Kyung J. Kwon-Chung

A new genus, Filobasidiella, the perfect state of Cryptococcus neoformans

In 1966, Shadomy and Utz (2) reported hyphal formation in a few strains of Cryptococcus neoformans, a pathogenic yeast. Shadomy (1) later observed clamplike connections in the hyphae-forming isolates similar to those produced in basidiomycetes. On the basis of such observations and the fact that all basidiomycetous yeasts belong to heterobasidiomycetes, she has proposed that C. neoformans be placed in the genus Leucosporidium of the Ustilaginaceae. Recently I have cultured 15 strains of C. neoformans isolated from different clinical specimens on malt extract and sporulation agar (3) in various pairs. A basidiomycetous state was found in four pairs after a total of 3 wk of incubation, 1 wk at 25 C, and 2 wk at 15 C. Sexual reproduction was initiated by conjugation of two yeast cells which was followed by the formation of hyphae with clamp connections. A group of long basidia (FIG. 1) each having a subglobose to flask-shaped apex with sessile basidiospores was produced terminally or laterally from the hyphae. In contrast to Leucosporidium, teliospores were lacking; the basidia were never septate and produced terminal basidiospores. Basidial structures of C. neoformans showed a close relationship with Filobasidium Olive (4) in the monogeneric family Filobasidiaceae Olive (3) of the Ustilaginales. However, there are enough differences in the manner of basidiospore formation to warrant description of a new genus. Formation of basidiospores in species of Filobasidium is not repetitious, whereas the new basidiomycete forms basidiospores in basipetal chains by repetitious budding. 1197

Morphogenesis of Filobasidiella neoformans, the sexual state of Cryptococcus neoformans.

SUMMARYMorphogenesis of Filobasidiella neoformans (= Cryptococcus neoformans) has been studied. A dikaryotic mycelium with clamp connections is formed after conjugation of two yeast cells of opposi...

A new species of Filobasidiella, the sexual state of Cryptococcus neoformans B and C serotypes.

King, D. S. 1976a. Systematics of Conidiobolus (Entomophtho,rales) using numerical taxonomy. I. Biology and cluster analysis. Canad. J. Bot. 54: 45-65. . 1976b. Systematics of Conidiobolus (Entomophthorales) using numerical taxonomy. II. Taxonomic considerations. Canad. J. Bot. 54: 1285-1296. Praserthphon, S. 1963. Conidial formation in Entomophthora coronata (Costantin) Kevorkian. J. Insect Pathol. 5: 318-335. Srinivasan, M. C., and M. J. Thirumalachar. 1967. Evaluation of taxonomic characters in the genus Conidiobolus, with key to known species. Mycologia 59: 698-713. White, W. L. 1937. Note on Conidiobolus. Mycologia 29: 148-149.

ASPERGILLUS NIDULANS INFECTION IN CHRONIC GRANULOMATOUS DISEASE

Chronic granulomatous disease (CGD) is a rare inherited disorder of the NADPH oxidase complex in which phagocytes are defective in generating reactive oxidants. As a result, patients with CGD suffer from recurrent bacterial and fungal infections. The most common fungal infections are caused by Aspergillus species. Aspergillus nidulans is a rare pathogen in most patient populations with quantitative or qualitative neutrophil defects. We have reviewed all cases in which A. nidulans was isolated from patients at the National Institutes of Health (Bethesda, MD) between 1976 and 1997. A. nidulans infection occurred in 6 patients with CGD, but was not a pathogen in any other patient group. Aspergillus fumigatus was a more common pathogen in CGD compared with A. nidulans, but A. nidulans was more virulent. A. nidulans was significantly more likely to result in death compared with A. fumigatus, to involve adjacent bone, and to cause disseminated disease. Patients with A. nidulans received longer courses of amphotericin B therapy than patients with A. fumigatus, and were treated with surgery more often. In contrast to A. fumigatus, A. nidulans was generally refractory to intensive antifungal therapy, suggesting that early surgery may be important. These data show that A. nidulans is a distinct pathogen in CGD and its isolation carries more severe implications than that of A. fumigatus.

Cryptococcus neoformans overcomes stress of azole drugs by formation of disomy in specific multiple chromosomes.

Cryptococcus neoformans is a haploid environmental organism and the major cause of fungal meningoencephalitis in AIDS patients. Fluconazole (FLC), a triazole, is widely used for the maintenance therapy of cryptococcosis. Heteroresistance to FLC, an adaptive mode of azole resistance, was associated with FLC therapy failure cases but the mechanism underlying the resistance was unknown. We used comparative genome hybridization and quantitative real-time PCR in order to show that C. neoformans adapts to high concentrations of FLC by duplication of multiple chromosomes. Formation of disomic chromosomes in response to FLC stress was observed in both serotype A and D strains. Strains that adapted to FLC concentrations higher than their minimal inhibitory concentration (MIC) contained disomies of chromosome 1 and stepwise exposure to even higher drug concentrations induced additional duplications of several other specific chromosomes. The number of disomic chromosomes in each resistant strain directly correlated with the concentration of FLC tolerated by each strain. Upon removal of the drug pressure, strains that had adapted to high concentrations of FLC returned to their original level of susceptibility by initially losing the extra copy of chromosome 1 followed by loss of the extra copies of the remaining disomic chromosomes. The duplication of chromosome 1 was closely associated with two of its resident genes: ERG11, the target of FLC and AFR1, the major transporter of azoles in C. neoformans. This adaptive mechanism in C. neoformans may play an important role in FLC therapy failure of cryptococcosis leading to relapse during azole maintenance therapy.

Cryptococcal Yeast Cells Invade the Central Nervous System via Transcellular Penetration of the Blood-Brain Barrier

ABSTRACT Cryptococcal meningoencephalitis develops as a result of hematogenous dissemination of inhaled Cryptococcus neoformans from the lung to the brain. The mechanism(s) by which C. neoformans crosses the blood-brain barrier (BBB) is a key unresolved issue in cryptococcosis. We used both an in vivo mouse model and an in vitro model of the human BBB to investigate the cryptococcal association with and traversal of the BBB. Exposure of human brain microvascular endothelial cells (HBMEC) to C. neoformans triggered the formation of microvillus-like membrane protrusions within 15 to 30 min. Yeast cells of C. neoformans adhered to and were internalized by the HBMEC, and they crossed the HBMEC monolayers via a transcellular pathway without affecting the monolayer integrity. The histopathology of mouse brains obtained after intravenous injection of C. neoformans showed that the yeast cells either were associated with endothelial cells or escaped from the brain capillary vessels into the neuropil by 3 h. C. neoformans was found in the brain parenchyma away from the vessels by 22 h. Association of C. neoformans with the choroid plexus, however, was not detected during up to 10 days of observation. Our findings indicate that C. neoformans cells invade the central nervous system by transcellular crossing of the endothelium of the BBB.

Cryptococcus neoformans Strains and Infection in Apparently Immunocompetent Patients, China

High incidence of cryptococcosis in these patients is in contrast to reports from other countries.

Gliotoxin Is a Virulence Factor of Aspergillus fumigatus: gliP Deletion Attenuates Virulence in Mice Immunosuppressed with Hydrocortisone

ABSTRACT Gliotoxin is an immunosuppressive mycotoxin long suspected to be a potential virulence factor of Aspergillus fumigatus. Recent studies using mutants lacking gliotoxin production, however, suggested that the mycotoxin is not important for pathogenesis of A. fumigatus in neutropenic mice resulting from treatment with cyclophosphomide and hydrocortisone. In this study, we report on the pathobiological role of gliotoxin in two different mouse strains, 129/Sv and BALB/c, that were immunosuppressed by hydrocortisone alone to avoid neutropenia. These strains of mice were infected using the isogenic set of a wild type strain (B-5233) and its mutant strain (gliPΔ) and the the glip reconstituted strain (gliPR). The gliP gene encodes a nonribosomal peptide synthase that catalyzes the first step in gliotoxin biosynthesis. The gliPΔ strain was significantly less virulent than strain B-5233 or gliPR in both mouse models. In vitro assays with culture filtrates (CFs) of B-5233, gliPΔ, and gliPR strains showed the following: (i) deletion of gliP abrogated gliotoxin production, as determined by high-performance liquid chromatography analysis; (ii) unlike the CFs from strains B-5233 and gliPR, gliPΔ CFs failed to induce proapoptotic processes in EL4 thymoma cells, as tested by Bak conformational change, mitochondrial-membrane potential disruption, superoxide production, caspase 3 activation, and phosphatidylserine translocation. Furthermore, superoxide production in human neutrophils was strongly inhibited by CFs from strain B-5233 and the gliPR strain, but not the gliPΔ strain. Our study confirms that gliotoxin is an important virulence determinant of A. fumigatus and that the type of immunosuppression regimen used is important to reveal the pathogenic potential of gliotoxin.

Human Polymorphonuclear Leukocytes Inhibit Aspergillus fumigatus Conidial Growth by Lactoferrin-Mediated Iron Depletion

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.

Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility.

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae.