capsules, which significantly contribute to virulence.
Low-iron minimal medium supported growth of Cryptococcus neoformans but spent medium contained no hydroxamates, organic acids or other iron chelators. Exogenous deferoxamine stimulated growth in extreme iron-limitation, while neither organic acids nor quartersaturated transferrins were stimulatory. These results suggest two iron uptake mechanisms, one dependent upon exogenous hydroxamate, the other hydroxamate-independent.
Cryptococcus neoformans is a major fungal pathogen for patients with debilitated immune systems. However, no information is available on the stability of virulence or of phenotypes associated with virulence for C. neoformans laboratory strains. A serendipitous observation in our laboratory that one isolate of C. neoformans ATCC 24067 (strain 52D) became attenuated after continuous in vitro culture prompted us to perform a comparative study of nine strain 24067 isolates obtained from six different research laboratories. Each isolate was characterized by DNA typing, virulence for mice, proteinase production, extracellular protein synthesis, melanin synthesis, carbon assimilation pattern, antifungal drug susceptibility, colony morphology, growth rate, agglutination titers, phagocytosis by murine macrophages, capsule size, and capsular polysaccharide structure. All isolates had similar DNA typing patterns consistent with their assignment to the same strain, although minor chromosome size polymorphisms were observed in the electrophoretic karyotypes of two isolates. Several isolates had major differences in phenotypes that may be associated with virulence, including growth rate, capsule size, proteinase production, and melanization. These findings imply that C. neoformans is able to undergo rapid changes in vitro, probably as a result of adaptation to laboratory conditions, and suggest the need for careful attention to storage and maintenance conditions. In summary, our results indicate that C. neoformans (i) can become attenuated by in vitro culture and (ii) is capable of microevolution in vitro with the emergence of variants exhibiting new genotypic and phenotypic characteristics.
Host defense mechanisms to the important fungal pathogen Cryptococcus neoformans are complex and incompletely understood. From in vitro studies, we could expect CD8+ T cells to have the potential for both protective and suppressive effects on defense against cryptococci. The current study used the technique of in vivo subset depletion to determine the net effect of CD8+ T cells during actual infection. Mice depleted of CD8+ T cells by monoclonal antibody (YTS 169.4) injections were infected with the moderately virulent cryptococcal strain 613D by the intratracheal route, which mimics natural pulmonary infection. To ensure adequacy of depletion, T cell subsets were enumerated by flow cytometry in blood, spleen, lymph node, and lung. Specific elements of host defense were measured by clearance of cryptococci in vivo and by the response to cryptococcal antigens, both in vivo by delayed-type hypersensitivity (DTH) and in vitro by lymphocyte proliferation. We found that depletion of CD8+ T cells decreased pulmonary clearance of C. neoformans. CD8 depletion abrogated DTH without affecting antigen recognition or lymphocyte proliferation in vitro. These data demonstrate that the net effect of CD8+ T cells to a moderately virulent strain of C. neoformans is to enhance rather than suppresses host defenses. Abrogation of DTH suggests that CD8+ T cells contribute to the protective immunologic response to C. neoformans. It is possible that in hosts deficient in CD4 cells, such as patients with AIDS, the protective effects of CD8+ T cells could be stimulated by selected immunizing epitopes.
Three hundred twenty-three clinical isolates of Cryptococcus neoformans of diverse geographic origins were biochemically serogrouped using glycine-cycloheximide-phenol red agar (GCP), the same medium less cycloheximide (GOP), and glycine-L-canavanine bromothymol blue agar (CGB). Twenty isolates gave positive reactions on all three media typical of the B and C serotypes. Three were from the Peoples' Republic of China; three each were from Michigan (two patients) and Louisiana; two each were from California, Georgia, and Virginia; and one each was from Alabama, Florida, North Carolina, Oklahoma, and Tennessee. Two hundred seventy-six isolates were identified as belonging to the A/D serogroup; 272 were of American origin and four were from China. Twenty-seven isolates were biochemically ungroupable. Evaluations of the reactions on all three media were open to subjective interpretations. Utilization of glycine was the most frequent atypical variable; 36 of 276 (13%) A/D isolates utilized glycine while being inhibited by either GCP or CGB or both. Significant differences between A/D and B/C serogroups in terms of susceptibility to 5-fluorocytosine but not to amphotericin B were observed; B/C serogroup isolates appeared to be less susceptible to 5-fluorocytosine in vitro than were the A/D serogroup isolates. These results provided new evidence on the distribution of B/C serogroup isolates of C. neoformans in America and demonstrate the difficulties of using biochemical tests for serotyping purposes. They also offer a possible explanation for the apparent more refractory therapeutic responses of infections caused by B and C serotypes to conventional antifungal chemotherapy.
We compared multiple isolates of the two varieties of Cryptococcus neoformans, as well as previously characterized representative isolates, for their electrophoretic karyotypes using pulsed-field electrophoresis. The two varieties could be clearly distinguished based upon the size of the smallest chromosome. The smallest chromosome for isolates of the gattii variety (serotypes B and C) was found to be 400-700 kb in size. The smallest chromosome for isolates of the neoformans variety was consistently found to be larger, approximately 770 kb in size. Isolates of the gattii variety averaged 13 chromosomes while the neoformans variety averaged 12. The size of the Cryptococcus genome was found to be approximately 23 megabases. Isolates of C. neoformans var. neoformans tended to be more conserved than those of var. gattii with regard to gene position.
The major virulence factor of the pathogenic fungus Cryptococcus neoformans is an extensive polysaccharide capsule which surrounds the cell. Almost 90% of the capsule is composed of a partially acetylated linear alpha-1,3-linked mannan substituted with D-xylose and D-glucuronic acid. A novel mannosyltransferase with specificity appropriate for a role in the synthesis of this glucuronoxylomannan is active in cryptococcal membranes. This membrane-associated activity transfers mannose in vitro from GDP-mannose to an alpha-1, 3-dimannoside acceptor, forming a second alpha-1,3 linkage. Product formation by the transferase is dependent on protein, time, temperature, divalent cations, and each substrate. It is not affected by amphomycin or tunicamycin but is inhibited by GDP and mannose-1-phosphate. The described activity is not detectable in the model yeast Saccharomyces cerevisiae, consistent with the absence of a similar polysaccharide structure in that organism. A second mannosyltransferase from C. neoformans membranes adds mannose in alpha-1,2 linkage to the same dimannoside acceptor. The two activities differ in pH optimum and cation preference. While the alpha-1,2 transferase does not have specificity appropriate for a role in glucuronoxylomannan synthesis, it may participate in production of mannoprotein components of the capsule. This study suggests two new targets for antifungal drug discovery.
A urease-negative serotype A strain of Cryptococcus neoformans (B-4587) was isolated from the cerebrospinal fluid of an immunocompetent patient with a central nervous system infection. The URE1 gene encoding urease failed to complement the mutant phenotype. Urease-positive clones of B-4587 obtained by complementing with a genomic library of strain H99 harboured an episomal plasmid containing DNA inserts with homology to the sudA gene of Aspergillus nidulans. The gene harboured by these plasmids was named URE2 since it enabled the transformants to grow on media containing urea as the sole nitrogen source while the transformants with an empty vector failed to grow. Transformation of strain B-4587 with a plasmid construct containing a truncated version of the URE2 gene failed to complement the urease-negative phenotype. Disruption of the native URE2 gene in a wild-type serotype A strain H99 and a serotype D strain LP1 of C. neoformans resulted in the inability of the strains to grow on media containing urea as the sole nitrogen source, suggesting that the URE2 gene product is involved in the utilization of urea by the organism. Virulence in mice of the urease-negative isolate B-4587, the urease-positive transformants containing the wild-type copy of the URE2 gene, and the urease-negative vector-only transformants was comparable to that of the H99 strain of C. neoformans regardless of the infection route. Virulence of the URE2 disruption stain of H99 was slightly reduced compared to the wild-type strain in the intravenous model but was significantly attenuated in the inhalation model. These results indicate that the importance of urease activity in pathogenicity varies depending on the strains of C. neoformans used and/or the route of infection. Furthermore, this study shows that complementation cloning can serve as a useful tool to functionally identify genes such as URE2 that have otherwise been annotated as hypothetical proteins in genomic databases.
Cryptococcus neoformans is an opportunistic fungus that causes life-threatening meningoencephalitis in 5-10% of patients with acquired immune deficiency syndrome. Cryptococcal meningoencephalitis is characterized by a lymphohistiocytic infiltrate, accumulation of encapsulated forms of C. neoformans, and varying degrees of glial reaction. Little is known about the contribution of endogenous central nervous system cells to the pathogenesis of cryptococcal infections. In this study, we investigated the role of astrocytes as potential effector cells against C. neoformans. Primary cultures of human fetal astrocytes, activated with interleukin 1 beta plus interferon gamma inhibited the growth of C. neoformans. The inhibition of C. neoformans growth was paralleled by production of nitrite, and reversed by the inhibitors of nitric oxide (NO.) synthase, NG-methyl-mono-arginine and NG-nitro-arginine methyl ester. The results suggest a novel function for human astrocytes in host defence and provide a precedent for the use of NO. as an antimicrobial effector molecule by human cells.
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.
A case of prosthetic valve endocarditis caused by Cryptococcus neoformans var. neoformans is described. The infection followed closed mitral valvotomy and insertion of a valvular prosthesis. Infection was manifested 2 weeks after the operation. The diagnosis was based on direct demonstration of the yeast with characteristic morphology in clinical material, isolation from an arterial thrombus and detection of cryptococcal antigen in the serum. The patient's infection could not be resolved despite institution of antifungal therapy.