Microbial pathogens use a number of genetic strategies to invade the host and cause infection. These common themes are found throughout microbial virulence factors. Secretion of enzymes, such as phospholipase, has been proposed as one of these themes which is used by bacteria, parasite, and pathogenic fungi. The role of extracellular phospholipase as a potential virulence factor in pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus has gained credence recently. In this address data implicating phospholipase as a virulence factor in Cryptococcus neoformans and Aspergillus fumigatus will be presented. This will be followed by a more detailed description of our molecular and biochemical approaches we used to more definitively delineate the role of phospholipase in the virulence of C. albicans. First, we purified the phospholipase B protein, the dominant phospholipase secreted by C. albicans, obtained the amino acid sequence of its N-terminus and an internal peptide fragment, and used this information to clone the gene encoding the protein using a PCR-based approach. Nucleotide sequence analysis revealed an ORF of 1818 bp that predicted for a pre-protein of 605 amino acid residues. The deduced amino acid sequences of the cloned gene (PLB1) showed 42.3%, 45%, and 47.8% overall sequence identity, with the reported sequences of phospholipase B cloned from Penicillium notatum, Saccharomyces cerevisiae, and Saccharomyces rosei, respectively. Second, using targeted gene disruption, URA blaster, we created C. albicans null mutants which failed to secrete phospholipase B. Third, we tested the ability of these isogenic strain pairs to cause lethality using a murine model of hematogenously disseminated candidiasis. Our data demonstrate that the parent phospholipase-producing strain caused more fatality in mice, while the null phospholipase-deficient strain was avirulent. Importantly, the parent and null mutants had similar growth and germination rates. These data prove that phospholipase B is essential for candidal virulence, and pave the way for studies directed at determining the mechanism/s through which phospholipase modulate candidal virulence. Understanding phospholipase as a common theme in fungal pathogenicity is critical for developing new antifungal strategies based on anti-virulence.

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Ethanol has been reported to cause mycelial growth in Candida tropicalis Pk233. Cultivation with ethanol in synthetic media containing glucose gave biphasic growth curves. During the first growth phase, there was an accumulation of swollen spherical yeast cells, instead of the oblong ones observed in the control culture, followed by the appearance of spherical daughter cells in chains. During the second growth phase, pseudohyphal cells appeared, projecting from the swollen yeast cells.
Subtractive cloning was performed on cDNAs from both cultures to isolate genes expressed during the first phase, correlating to the process of ethanol induced hyphal growth. Subtracted cDNAs identified by homology search included a homologue of URP2 coding ribosomal protein S20, a homologue of nmt1 coding a regulator gene working on thiamine metabolism, and a homologue of MSG5 coding tyrosine phosphatase. Roles of these cloned homologues were discussed on the process of mycelial growth in this organism.

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The major fungal pathogen for fungal diseases which have become a major medical problem in the last few years is Candida albicans, which can grow both in yeast and hyphae forms. This ability of C. albicans is thought to contribute to its colonization and dissemination within host tissues. In a recent few years, accompanying the introduction of molecular biological tools into C. albicans organism, several factors involved in the signal transduction pathway for yeast-hyphal transition have been identified. One MAP kinase pathway in C. albicans, similar to that leading to STE12 activation in Saccharomyces cerevisiae, has been reported. C. albicans strains mutant in these genes show retarded filamentous growth on a solid media but no impairment of filamentous growth in mice. These results suggest two scenarios that a kinase signaling cascade plays a part in stimulating the morphological transition in C. albicans, and that there would be another signaling pathway effective in animals. In this latter true hyphal pathway, although some candidate proteins, such as Efg1 (transcription factor), Int1 (integrin-like membrane protein), or Phr1 (pH-regulated membrane protein), have been identified, it is still too early to say that we understand the whole picture of that cascade.
We have cloned a C. albicans gene encoding a novel DNA binding protein, Rbf1, that predominantly localizes in the nucleus, and shows transcriptional activation capability. Disruption of the functional RBF1 genes of C. albicans induced the filamentous growth on all solid and liquid media tested, suggesting that Rbf1 might be another candidate for the true hyphal pathway. Relationships with other factors described above, and the target (regulated) genes of Rbf1 is under investigation.

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Multiple drug resistance is becoming a major problem in the treatment of AIDS patients with oropharyngeal candidosis. Candida albicans strains isolated from candidosis patients who do not respond to fluconazole therapy often show azole drug resistance which usually correlates with the expression of C. albicans CDR1, CDR2 or BEN^r genes, encoding potential drug efflux pumps. The objective of this study was to develop a yeast secretory vesicle transport assay and use this system to study the pumping function of Cdr1 and Ben^r.
The C. albicans CDR1 and BEN^r genes were cloned separately into plasmid pVT101-U, to form plasmids pKY1011 and pKN5001 respectively. Plasmids pVT101-U, pKY1011 and pKN5001 were transformed into Saccharomyces cerevisiae SY1, a sec6-4 mutant with a temperature-sensitive mutation in the secretory pathway. SY1 cells transformed with pKY1011 or pKN5001, were more resistant to fluconazole (MICs in both cases 64μg/ml) than SY1 cells (MIC 32μg/ml). In addition, cells transformed with pKY1011 were more resistant to cycloheximide (MIC 16μg/ml) than SY1 cells (MIC 2μg/ml).
Intact secretory vesicles were isolated from SY1 cells expressing Cdr1 and these vesicles accumulated fluconazole in a time dependent manner. These experiments demonstrated that S. cerevisiae secretory vesicles can be used to examine the mechanism of fluconazole transport by putative C. albicans membrane pumps.

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Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine, a major substrate, to phosphatidic acid and choline, and its activity is regulated by a variety of hormones, growth factors, and other extracellular signals in mammalian cells. Thus, it is now recognized as a signal transducing enzyme such as phosphatidylinositol-specific phospholipase C, adenylate cyclase, or protein tyrosine kinases. Furthermore, recent findings that regulation by members of the ADP-ribosylation factor (ARF) and Rho families of monomeric GTP-binding protein suggest roles of PLD in intracellular vesicle trafficking, morphological changes, and mitogenic signaling process. In Saccharomyces cerevisiae, PLD gene has been cloned and revealed to be essential for meiosis. In contrast, little is known about PLD in Candida albicans. As a first step to understand possible physiological roles of PLD in C. albicans, we cloned a PLD gene from a C. albicans genomic DNA library. Deduced amino acid sequence analysis showed the structural similarity to mammalian, yeast, and plant PLDs. It was also suggested employing RT-PCR (reverse transcriptase polymerase chain reaction) that an isozyme of C. albicans PLD was present.

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The Aspergillus oryzae Taka-amylase A (TAA) gene has been used as a model gene to characterize the regulatory mechanisms of gene expression in Aspergilli. TAA gene contained a typical eukaryotic promoter with a TATA box and putative regulatory elements such as a CCAAT sequence in its 5′-noncoding region. A nuclear protein designated AnCP bound to the CCAAT sequence. Replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. Although AnCP was not purified to homogeneity, AnCP appeared to have an apparent molecular mass of approximately 120 kDa.
In Saccharomyces cerevisiae, the CCAAT-binding HAP complex is a heteromeric protein comprising at least four subunits (yHAP2, yHAP3, yHAP4 and yHAP5) and is involved in the regulation of genes responsible for oxidative phosphorylation. A gene designated hapC with significant homology to yHAP3 has been isolated from A. nidulans by Hynes et al. We expressed hapC gene as a fusion protein with MalE in E. coli, purified HapC protein and prepared anti-HapC antiserum. The MalE-HapC fusion protein was able to substitute for the authentic HapC in AnCP. Furthermore, addition of the anti-HapC antiserum to the DNA binding reaction mixture retarded the mobility of the shifted band. These indicate clearly that HapC protein acts as a subunit of AnCP and that AnCP is a counterpart of the yeast Hap complex.

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We report two cases of dermatophytosis of the external auditory meatus.
Case 1: A 44-year-old man suffered from severe itching in the right external auditory meatus for a year, and had also had tinea unguium for several years. He visited our outpatient clinic because of scaly erythema which had developed on the auricle. Otoscopic examination revealed yellow-brown dry cerumen and redness from the cartilaginous to the bony portion of the external auditory meatus.
Case 2: A 14-year-old boy, the son of Case 1, suffered from severe itching in the left external auditory meatus. He scratched the auditory meatus with an earpick which his father had used. Otoscopic examination revealed a similar lesion as in the father's case, although he had no history of dermatophytosis elsewhere on his body, including the auricle.
Direct examination using a KOH method of the cerumen from both cases demonstrated numerous fungal elements. Fungal culture identified Trichophyton rubrum. Both cases were successfully treated with oral itraconazole. We suggest that infection from father to son was transferred by the earpick.

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The combined effects of lysozyme and various antifungal agents were investigated by microbroth dilution method against Candida albicans in vitro. Synergistic anti-Candida activity was observed between egg white lysozyme and itraconazole, clotrimazole, miconazole and lanoconazole. Similar, although not as clear, combination anti-Candida activities were seen in the cases of ketoconazole, bifonazole, amphotericin B and nystatin. Anti-Candida activity of fluconazole was not affected by the addition of lysozyme, however. Physiological roles of this combination effect in anti-Candida therapy by azole antifungals were discussed.

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We report cases of two 4-year-old boys with tinea capitis who attend the same daynursery. Clinically, on the first visit both lesions were a superficial type, however we initiated treatment with a dose of 50mg/day of oral itraconazole. Althougth the lesion of case 2 improved after 11 weeks of treatment, case 1 developed to kerion celsi and required continued treatment for 22 weeks for cure. Microsporum canis was isolated from the hairs of both cases and MIC of itraconazole against both isolates was 1μg/ml. We specurate that secondary bacterial infection was responsible for the development to kelion celsi in case 2.

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