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1.  Genome Variation in Cryptococcus gattii, an Emerging Pathogen of Immunocompetent Hosts 
mBio  2011;2(1):e00342-10.
Cryptococcus gattii recently emerged as the causative agent of cryptococcosis in healthy individuals in western North America, despite previous characterization of the fungus as a pathogen in tropical or subtropical regions. As a foundation to study the genetics of virulence in this pathogen, we sequenced the genomes of a strain (WM276) representing the predominant global molecular type (VGI) and a clinical strain (R265) of the major genotype (VGIIa) causing disease in North America. We compared these C. gattii genomes with each other and with the genomes of representative strains of the two varieties of Cryptococcus neoformans that generally cause disease in immunocompromised people. Our comparisons included chromosome alignments, analysis of gene content and gene family evolution, and comparative genome hybridization (CGH). These studies revealed that the genomes of the two representative C. gattii strains (genotypes VGI and VGIIa) are colinear for the majority of chromosomes, with some minor rearrangements. However, multiortholog phylogenetic analysis and an evaluation of gene/sequence conservation support the existence of speciation within the C. gattii complex. More extensive chromosome rearrangements were observed upon comparison of the C. gattii and the C. neoformans genomes. Finally, CGH revealed considerable variation in clinical and environmental isolates as well as changes in chromosome copy numbers in C. gattii isolates displaying fluconazole heteroresistance.
Isolates of Cryptococcus gattii are currently causing an outbreak of cryptococcosis in western North America, and most of the cases occurred in the absence of coinfection with HIV. This pattern is therefore in stark contrast to the current global burden of one million annual cases of cryptococcosis, caused by the related species Cryptococcus neoformans, in the HIV/AIDS population. The genome sequences of two outbreak-associated major genotypes of C. gattii reported here provide insights into genome variation within and between cryptococcal species. These sequences also provide a resource to further evaluate the epidemiology of cryptococcal disease and to evaluate the role of pathogen genes in the differential interactions of C. gattii and C. neoformans with immunocompromised and immunocompetent hosts.
PMCID: PMC3037005  PMID: 21304167
2.  Death by Edible Mushroom: First Report of Volvariella volvacea as an Etiologic Agent of Invasive Disease in a Patient following Double Umbilical Cord Blood Transplantation▿  
Journal of Clinical Microbiology  2010;48(11):4329-4332.
We describe a case of invasive fungal infection caused by Volvariella volvacea following double umbilical cord blood transplantation (UCBT). Although infections caused by several mushroom species have been documented, we believe this to be the first published report of invasive infection with Volvariella volvacea, an edible mushroom belonging to Agaricales.
PMCID: PMC3020845  PMID: 20826647
3.  Ultrastructure of the septal complex in hyphae of Cryptococcus laurentii. 
Journal of Bacteriology  1981;145(3):1410-1412.
Electron microscopy of hyphae produced by Cryptococcus laurentii revealed typical basidiomycetous dolipore septa between the cells. Parenthesomes were not observed.
PMCID: PMC217146  PMID: 7204345
4.  Heteroresistance of Cryptococcus gattii to Fluconazole▿  
We analyzed 71 clinical and environmental Cryptococcus gattii strains that had been isolated before or after the advent of azole antifungals to determine their level of heteroresistance to fluconazole (LHF). All strains of C. gattii manifested heteroresistance, with LHFs that ranged between 4 μg/ml and 32 μg/ml. A considerably higher proportion of the C. gattii strains (86%) than Cryptococcus neoformans strains (46%) exhibited LHFs that were ≥16 μg/ml. No significant correlation was observed between the molecular type or serotypes of strains and their respective LHF. The strains which expressed a higher LHF were also more resistant to xenobiotics than the strains with a low LHF, and the level of resistance to xenobiotics was significantly higher than that reported for C. neoformans. The heteroresistant subpopulation, whose level of drug resistance had been raised in a stepwise manner to 64 μg/ml, reverted to the original LHF upon daily transfers in drug-free medium. Importantly, the strains with high LHFs were significantly more virulent than those with low LHFs. Since all the clinical isolates that had not been exposed to azole drugs as well as the environmental strains manifested heteroresistance to fluconazole, heteroresistance of C. gattii to azoles is an intrinsic mechanism as in C. neoformans and is associated with the strain's virulence.
PMCID: PMC2876399  PMID: 20385871
5.  Neosartorya udagawae (Aspergillus udagawae), an Emerging Agent of Aspergillosis: How Different Is It from Aspergillus fumigatus?▿  
Journal of Clinical Microbiology  2009;48(1):220-228.
A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus. This prompted us to characterize N. udagawae in comparison to A. fumigatus. Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55°C but not at 10°C, N. udagawae is able to grow at 10°C but fails to grow at >42°C. Furthermore, compared to A. fumigatus, the conidia of N. udagawae require longer incubation periods to germinate at 37°C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91phox−/− mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.
PMCID: PMC2812273  PMID: 19889894
6.  Polyene-Resistant Mutants of Aspergillus fennelliae: Sterol Content and Genetics 
Mutants of Aspergillus fennelliae (Neosartorya fennelliae) resistant to relatively high levels of amphotericin B and low levels of nystatin were obtained by successive transfers of wild type in the presence of increasing concentrations of the polyenes. The resistance of the mutants to the polyenes was accompanied by both qualitative and quantitative changes in the sterol composition of the cells. Those resistant to amphotericin B (AF5-AB1 and p-AB1) lacked ergosterol, the major sterol of the wild type, but contained a new sterol clearly distinguished by the pattern of ultraviolet spectrophotometry and thin-layer chromatography. The mutants resistant to nystatin, however, contained both ergosterol and a new sterol, but the former was produced in a much reduced amount, as compared with the wild type. Genetic analysis indicated that the lack of ergosterol is closely associated with a reduced growth rate, poor asexual reproduction, and the loss of sexual reproduction. Growth studies revealed that the addition of ergosterol to the media did not affect the growth pattern of the mutants. Mutants resistant to amphotericin B showed an increased minimal inhibitory concentration for nystatin, pimaricin, and filipin. Mutants resistant to nystatin, however, conferred increased minimal inhibitory concentration for pimaricin and filipin but not for amphotericin B.
PMCID: PMC429054  PMID: 15828178
7.  The Presence of Capsule in Cryptococcus neoformans Influences the Gene Expression Profile in Dendritic Cells during Interaction with the Fungus▿ †  
Infection and Immunity  2008;76(4):1581-1589.
The aim of this investigation was to study the effect of polysaccharide capsule on the gene expression in dendritic cells (DC) during their interaction with Cryptococcus neoformans. To this end, we used an encapsulated virulent strain of C. neoformans and a cap59 gene-disrupted acapsular avirulent strain derived from the same genetic background. DC were exposed to encapsulated and acapsular C. neoformans strains for 4 h and 18 h, and their transcriptional profiles were analyzed using the Affymetrix mouse gene chip U74Av2. A large number of DC genes were up-regulated after treatment with the acapsular strain. In particular, we observed the up-regulation of the genes involved in DC maturation, such as cell surface receptors, cytokines, and chemokines (interleukin-12 [IL-12], IL-2, IL-1α, IL-1β, IL-6, IL-10, tumor necrosis factor alpha, CCR7, CCL17, CCL22, CCL3, CCL4, CCL7, and CXCL10), membrane proteins, and the genes involved in antigen processing and presentation as well as cell cycle or apoptosis. The chemokine gene expression data were confirmed by real-time reverse transcription-PCR, while the expression of cytokine genes was correlated with their secretion. A completely different pattern of gene expression was observed for DC treated with an encapsulated strain of C. neoformans. In particular, no significant induction was observed in the expression of the genes mentioned above. Moreover, a number of genes, such as those coding for chemokines, were down-regulated. These results suggest that the polysaccharide capsule shrouding the cell wall of C. neoformans plays a fundamental role in inducing DC response, highlighting the molecular basis of the true nature of immune silencing exerted by capsular material.
PMCID: PMC2292858  PMID: 18250173
8.  CPS1, a Homolog of the Streptococcus pneumoniae Type 3 Polysaccharide Synthase Gene, Is Important for the Pathobiology of Cryptococcus neoformans  
Infection and Immunity  2006;74(7):3930-3938.
The polysaccharide capsule is known to be the major factor required for the virulence of Cryptococcus neoformans. We have cloned and characterized a gene, designated CPS1, that encodes a protein containing a glycosyltransferase moiety and shares similarity with the type 3 polysaccharide synthase encoded by the cap3B gene of Streptococcus pneumoniae. Cps1p also shares similarity with hyaluronan synthase of higher eukaryotes. Deletion of the CPS1 gene from a serotype D strain of C. neoformans resulted in a slight reduction of the capsule size as observed by using an India ink preparation. The growth at 37°C was impaired, and the ability to associate with human brain endothelial cells in vitro was also significantly reduced by the deletion of CPS1. Using site-specific mutagenesis, we showed that the conserved glycosyltransferase domains are critical for the ability of the strain to grow at elevated temperatures. A hyaluronan enzyme-linked immunosorbent assay method demonstrated that CPS1 is important for the synthesis of hyaluronan or its related polysaccharides in C. neoformans. Comparisons between the wild-type and the cps1Δ strains, using three different transmission electron microscopic methods, indicated that the CPS1 gene product is involved in the composition or maintenance of an electron-dense layer between the outer cell wall and the capsule. These and the virulence studies in a mouse model suggested that the CPS1 gene is important in the pathobiology of C. neoformans.
PMCID: PMC1489683  PMID: 16790766
9.  Chromosomal rearrangement in Candida stellatoidea results in a positive effect on phenotype. 
Infection and Immunity  1991;59(5):1762-1771.
When type I Candida stellatoidea is plated onto sucrose agar at levels in excess of 10(8) cells, some isolates spontaneously form sucrose-positive colonies. These isolates do not display typical type I phenotypes but instead exhibit phenotypes intermediate between type I C. stellatoidea and C. albicans. Also, this phenotypic change only occurs in conjunction with a chromosomal rearrangement. These rearrangements have been studied in a strain naturally marked for methionine auxotrophy. Chromosome-size DNA bands separated by pulsed-field gel electrophoresis were probed with genes cloned from C. albicans. The hybridization pattern indicated that the genes on several chromosomes underwent extensive rearrangement.
PMCID: PMC257913  PMID: 2019440
10.  Agrobacterium tumefaciens-Mediated Transformation of Aspergillus fumigatus: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption 
Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/107 conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Δalb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.
PMCID: PMC1082565  PMID: 15812003
11.  Phenoloxidase activity and virulence in isogenic strains of Cryptococcus neoformans. 
Infection and Immunity  1982;36(3):1175-1184.
A naturally occurring Mel- variant of Cryptococcus neoformans was isolated from the wild type. The effect of phenoloxidase activity on virulence was analyzed on genetically constructed Mel+ and Mel- isolates. The traits Mel+ and virulence in mice, as measured by cumulative mortality and replication potential in brain tissue, cosegregated among the progeny of a Mel+ X Mel- cross. Revertants (MelR) isolated during the course of the cumulative mortality experiment were used to compare virulence in isogenic sets of Mel- and MelR. In two separate sets of such isolates, Mel+ phenotype and virulence coreverted. Measurement of substrate uptake and phenoloxidase activity showed that loss of detectable phenoloxidase was the basis for the Mel- phenotype and that enzyme activity reappeared in the MelR isolates. An intermediate phenotype, Melbg, was also described. Cosegregation and coreversion of the melanin phenotype and virulence suggest that phenoloxidase is a virulence factor in C. neoformans.
PMCID: PMC551454  PMID: 6807845
12.  Ultrastructure of septal complex in Filobasidiella neoformans (Cryptococcus neoformans). 
Journal of Bacteriology  1976;126(1):524-528.
Electron microscopy of Filobasidiella neoformans, the perfect state of Cryptococcus neoformans, revealed basidiomycete doliporesepta between hyphal cells and also between clamp connections and adjacent cells. The pore-occluding material was a heterogeneous flattened plate with dark margins and a lighter center, as seen in the species of Filobasidium. Representative basidiomycete parenthesomes were lacking, and endoplasmic reticulum was seen in the dolipore region.
PMCID: PMC233310  PMID: 770437
13.  Cryptococcus neoformans CAP59 (or Cap59p) Is Involved in the Extracellular Trafficking of Capsular Glucuronoxylomannan 
Eukaryotic Cell  2004;3(2):385-392.
Several genes are essential for Cryptococcus neoformans capsule synthesis, but their functions are unknown. We examined the localization of glucuronoxylomannan (GXM) in strain B-3501 and in cap59 mutants B-4131 and C536. Wild-type strain B-3501 showed a visible capsule by India ink staining and immunofluorescence with anticapsular monoclonal antibodies (MAbs) 12A1 and 18B7. B-4131, a mutant containing a missense mutation in CAP59, showed no capsule by India ink staining but revealed the presence of capsular polysaccharide on the cell surface by immunofluorescence. The cap59 gene deletion mutant (C536), however, did not show a capsule by either India ink staining or immunofluorescence. Analysis of cell lysates for GXM by enzyme-linked immunosorbent assay revealed GXM in C536 samples. Furthermore, the epitopes recognized by MAbs 12A1, 2D10, 13F1, and 18B7 were each detected in the cytoplasm of all strains by immunogold electron microscopy, although there were differences in location consistent with differences in epitope synthesis and/or transport. In addition, the cells of B-3501 and B-4131, but not those of the cap59 deletant, assimilated raffinose or urea. Hence, the missense mutation of CAP59 in B-4131 partially hampered the trafficking of GXM but allowed the secretion of enzymes involved in hydrolysis of raffinose or urea. Furthermore, the cell diameter and volume for strain C536 are higher than those for strain B-3501 or B-4131 and may suggest the accumulation of cellular material in the cytoplasm. Our results suggest that CAP59 is involved in capsule synthesis by participating in the process of GXM (polysaccharide) export.
PMCID: PMC387637  PMID: 15075268
14.  Importance of a Developmentally Regulated Pheromone Receptor of Cryptococcus neoformans for Virulence  
Infection and Immunity  2003;71(9):4953-4960.
Cryptococcus neoformans is the etiologic agent of cryptococcosis. Two mating types exist in this fungus, MATα and MATa. The CPRa gene of C. neoformans is a MATa strain-specific gene and encodes a putative seven-transmembrane domain pheromone receptor. Unlike the other reported fungal pheromone receptors, CPRa shows functional diversity. Deletion of CPRa drastically affects mating efficiency but does not abolish mating. CPRa expression is developmentally regulated and is not affected by deletion of the transcriptional regulator STE12a. The expression of CPRa is markedly increased by shifting cultures from liquid to solid media. CPRa also plays a significant role in virulence. Δcpra cells produce smaller capsules in the brains of mice than the wild-type cells, and the mice infected with Δcpra survive significantly longer than those receiving the wild-type strain. Our results suggest that the MATa pheromone receptor of C. neoformans is not only required for mating but also important for survival and growth of the fungus in host tissue.
PMCID: PMC187348  PMID: 12933837
15.  Mapping of the Cryptococcus neoformans MATα Locus: Presence of Mating Type-Specific Mitogen-Activated Protein Kinase Cascade Homologs 
Journal of Bacteriology  2000;182(21):6222-6227.
In this study we investigated the relationship between the MATα locus of Cryptococcus neoformans and several MATα-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including STE12α, STE11α, and STE20α. To resolve the location of the genes, we screened a cosmid library of the MATα strain B-4500 (JEC21), which was chosen for the C. neoformans genome project. We isolated several overlapping cosmids spanning a region of about 71 kb covering the entire MATα locus. It was found that STE12α, STE11α, and STE20α are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of MFα, the mating type α-pheromone gene, a MATα-specific myosin gene, and a pheromone receptor (CPRα) were identified within the locus. We created a physical map, based on the restriction enzyme BamHI, and identified both borders of the MATα locus. The MATα locus of C. neoformans is approximately 50 kb in size and is one of the largest mating type loci reported among fungi with a one-locus, two-allele mating system.
PMCID: PMC94759  PMID: 11029445
16.  Cryptococcus neoformans STE12α Regulates Virulence but Is Not Essential for Mating 
The Cryptococcus neoformans STE12α gene, a homologue of Saccharomyces cerevisiae STE12, exists only in mating type (MAT)α cells. In S. cerevisiae, STE12 was required for mating and filament formation. In C. neoformans, haploid fruiting on filament agar required STE12α. The ability to form hyphae, however, was not affected by deletion of STE12α when convergently growing MATa strains were present. Furthermore, ste12α disruptants were fertile when mated with MATa strains, albeit with reduced mating frequency. Most importantly, the virulence of a ste12α disruptant of serotype D strain was significantly reduced in a mouse model. When the ste12α locus was reconstituted with the wild-type allele by cotransformation, virulence was restored. Histopathological analysis demonstrated a reduction in capsular size of yeast cells, less severe cystic lesions, and stronger immune responses in meninges of mice infected with ste12α cells than those of mice infected with STE12α cells. Using reporter gene constructs, we found that STE12α controls the expression of several phenotypes known to be involved in virulence, such as capsule and melanin production. These results demonstrate a clear molecular link between mating type and virulence in C. neoformans.
PMCID: PMC2195848  PMID: 10704467
haploid fruiting; mating assay; STE12; cotransformation; virulence factor
17.  Temperature-Sensitive Strain of Cryptococcus neoformans Producing Hyphal Elements in a Feline Nasal Granuloma 
Journal of Clinical Microbiology  2000;38(2):926-928.
We report the isolation of a temperature-sensitive, serotype A, mating type α strain of Cryptococcus neoformans from a case of nasal cryptococcosis in a cat. The strain grew extremely slowly at 35°C and failed to grow at 37°C in vitro. Histopathological sections of the infected tissue revealed yeast cells producing hyphae up to several hundred micrometers in length, in addition to numerous encapsulated yeast cells typical of C. neoformans. The cultures grown on yeast extract-peptone-glucose agar at 35°C also produced some yeast cells with germ tube-like hyphal elements up to 100 μm in length.
PMCID: PMC86250  PMID: 10655419
18.  A Developmentally Regulated Gene Cluster Involved in Conidial Pigment Biosynthesis in Aspergillus fumigatus 
Journal of Bacteriology  1999;181(20):6469-6477.
Aspergillus fumigatus, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of A. fumigatus significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in A. fumigatus. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of alb1 (for “albino 1”), arp1 (for “aspergillus reddish-pink 1”), and arp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The abr1 gene (for “aspergillus brown 1”) encodes a putative protein possessing two signatures of multicopper oxidases. The abr2 gene product has homology to the laccase encoded by the yA gene of Aspergillus nidulans. The function of ayg1 (for “aspergillus yellowish-green 1”) remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in A. fumigatus. The presence of a DHN-melanin pathway in A. fumigatus was supported by the accumulation of scytalone and flaviolin in the arp1 deletant, whereas only flaviolin was accumulated in the arp2 deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in A. fumigatus. However, the presence of abr1, abr2, and ayg1 in addition to alb1, arp1, and arp2 suggests that conidial pigment biosynthesis in A. fumigatus is more complex than the known DHN-melanin pathway.
PMCID: PMC103784  PMID: 10515939
19.  Isolation, Characterization, and Localization of a Capsule-Associated Gene, CAP10, of Cryptococcus neoformans 
Journal of Bacteriology  1999;181(18):5636-5643.
Cryptococcus neoformans is a pathogenic fungus which most commonly affects the central nervous system and causes fatal meningoencephalitis primarily in patients with AIDS. This fungus produces a thick extracellular polysaccharide capsule which is well recognized as a virulence factor. Here, we describe the isolation and characterization of a novel gene, CAP10, which is required for capsule formation. Complementation of the acapsular cap10 mutant produced an encapsulated strain and the deletion of CAP10 from a wild strain resulted in an acapsular phenotype. The molecular mass of the hemagglutinin epitope-tagged Cap10p is about 73 kDa, which is similar to the size predicted from sequence analysis. When CAP10 was fused with a hybrid green fluorescent protein construct, the fluorescence signals appeared as patches in the cytoplasm. Using a reporter gene construct, we found that CAP10 was expressed at high levels in late-stationary-phase cells. In addition, we found that the expression levels of CAP10 are modulated by the transcriptional factor STE12α. Deletion of STE12α downregulated the expression levels of CAP10 while overexpression of STE12α upregulated the expression levels of CAP10. Animal model studies indicate that deletion of the CAP10 gene results in the loss of virulence, and complementation of the acapsular phenotype of cap10 restores virulence. Thus, CAP10 is required for capsule formation and virulence.
PMCID: PMC94082  PMID: 10482503
20.  Heteroresistance to Fluconazole and Voriconazole in Cryptococcus neoformans 
Cryptococcus neoformans isolates that exhibited unusual patterns of resistance to fluconazole and voriconazole were isolated from seven isolates from two different geographical regions: one isolate from an Israeli non-AIDS patient and six serial isolates from an Italian AIDS patient who had suffered six recurrent episodes of cryptococcal meningitis. Each isolate produced cultures with heterogeneous compositions in which most of the cells were susceptible, but cells highly resistant to fluconazole (MICs, ≥64 μg/ml) were recovered at a variable frequency (7 × 10−3 to 4.6 × 10−2). Evidence showed that this type of resistance is innate and is unrelated to drug exposure since the Israeli patient had never been treated with azoles or any other antimycotic agents. Analysis of clonal subpopulations of these two strains showed that they exhibited heterogeneous patterns of resistance. The number of subpopulations which grew on fluconazole or voriconazole agar declined progressively with increasing azole concentration without a sharp cutoff point. For the Italian serial isolates, the number of clonal populations resistant to fluconazole (64 μg/ml) and voriconazole (1 μg/ml) increased steadily, yielding the highest number for the isolate from the last episode. Attempts to purify a sensitive subpopulation failed, but clones highly resistant to fluconazole (100 μg/ml) and moderately resistant to voriconazole (1 μg/ml) always produced a homogeneous population of resistant cells. Upon maintenance on drug-free medium, however, the majority of the homogeneously resistant cells of these subclones lost their resistance and returned to the stable initial heteroresistant phenotype. The pattern of heteroresistance was not affected by the pH or osmolarity of the medium but was influenced by temperature. The resistance appeared to be suppressed at 35°C and was completely abolished at 40°C. Although heterogeneity in azole resistance among subpopulations of single isolates has been reported for Candida species, the transient changes in expression of resistance under different growth conditions reported here have not been observed in fungal pathogens.
PMCID: PMC89380  PMID: 10428902
21.  The Developmentally Regulated alb1 Gene of Aspergillus fumigatus: Its Role in Modulation of Conidial Morphology and Virulence 
Journal of Bacteriology  1998;180(12):3031-3038.
Aspergillus fumigatus, an important opportunistic pathogen which commonly affects neutropenic patients, produces conidia with a bluish-green color. We identified a gene, alb1, which is required for conidial pigmentation. The alb1 gene encodes a putative polyketide synthase, and disruption of alb1 resulted in an albino conidial phenotype. Expression of alb1 is developmentally regulated, and the 7-kb transcript is detected only during the conidiation stage. The alb1 mutation was found to block 1,3,6,8-tetrahydroxynaphthalene production, indicating that alb1 is involved in dihydroxynaphthalene-melanin biosynthesis. Scanning electron microscopy studies showed that the alb1 disruptant exhibited a smooth conidial surface, whereas complementation of the alb1 deletion restored the echinulate wild-type surface. Disruption of alb1 resulted in a significant increase in C3 binding on conidial surfaces, and the conidia of the alb1 disruptant were ingested by human neutrophils at a higher rate than were those of the wild type. The alb1-complemented strain producing bluish-green conidia exhibited inefficient C3 binding and neutrophil-mediated phagocytosis quantitatively similar to those of the wild type. Importantly, the alb1 disruptant had a statistically significant loss of virulence compared to the wild-type and alb1-complemented strains in a murine model. These results suggest that disruption of alb1 causes pleiotropic effects on conidial morphology and fungal virulence.
PMCID: PMC107801  PMID: 9620950
22.  Isolation of the Third Capsule-Associated Gene, CAP60, Required for Virulence in Cryptococcus neoformans 
Infection and Immunity  1998;66(5):2230-2236.
A polysaccharide capsule is one of the most important virulence factors for the pathogenic fungus Cryptococcus neoformans. We previously characterized two capsule-associated genes, CAP59 and CAP64. To further dissect the molecular mechanism of capsule synthesis, 16 acapsular mutants induced by 4-nitroquinoline-1-oxide were obtained. The acapsular phenotype of one of these mutants was complemented. The cloned gene was designated CAP60, and deletion of this newly described capsule-associated gene resulted in an acapsular phenotype. The proposed 67-kDa Cap60p contains 592 amino acids and appears to have a putative transmembrane domain close to the N terminus. DNA sequence analysis revealed that CAP60 has similarity to CAP59 at the center portion of its coding regions. Contour-clamped homogeneous electric field blot analysis suggested that these two genes are on the same chromosome. CAP60 and CAP59, however, could not be functionally substituted for each other by direct complementation or by domain swap experiments. In addition, CAP60 is closely linked to a gene which is similar to a cellulose growth-specific gene of Agaricus bisporus, CEL1. Immunogold electron microscopy studies of the epitope-tagged CAP60 gene revealed that Cap60p was primarily localized to the nuclear membrane. Animal model studies indicated that CAP60 is essential for virulence. Thus, CAP60 is required for both capsule formation and virulence.
PMCID: PMC108186  PMID: 9573112
23.  Virulence of catalase-deficient aspergillus nidulans in p47(phox)-/- mice. Implications for fungal pathogenicity and host defense in chronic granulomatous disease. 
Journal of Clinical Investigation  1998;101(9):1843-1850.
Chronic granulomatous disease (CGD) is a rare genetic disorder in which phagocytes fail to produce superoxide because of defects in one of several components of the NADPH oxidase complex. As a result, patients develop recurrent life-threatening bacterial and fungal infections. The organisms to which CGD patients are most susceptible produce catalase, regarded as an important factor for microbial pathogenicity in CGD. To test the role of pathogen-derived catalase in CGD directly, we have generated isogenic strains of Aspergillus nidulans in which one or both of the catalase genes (catA and catB), have been deleted. We hypothesized that catalase negative mutants would be less virulent than the wild-type strain in experimental animal models. CGD mice were produced by disruption of the p47(phox) gene which encodes the 47-kD subunit of the NADPH oxidase. Wild-type A. nidulans inoculated intranasally caused fatal infection in CGD mice, but did not cause disease in wild-type littermates. Surprisingly, wild-type A. nidulans and the catA, catB, and catA/catB mutants were equally virulent in CGD mice. Histopathological studies of fatally infected CGD mice showed widely distributed lesions in the lungs regardless of the presence or absence of the catA and catB genes. Similar to the CGD model, catalase-deficient A. nidulans was highly virulent in cortisone-treated BALB/c mice. Taken together, these results indicate that catalases do not play a significant role in pathogenicity of A. nidulans in p47(phox)-/- mice, and therefore raise doubt about the central role of catalases as a fungal virulence factor in CGD.
PMCID: PMC508769  PMID: 9576747
24.  A gene homologous to Saccharomyces cerevisiae SNF1 appears to be essential for the viability of Candida albicans. 
Infection and Immunity  1997;65(12):4909-4917.
The SNF1 gene of Saccharomyces cerevisiae (ScSNF1) is essential for the derepression of catabolic repression. We report here the isolation and characterization of an SNF1 homolog from Candida albicans (CaSNF1) which is apparently essential for the viability of this organism. The putative amino acid sequence of CaSNF1 has 68% identity with that of ScSNF1 and can restore the S. cerevisiae snf1 delta mutant's ability to utilize sucrose. Disruption of one of the CaSNF1 alleles resulted in morphological changes and decreased growth rates but did not modify the carbon source utilization pattern. Repetitive unsuccessful attempts to generate a snf1/snf1 homozygote by disruption of the second allele, using various vectors and approaches, suggest the lethal nature of this mutation. Integration into the second allele was possible only when a full-length functional SNF1 sequence was reassembled, further supporting this hypothesis and indicating that the indispensability of Snf1p prevented the isolation of snf1/snf1 mutants. The mutant bearing two disrupted SNF1 alleles and the SNF1 functional sequence maintained its ability to utilize sucrose and produced stellate colonies with extensive hyphal growth on agar media. It was demonstrated that in a mouse model, the virulences of this mutant and the wild-type strain are similar, suggesting that hyphal growth in vitro is not an indicator for higher virulence.
PMCID: PMC175708  PMID: 9393775
25.  Stereoselective interaction of the azole antifungal agent SCH39304 with the cytochrome P-450 monooxygenase system isolated from Cryptococcus neoformans. 
We investigated the stereoselective inhibition of growth and ergosterol biosynthesis by SCH39304 in the pathogenic fungus Cryptococcus neoformans obtained from four AIDS patients who failed fluconazole therapy and compared the results to those obtained with a wild-type strain. For all strains, the MICs of the RR isomer were approximately half those of the racemate, with the SS enantiomer showing no inhibitory activity. The 50% inhibitory concentrations for in vitro ergosterol biosynthesis correlated with the MIC data, indicating stereoselective inhibition of their target P-450 enzyme, sterol 14alpha-demethylase, as the cause of this difference. The RR enantiomer produced classical type II spectra on addition to microsomal extracts of the strains, whereas the SS enantiomer showed an absence of binding. Stereo- and regio-specific localization of N-1 substituent groups of SCH39304 within the active site of the enzyme determined the unique discrimination between its two enantiomers, and the inability to bind to sterol 14alpha-demethylase is also true of other P-450 enzymes contained in the microsomal fraction. As previously observed for other antifungal azoles, isolates obtained following failure of fluconazole therapy showed resistance to SCH39304 and its RR enantiomer. This resistance could be associated with an alteration in the sensitivity of ergosterol biosynthesis in vitro. These alterations did not cause any changes allowing the SS enantiomer to bind to the P-450 mediating sterol 14alpha-demethylation.
PMCID: PMC163941  PMID: 9210667

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