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Summary: Conidial pigment is an important virulence factor in Aspergillus fumigatus, a human fungal pathogen. The biosynthetic gene cluster for 1,8‐dihydroxynaphthalene (DHN)‐melanin in A. fumigatus consists of six genes, alb1, ayg1, arp1, arp2, abr1 and abr2. In contrast to black DHN‐melanin fungi such as Magnaporthe grisea, the polyketide synthase Alb1p in A. fumigatus produces naphthopyrone YWA1 instead of 1,3,6,8‐THN (T4HN) and YWA1 is converted to T4HN by Ayg1p. The yeast transformant expressing Alb1p and Arp1p dehydratase produced an unknown compound which was identified to be a novel angular naphthopyrone named YWA3 formed from YWA1. In addition, the amount of YWA3 produced was much more than that of YWA2 formed by non‐enzymatic dehydration from YWA1. To further analyse the reaction in vitro, Arp1p was overexpressed in E. coli and purified. Kinetic analysis revealed Km value of Arp1p for YWA1 to be 41 μM which is comparable with that of Ayg1p for YWA1 in conversion to T4HN. The complex structure modelling well explained the mechanism of YWA3 generation by the dehydration of angular YWA1 by Arp1p. These results indicated the possibility that polymerization of angular naphthopyrone YWA3 but not YWA2 could be involved in the characteristic bluish‐green conidial pigmentation of A. fumigatus. [ABSTRACT FROM AUTHOR]

Cryptococcus neoformansexists in two mating typesMATaandMATα.Although the morphology, growth characteristics and genetic segregation patterns amongMATaandMATα strains are indistinguishable in the laboratory, the predominance ofMATα strains in nature suggests thatMATα strains are better suited for survival in nature. We disrupted theTUP1gene, a global repressor, to find the possible biological differences in congenicMATα andMATacells ofC. neoformans.Disruption ofTUP1affected neither the yeast nor the hyphal cell morphology but resulted in a similar reduction of mating frequencies in bothMATα andMATacells. Disruption ofTUP1, however, functionally manifested itself in several mating type-dependent phenotypes: (i)MATα cells became more sensitive to 0.8 M KCl whileMATacells showed no change in sensitivity, (ii) a temperature-dependent growth reduction was exhibited at both 30°C and 25°C inMATabut a similar growth reduction was not observed inMATα cells until the temperature was lowered to 25°C and (iii) the transcriptional level of genes in several different biological pathways was markedly altered in a mating type-dependent manner. This work is the first case in which non-mating-related biological differences are observed between two congenic mating partners in yeast. [ABSTRACT FROM AUTHOR]

Ctyptococcus gattii causes life-threatening infection of the pulmonary and central nervous systems in hosts with normal immunity and traditionally has been considered to be restricted geographically to tropical and subtropical climates. The recent outbreak of C gaitii in the temperate climate of Vancouver Island, BC, Canada, led to a collaborative investigation. The objectives of the current study were to ascertain the environmental source of the outhreak infections, survey the molecular types of the outbreak and environmental cryptococcal isolates, and determine the extent of genetic diversity among the isolates. PCR-fingerprinting and amplified fragment length polymorphism (AFLP) were used to examine the genotypes, and mating assays were performed to determine the mating type of the isolates. All outbreak and environmental isolates belonged to C gattii Concordant results were obtained by using PCR-fingerprinting and AFLP analysis. The vast majority of clinical and veterinary infections were caused by isolates of the molecular type VGII/AELP6, but two were caused by molecular type VGI/AFLP4. All environmental isolates belonged to molecular type VGII/AFLP6. Two or three subtypes were observed within VGII/AFLP6 among outbreak and environmental isolates. All mating-competent isolates were of the α-mating type. The emergence of this usually tropical pathogen on Vancouver Island highlights the changing distribution of this genotype and emphasizes the importance of an ongoing collaborative effort to monitor the global epidemiology of this yeast. [ABSTRACT FROM AUTHOR]

The STE12α gene of Cryptococcus neoformans encodes a protein containing both homeodomain and zinc finger regions. As homeodomains and zinc finger regions are important domains for the function of many transcription factors, we used site-specific mutagenesis to delineate the roles of these two domains. The homeodomain and zinc finger regions are each important for the function of Ste12αp. DNA binding ability, mating frequency, and haploid fruiting capability were reduced in strains with mutations in the homeodomain, whereas virulence and capsule size in the mouse brain were increased. In contrast, mutations in the zinc fingers region resulted in decreased virulence, reduced capsule size in the mouse brain and decreased gene expression of capsule associated genes. In addition, phospholipase activity was increased in the zinc finger mutants. Taken together, most of the phenotypes previously observed in the ste12α deletion strains were reproduced in these two types of mutants. However, unlike mutations in the homeodomain/zinc finger region, complete deletion of STE12α caused a severe reduction in virulence and a decrease in phospholipase activity. These data suggest that region(s) other than the homeodomain and zinc finger regions of Ste12αp contribute to the variable influences on the different phenotypes observed in C. neoformans. [ABSTRACT FROM AUTHOR]

An 8-year-old boy who had chronic granulomatous disease developed a soft tissue infection of the right heel after riding on a motor scooter. Infection was insidious, and minor heel pain and fevers occurred only on the day interferon-γ was injected. Soft tissue biopsy showed hyphal elements, and Paecilomyces varioti grew in culture. The infection was treated with amphotericin B for 7 weeks (total dose, 40 mg/kg) followed by 1 year of therapy with itraconazole (100 mg twice daily). Complete cure was achieved during the follow-up period of 10 months. [ABSTRACT FROM PUBLISHER]

Exophiala dermatitidis, one of the saprophytic dematiaceous fungi, is a rare cause of human infection that, when invasive, is nearly always fatal. Besides the more common subcutaneous infection usually caused by traumatic inoculation, infection can also spread hematogenously, in which case the organism has a distinct neurotropism. A patient with autosomal recessive chronic granulomatous disease of childhood who was found to have a progressive pulmonary and central nervous system infection with E. dermatitidis responded to an aggressive, multifaceted therapeutic approach. Scanning electron microscopy of the cultured conidiogenous cells confirmed that the manner of conidiogenesis is typical of the genus Exophiala. We report the first successful treatment of an infection involving the lungs and central nervous system by a combination of surgical resection of the pulmonary source and medical therapy with amphotericin B, flucytosine or ketoconazole, and transfused white cells, followed by a prolonged course of fluconazole. [ABSTRACT FROM PUBLISHER]

Aspergillus fumigatus is an important pathogen causing invasive pulmonary aspergillosis in immunocompromised patients. The fungus propagates by conidia, which are the infectious structures inhaled by the human host. Opsonophagocytosis is thought to contribute to clearance of the inhaled conidia, a process that is facilitated by complement deposition on conidial surfaces. We now show that conidial colour mutants exhibit significant increases in C3 binding capacity compared with wild type. A reddish-pink mutation that led to enhanced C3 binding was complemented by a cosmid clone. A 3.3 kb DNA fragment from the subsequently rescued cosmid was sufficient to restore the bluish-green conidial pigment. The bluish-green transformant exhibited a level of C3 binding similar to that of the parental strain. A gene, designated arp1, was responsible for the complementation. Comparison of the genomic and cDNA sequences of arp1 revealed that it has two introns and encodes a putative protein of 168 amino acids. Arp1 is very similar to scytalone dehydratase, an enzyme involved in 1,8-dihydroxynaphthalene-melanin synthesis in Colletotrichum lagenarium and Magnaporthe grisea. Northern hybridization analysis revealed that arp1 is developmentally regulated, being expressed during conidiation. Disruption of arp1 resulted in reddish-pink conidia and increased C3 binding. Our studies suggest that arp1 modulates the bluish-green pigmentation of conidia as well as complement deposition. [ABSTRACT FROM AUTHOR]

To determine whether Candida albicans in patients with AIDS represents a unique strain, C. albicans isolated from 24 patients with AIDS was compared with Candida isolated from 23 healthy adults. Resistance to 5-fluorocytosine, synthesis of amino acids and nucleotides, sugar use, and enzyme activity patterns were similar among isolates from the two groups. Molecular analysis revealed similar banding patterns of EeoR! restriction fragments of DNA between 2.5–3 and 6–7 kb. In addition, the frequency of a dimorphic 3.7- versus 4.2-kb band, identified by agarose gel electrophoresis and by probing Southern transfers of EcoRI digests with a cloned fragment of C. albicans DNA encoding 25S ribosomal RNA, was not significantly different between the AIDS-derived and control C. albicans. C. albicans isolated at different time points in the course ofdisease and from different sites in individual patients showed identical DNA fingerprints. The similarity in isolates of C. albicans that cause disease in AIDS patients and those present in healthy subjects suggests that the candidiasis associated with AIDS is not due to the presence ofa unique or particularly virulent strain but is likely the consequence of a defect in host defense mechanisms. [ABSTRACT FROM PUBLISHER]

Thirty-four clinical isolates of Sporothrix schenckii were studied for their temperature sensitivity and morphology in vitro. Four selected isolates were also studied for their morphology and growth rate in vivo. Conidia from 26 clinical isolates obtained from lymphocutaneous or extracutaneous lesions formed colonies at both 35 C and 37 C in vitro within five days. Conidia from eight isolates obtained from fixed cutaneous lesions (nonlymphangitic) formed colonies at 35 C but failed to do so at 37 C during a two-week incubation period. Studies of the growth rate at 25 C and morphology in vitro did not show any clear differences between the two groups of isolates. When the isolates that grew at 37 C were injected into mice by the intraperitoneal route, the organisms multiplied well in the internal organs but multiplied even better in the testes. Mice developed skin lesions on paws and tails within 72 days after injection. When the isolates that did not grow at 37 C were injected into mice by the same route, the organisms did not multiply in the internal organs but grew well in the testes. These mice did not develop any skin lesions during the 72day observation period. Epidermal or intradermal inoculation of conidia into guinea pigs showed that the isolates capable of growth at 37 C produced skin lesions with a lower concentration of conidia than the isolates not growing at 37 C. Of four additional isolates from natural sources, three grew at 37 C in vitro. [ABSTRACT FROM PUBLISHER]

We report the generation of stable plasmids constructed by inserting specific DNA sequences into previously known unstable vectors. These sequences were obtained from a DNA library recovered from a previously reported stable minichromosome created by electroporative transformation in Cryptococcus neoformans (Varma and Kwon-Chung 1994 ). A 6-kb insert from this minichromosome significantly enhanced both the frequencies at which URA5 transformants were obtained as well as the stability of their uracil prototrophy on non-selective media. A 1.5-kb sequence of this insert contained telomeric sequence repeats which when introduced into plasmids resulted in significant increases in transformation frequency. A 1081-bp sequence (STAB), present in the remainder of the insert, had an ARS-like function enhancing the episomal maintenance of plasmids in the transformants regardless of the gene ( ADE2/ URA5) used as a selection marker. [ABSTRACT FROM AUTHOR]

Invasive aspergillosis (IA) is the most serious mold infection encountered in patients with iatrogenic immunosuppression. IA is also a major cause of mortality and morbidity in individuals with primary immunodeficiency (PID). Although Aspergillus fumigatus is the most common etiologic agent of IA reported in PID patients, followed by A. nidulans, multiple poorly recognized Aspergillus species such as A. udagawae, A. quadrilineatus, A. pseudoviridinutans, A. tanneri, A. subramanianii, and A. fumisynnematus have been reported almost exclusively from patients with inborn defects in host antifungal defense pathways. Infection in PID patients exhibits patterns of disease progression distinct from those in iatrogenic immunosuppression. Specifically, the disease can be extrapulmonary and chronic with a tendency to disseminate in a contiguous manner across anatomical planes. It is also more refractory to standard antifungal therapy. This synopsis summarizes our understanding of emerging rare Aspergillus species that primarily affect patients with PIDs but not those with acquired immunodeficiencies. [ABSTRACT FROM AUTHOR]

Recent phylogenetic studies based on ribosomal RNA sequences have confirmed that the organisms traditionally treated as fungi include those that have evolved from several different lines (multiphyletic organisms), as has been suspected. Even organisms causing disease in humans represent at least two evolutional lines. Pythium insidiosum and Prototheca species are both believed to have evolved from one line, while the rest of the pathogens have evolved from another line. P. insidiosum is more closely related to red algae and diatoms than to fungi. Prototheca species, as has been previously postulated, are closer to blue-green algae and plants than to fungi. Pythiosis and protothecosis, however, will still be dealt with by medical mycologists because of the morphological and in vivo staining characteristics of the causative organisms. Molecular genetic studies have revealed that Pneumocystis carinii can best be categorized as a fungus, although questions regarding its fungal status may remain unanswered until additional information becomes available on its life cycle, nuclear division, cell-wall chemistry, nutritional uptake pattern, and lysine biosynthetic pathway as well as the ultrastructural characteristics of its cellular components such as the Golgi complex. The phylogeny of the agents of lobomycosis and rhinosporidiosis, although they are treated as fungi, remains unknown. Although there is no in vitro culture system for Loboa loboi and Rhinosporidium seeberi at present, a molecular approach would allow us to reveal their phylogenetic relationship, and we can hope that such attempts are forthcoming. [ABSTRACT FROM PUBLISHER]