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Aspergillus fumigatus grows optimally from 37 to 42 degrees C but can grow at temperatures up to 55 degrees C. To study the genetic basis of thermotolerance and its role in virulence of A. fumigatus, temperature sensitive mutants were isolated. One of the mutants that grew at 42 degrees C but not at 48 degrees C was complemented and the gene, THTA, was identified. Deletion of THTA showed the same temperature sensitivity as the original mutant. THTA encodes a putative protein of 141 kDa with unknown function and the HA-tagged ThtAp accumulated to similar levels in cultures grown at either 37 or 48 degrees C. Southern blot analysis and database searches revealed the presence of THTA-related sequences in several other ascomycetous fungi. No difference in virulence was observed between the deltathtA and wild-type strains. Thus, THTA is essential for growth of A. fumigatus at high temperatures but does not contribute to the pathogenicity of the species.

Under appropriate conditions, haploid Cryptococcus neoformans cells can undergo a morphological switch from a budding yeast form to develop hyphae and viable basidiospores, which resemble those produced by mating. This process, known as haploid fruiting, was previously thought to occur only in MATalpha strains. We identified two new strains of C. neoformans var. neoformans serotype D that are MATa type and are able to haploid fruit. Further, a MATa reference strain, B-3502, also produced hyphae and fruited after prolonged incubation on filament agar. Over-expression of STE12a dramatically enhanced the ability of all MATa strains tested to filament. Segregation analysis of haploid fruiting ability confirmed that haploid fruiting is not MATalpha-specific. Our results indicate that MATa cells are intrinsically able to haploid fruit and previous observations that they do not were probably biased by the examination of a small number of genetically related isolates that have been maintained in the laboratory for many years.

In this study we investigated the relationship between the MATalpha locus of Cryptococcus neoformans and several MATalpha-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including STE12alpha, STE11alpha, and STE20alpha. To resolve the location of the genes, we screened a cosmid library of the MATalpha 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 MATalpha locus. It was found that STE12alpha, STE11alpha, and STE20alpha are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of MFalpha, the mating type alpha-pheromone gene, a MATalpha-specific myosin gene, and a pheromone receptor (CPRalpha) were identified within the locus. We created a physical map, based on the restriction enzyme BamHI, and identified both borders of the MATalpha locus. The MATalpha 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.

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.

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.

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.

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.

The SNF1 gene product, a serine/threonine protein kinase, is a global regulatory protein which has been isolated from several organisms. In Saccharomyces cerevisiae the SNF1 gene product is essential for the derepression of glucose repression since snf1 strains are unable to utilize sucrose, galactose, maltose, melibiose, or nonfermentable carbohydrates. Moreover, the SNF1 gene product was suggested to interact with additional regulatory pathways and to affect the expression of multiple target genes as reflected by the pleiotropic nature of the snf1 mutation. Here we report the characterization of the SNF1 homolog of Candida glabrata, a pathogenic yeast phylogenetically related to S. cerevisiae. The carbon utilization spectrum of C. glabrata is considerably narrower than that of other pathogenic yeasts, and the majority of the strains utilize solely glucose and trehalose from among 20 of the most commonly tested carbohydrates. Disruption of the C. glabrata SNF1 homolog resulted in the loss of the ability to utilize trehalose, indicating that even in an organism with such a limited carbon utilization spectrum, the regulatory mechanism governing catabolic repression is preserved.

The extracellular polysaccharide capsule produced by Cryptococcus neoformans is essential for its pathogenicity. We have isolated and characterized a gene, (AP64, which is required for capsule formation. An encapsulated strain created by complementation of the cap64 mutation produced fatal infection of mice within 25 days, while the cap64 acapsular strain was avirulent. Gene deletion of CAP64 from a wild-type strain resulted in the loss of capsule as well as virulence. Contour-clamped homogeneous electric field gel analysis indicates that CAP64 is located on chromosome III which is different from the localization of another capsule-related gene, CAP59. The nonlinkage between CAP64 and CAP59 was also supported by classical recombinational analysis. Database searches did not reveal any sequence with high similarity to CAP64. We also found that the CAP64 locus is contiguous to a convergently transcribed gene which has significant similarity to the gene encoding the yeast proteasome subunit, PRE1. The distance between the cDNA ends of these two genes is only 22 bp. This study confirms the previous molecular genetic evidence that capsule is an essential factor for the virulence of C. neoformans in the murine model.

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.

Aspergillus fumigatus produces conidia that are highly dispersable and resistant to degradation. We have sought to analyze these properties by studying the rodlets which form the outer spore coat protein. Degenerate primers based on hydrophobins in other fungi were applied to genomic DNA from A. fumigatus in PCR. A product of this reaction with similarity to an Aspergillus nidulans gene as judged by Southern hybridization was chosen for further study. Cloning and sequencing revealed a gene with two introns which encodes a protein of 159 amino acids. Structural characteristics consistent with those of other fungal hydrophobin genes, especially conserved cysteine residues, are present. The expression of the gene is limited to the developmental stages in which maturing conidiophores are present. This A. fumigatus gene, HYP1, was used to transform a mutant strain of A. nidulans that lacks rodlets. Transformants with a single copy of HYP1 expressed a rodlet layer on their conidia as observed by freeze-fracture electron microscopy.

Capsule formation plays a significant role in the pathogenicity of Cryptococcus neoformans. To study the molecular basis of capsule synthesis, the capsule-deficient phenotype of a mutant strain was complemented by transformation. A plasmid rescued from the resulting Cap+ transformant complemented a cap59 mutation which was mapped previously by classical recombination analysis. Gene deletion by homologous integration resulted in an acapsular phenotype, indicating that we have identified the CAP59 gene. The CAP59 gene was assigned to chromosome I by Southern blot analysis of contour-clamped homogeneous electric field gel electrophoresis-resolved chromosomes of C. neoformans var. neoformans. Sequence comparison of genomic and cDNA clones indicated the presence of six introns. CAP59 encoded a 1.9-kb transcript and a deduced protein of 458 amino acids. Analysis of the nucleotide sequence revealed little similarity to existing sequences in the data bank. When the capsule-deficient phenotype was complemented, the originally avirulent C. neoformans strain became virulent for mice. In addition, the acapsular strain created by gene deletion of CAP59 lost its virulence. This work demonstrates the molecular basis for capsule-related virulence and that the CAP59 gene is required for capsule formation.

We cloned the 5S rRNA gene and determined its organization in the four genes encoding rRNAs in a ribosomal DNA repeat unit of Filobasidiella neoformans, the teleomorph of Cryptococcus neoformans. The 5S rRNA gene contained 118 nucleotides and was located 1 kb upstream from the 18S rRNA gene within the 8.6-kb fragment of the ribosomal DNA repeat unit. The sequence of the 5S rRNA gene from F. neoformans was more similar to the sequence of the 5S rRNA gene from Tremella mesenterica than to the sequences of the 5S rRNA genes from Filobasidium species. The arrangement of the rRNA genes in F. neoformans closely resembles the arrangement of the rRNA genes in mushrooms such as Schizophyllum commune, Agaricus bisporus, and Coprinus cinereus in that the 5S rRNA-coding region not only is located within the repeat unit that encodes the other rRNAs but also is transcribed in the same direction as the other rRNA genes. This is the first description of the arrangement of rRNA genes in a species belonging to the Heterobasidiomycetes.

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.

Cryptococcus neoformans var. neoformans ura5 mutants were transformed with linearized or circular plasmids containing the C. neoformans orotidine monophosphate pyrophosphorylase gene. Following electroporation, randomly isolated transformants were analyzed for the mitotic and meiotic stability of uracil prototrophy. All stable transformants tested showed nonspecific ectopic integration. Uracil prototrophy in these transformants was stable through meiosis. Some of the stable transformants showed integration of both URA5 and vector sequences, while others lacked any vector sequences. Unstable transformants exhibited the presence of an autonomously replicating plasmid which had undergone significant sequence rearrangement. The autonomously replicating plasmid in the transformants was observed to be the same size or smaller than the transforming plasmid, was maintained in a linear form, and had acquired a genomic sequence(s) with homology to a sequence(s) on all the chromosomes. The conservation of a 300-bp sequence at the 5' end of the URA5 gene was observed in all the rearranged plasmids. These results suggest mechanisms of plasmid maintenance in C. neoformans that are different from those reported for other yeasts. The ura5 mutant was significantly less virulent than the wild type. The transformants did not recover virulence regardless of prototrophic stability.

A pair of congenic Cryptococcus neoformans var. neoformans strains, B-4476 (a mating type) and B-4500 (alpha mating type), that presumably differ only in mating type was constructed. This pair and their progeny, five alpha type and five a type, were tested for virulence in mice. In the parent strains as well as the progeny, alpha type was clearly more virulent than a type. In addition, death tended to occur earlier among the alpha-strain-infected mice that died than among the mice that died by infection caused by a strains. These data strongly suggest the genetic association of virulence with mating type in this human fungal pathogen.

Analysis of the karyotypes of multiple Candida albicans isolates by pulsed-field electrophoresis confirms the observation by Lasker et al. of eight chromosomes. The genes previously assigned to chromosome 1 in fact fall into two groups, one (including ADE1, SOR9, and CDC10) is linked to the ribosomal DNA genes on a chromosome called R, whereas the others are found on chromosome 1. Chromosome R varies in electrophoretic mobility among strains, usually running equal to or faster than chromosome 1 but in rare cases running slower than chromosome 1. In strain 1012A, the decreased mobility of one homolog is associated with the very large majority of the rDNA genes being on that homolog; the second homolog, with only a few copies, migrates with chromosome 2. Linkage analysis by using spheroplast fusion confirms the gene assignments made by hybridization to blots of the electrophoretic karyotype. A newly cloned gene, LYS2, hybridizes to chromosome 1.

A cDNA encoding Cryptococcus neoformans orotidine monophosphate pyrophosphorylase (OMPPase) has been isolated by complementation of the cognate Escherichia coli pyrE mutant. The cDNA was used as a probe to isolate a genomic DNA fragment encoding the OMPPase gene (URA5). By using electroporation for the introduction of plasmid DNA containing the URA5 gene, C. neoformans ura5 mutants could be transformed at low efficiency. Ura+ transformants obtained with supercoiled plasmids containing the URA5 gene showed marked mitotic instability and contained extrachromosomal URA5 sequences, suggesting limited ability to replicate within C. neoformans. Transformants obtained with linear DNA were of two classes: stable transformants with integrated URA5 sequences, and unstable transformants with extrachromosomal URA5 sequences.