Glycosylphosphatidyl-inositol (GPI) -anchored mannoproteins are one of the major cell wall components of eukaryotic microorganisms, including yeast and fungi. Some GPI-anchored proteins are localized at the plasma membrane, but others are processed at the plasma membrane and are covalently linked to beta-1, 6-glucan of the cell wall through the GPI portion. The genes and enzymes responsible for their biosynthesis and cell wall assembly are potential targets of anti-fungal reagents. We identified GWT1 as a new anti-fungal drug candidate target and elucidated its function as being involved in the acylation of the inositol ring. We also found a new function of GPI7 , which is involved in transfer of ethanolamine phosphate to Man2 of GPI. Our results indicate that the localization of GPI-anchored endoglucanase Egt2p is displaced from the septal region to the cell cortex at the restrictive temperature in gpi7 mutant cells, suggesting that GPI7 is involved in the separation of mother and daughter cells and its defective phenotype is a good marker to select a new inhibitor of Gpi7 function.
We have also reported that PER1 is involved in lipid remodeling of GPI-anchored proteins, indicating that Per1p has a GPI-phospholipase A2 activity to eliminate the unsaturated fatty acyl chain at the sn-2 position of PI moiety. We further found that human PERLD1 , which is now known as an oncogene, is a functional homologue of yeast PER1 , indicating that this is a potential target for new anti-cancer drugs.

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The number of patients with invasive fungal infection (IFI) has dramatically increased since the beginning of the 1980s. Aspergillus fumigatus, the most common species recovered from aspergillosis, is an important pathogen of IFI. Recently, new antifungal agents have become available in Japan, but mortality from aspergillosis is still high. Early initiation of therapy seems to improve the survival rate. Study of virulence factors of the fungus may lead to the development of novel diagnostic tools or advancements in therapy. Many candidates of the fungal virulence factors have been studied including proteases and mycotoxins. We previously discussed the influence of fungal secondary metabolites such as gliotoxin and other low molecular components on the virulence, and showed that A. fumigatus produces potent cytotoxic substances other than gliotoxin. Studies are in progress to clarify the significance of the unknown substances.

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A pathological diagnosis can be a decisive diagnosis for deep-seated mycosis. HE stain is used to look for structural changes of the infected lesion and then various special stains are used to visualize the fungus in sections. When viability of the fungus is low, it is faintly stained with HE or PAS. Grocott stain can clearly demonstrate the fungus regardless of viability. Overstaining and understaining sometimes occur. Grocott stain is not suitable to detect structural changes of an infected lesion. Cell walls of Cryptococcus and dematiaceous fungi are stained brown with Fontana-Masson stain because of the existence of melanin. However, it is noteworthy that some Aspergillus and zygomycetes also turn brown with this stain. Fungiflora Y with a fluorescence microscope can readily demonstrate most fungal hyphae except zygomycetes. Immunohistochemistry with antibody against various fungi and in situ hybridization are useful to confirm a fungal genus on paraffin sections. Furthermore, probes to detect specific species of Aspergillus for in situ hybridization are now available.

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The number of systemic fungal infection (SFI) cases is going due to the increase in immunocompromised hosts associated with advanced chemotherapy and high-tech medical devices. Aspergillus, Candida and Cryptococcus are major etiologies of SFIs and a remarkable increase of aspergillosis has been noted. The problems of SFIs in general are lower diagnostic rate and worse prognosis compared to infections by other pathogens. Clinical concerns of SFIs are the low diagnostic rate of aspergillosis, lack of evidence for treatment of chronic aspergillosis, poor outcome of cryptococcal encephalomeningitis of immunocompromised hosts, and increasing low azole-susceptible Candida. Our final goal is to overcome these problems and to develop a newer rapid diagnostic tool for aspergillosis, establish clinical trials for chronic aspergillosis, discover new pathogenic factors of Cryptococcus and evaluate alternative treatment for low azole susceptible Candida infections. We believe research advances in these areas will be useful in the diagnosing and treatment of SFIs in the future.

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Farnesol is one of the quorum sensing molecules of Candida albicans. In this report, we discuss the effects of farnesol on: 1. growth of Candida albicans in vitro and in vivo; 2. the incorporation of biomolecules into the cell wall of Candida albicans; and 3. cytokine expression by the immune system. Our results indicate genes of Candida albicans expressed at an early stage of quorum-sensing. Half of these genes are known and two-thirds of known genes are up-regulated by two types of transcription factors.

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The intraperitoneal administration of CAWS (water-soluble extracellular polysaccharide fraction obtained from the culture supernatant of Candida albicans NBRC 1385) to mice induces coronaritis similar to Kawasaki disease. We analyzed differences in the occurrence of coronary arteritis among mouse strains, inbred strains, a closed colony, hybrids and mutants. CAWS vasculitis was induced in almost all of the inbred and closed colony strains tested, except for CBA ⁄ J mice; it was induced also in hybrids, CDF1 and BDF1. In mutant strains of various immunological defects, such as C57BL ⁄ 6J Ham Slc-bg , Balb ⁄ c nu ⁄ nu , C.B.17 ⁄ Icr-scid ⁄ scid , WBB6F1-W ⁄ W ^v mice, all induced CAWS vasculitis but a relatively weak phenotype. It has already been postulated that CAWS vasculitis is regulated by various genes, those related to acute as well as chronic inflammation. This might well reflect the clinical situation in human disease.

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Molecular biological studies of the host invasion mechanisms and possible virulence-related factors of dermatophytes have just begun. The identification of individual genes and large-scale investigations of transcripts expressed under different experimental culture conditions have provided useful information on the structure, expression, and regulation of the genes of major dermatophyte species such as Trichophyton rubrum . The next goal of dermatophytosis research will be to elucidate the functions and roles of the identified fungal genes during the infection process. It will also be necessary to investigate the host immune responses to fungal gene expression and regulation during infection. For such research, genetic manipulation techniques for dermatophytes, such as exogenous gene transfer into fungal cells and targeted gene disruption, are indispensable. However, such methods are not yet well established. We have developed an efficient dermatophyte genetic manipulation system using T. mentagrophytes . Here, we present our current research findings, mainly with regard to the system for exogenous gene transfer into T. mentagrophytes cells.

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The fungicidal activity of Cassia spectabilis leaf extracts was investigated using the disk diffusion technique and the broth dilution method. The extract showed a favorable antimicrobial activity against Candida albicans with a minimum inhibition concentration(MIC)value of 6.25 mg ⁄ ml. Apart from the fungicidal effects, imaging using scanning electron microscopy(SEM)was done to determine the major alterations in the microstructure of the C. albicans. The main abnormalities noted in the SEM studies were the alterations in morphology and complete collapse of the yeast cells after 36 h of exposure to the extract. The in vitro time-kill study performed using the leaf extract at 1 ⁄ 2, 1 or 2 times of the MIC significantly inhibited the yeast growth with a noticeable drop in optical density (OD) of yeast culture, thus confirming the fungicidal effect of the extract on C. albicans. In addition, in vivo antifungal activity studies on candidiasis in mice showed a 5-fold decrease in Candida in kidneys and blood samples in the groups of animals treated with the extract(2.5 g ⁄ kg body weight). In an acute toxicity study using mice, the acute minimum fatal dose of the extract was greater than 2000 mg ⁄ kg, and we found no histopathological changes in macroscopic examination by necropsy of mice treated with extract. We conclude that the extract may be safely used as an anticandidal agent.

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This paper presents the results of an examination for Trichophyton tonsurans(T. tonsurans)performed by the hairbrush(HB; 90 bristles)method at the All Japan Inter High School Championships, Saga 2007. Samples were taken from 487 Judo practitioners(265 males and 222 females)out of a total of 951. The areas with the highest positive rates were: Kyushu 21%(15 participants out of 73 sampled), Tohoku 17%(13 out of 77), Kinki 16%(14 out of 89), and Chubu 13%(12 out of 89). Four participants from Kyushu, four from Tohoku, two from Kinki, and two from Chubu were strongly positive carriers, their samples developing more than 30 colonies per dish. This finding is in concordance with the high HB-positive rates in these areas. The results of a questionnaire distributed during sampling showed that 90% of the examinees had heard of T. tonsurans infection, 11% had been sampled by the HB method previously, and 37% declined to receive the results of the medical examination. The low percentage of participants who had experienced a HB sampling before could be explained by the insufficient penetration of this test among dermatologists, as well as by the fact that team trainers are reluctant to expose their athletes to sampling. Although trainers' education concerning T. tonsurans is also an important factor, we strongly recommend that dermatologists take the initiative to perform medical examinations such as HB sampling in schools or at other public organizations. As for the reason why so many practitioners refused to be informed about the diagnosis, many of them mentioned being afraid that it might be overheard by trainers or fellow practitioners. It can be easily inferred that this type of concern leads Judo practitioners to avoid participation in sampling. Therefore, we concluded that substantial care to protect personal information is essential when communicating the results of the examination.

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To determine the relationships among Trichophyton species we constructed three phylogenies, based on the nucleotide sequences of the actin, rRNA and DNA topoisomerase II genes. These phylogenies showed several conflicting branch points. For example, strains of T. verrucosum,T. concentricum and T. mentagrophytes var. erinacei were mingled with strains of Arthroderma benhamiae and could not be separated into their own phylogenic groups. In addition, strains of A. vanbreuseghemii, T. tonsurans, T. mentagrophytes var. interdigitale and T. mentagrophytes var. quinckeanum were mingled with strains of A. simii and could not be separated into their own phylogenic groups. T. rubrum and T. violaceum made up a clade, which was phylogenetically related to the A. benhamiae clade or A. simii clade, depending on the gene examined. These findings indicate the need to reevaluate the boundaries among Trichophyton species using an alternative to morphological or molecular biological methods.

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Dermatophytes reside in the stratum corneum of the epidermis, and one scenario in superficial dermatophytosis is that fungi stimulate keratinocytes to secrete chemokines, thereby attracting inflammatory cells. We investigated the effect of the cytokine ⁄ chemokine production of keratinocytes solely stimulated by fungal elements. The fungal elements β -D-glucan and trichophytin from Trichophyton rubrum and Trichophyton mentagrophytes augmented production of IL-8 and IL-1 α of cultured normal human epidermal keratinocytes. It was found that keratinocytes can recognize elements of dermatophytes as a pathogen.
Next we examined the effect of liranaftate, a representative Japanese thiocarbamate antifungal agent, on the production of IL-8 and IL-1 α. Keratinocytes were incubated with this antifungal drug in the presence of β -glucan or trichophytin. Augmented production of IL-8 was profoundly suppressed by the addition of liranaftate to the culture in a dose-dependent manner. Clinically, liranaftate an antifungal drug with IL-8-decreasing activity may reduce infiltration of neutrophils in the skin and their invasion into the epidermis.

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[in Japanese]

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