Pathogenic roles of extracellular proteinases from fungi are of great interest in human mycoses. In this article, our recent studies on the properties of isolated or purified extracellular proteinases of various fungi and their relationship with pathogenecity were reviewed.
(1) Candida albicans produced and released a carboxyl proteinase (CAPP) with pH optima 4.0 and Mr 42, 000. Its production was induced in the culture medium containing human stratum corneum as a nitrogen source. Cell growth was suppressed by the addition of a specific inhibitor (pepstatin). These results suggested the pathogenic roles of cell invasion and growth in candidiasis.
(2) Sporothrix schenckii produced two proteinases; proteinase I: pH optima 6.0, Mr 36, 500, serine proteinase, and proteinase II; pH optima 3.5, Mr 39, 000, carboxyl proteinase. Proteinases I and II, each other, urged cell growth compensatively both in vitro and in vivo. Topical application of proteinase inhibitors (pepstatin and chymostatin) was found to be therapeutic for the cutaneous sporotrichosis.
(3) Some properties of a proteinase from Hendersonula toluroidea (pH optima 9.0, Mr 34, 000, serine proteinase) and acid serine proteinasis from Trichophyton mentagrophytes et rubrum were also described. It is of interest that acid and alkaline proteinasis from T. rubrum were produced extracellularly in the different time of culture. Proteinase activity as a pathogenic factor may explain T. rubrum granuloma and different clinical manifestations by Nocardia asteroides et brasiliensis. Enzyme activity of T. rubrum isolated from granuloma was higher than that from superficial infection. In addition, enzyme activity of N. brasiliensis was higher than that of N. asteroides which is thought to be less pathogenic. On the other hand, a strain of N. asteroides presenting strongly pathogenic clinical features produced higher proteinase activity.

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Host defenses against Cryptococcus neoformans and Sporothrix schenckii infections were studied using congenitally athymic nude mice, their heterozygous littermates and ddY mice. In addition, effect of splenectomy upon these fungal infections is studied.
In conclusion, mononuclear cells activated with T-lymphocytes played a leading role as the defense mechanisms of mice against these fungal infections. Concerning the effect of splenectomy, yeast cells of S. schenckii began to be destroyed in the splenectomized mice approximately 5 days earlier than in the intact or sham-operated mice. On the other hand, in the mice inoculated with Cr. neoformans there was a delay of approximately 5 days in the killing functions of the granuloma in the liver of the splenectomized mice compared to the intact or sham-operated mice.

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A total of 200 soil samples from a fox reservation and 24 wild fox burrows in the eastern part of Hokkaido Prefecture were examined for keratinophilic fungi using Vanbreuseghem's hair-baiting technique. The fungi isolated were as follows: 112 strains of Trichophyton ajelloi, 48 of Microsporum cookei, 29 of Arthroderma cuniculi, 18 of Chrysosporium anamorph of A. tuberculatum, 4 of Chrysosporium tropicum, 4 of C. keratinophilum and 2 of Trichophyton terrestre.
Thity captured wild foxes in the eastern part of Hokkaido Prefecture were examined for keratinophilic fungi by hair-brushing method. Arthroderma cuniculi was obtained from the hair of 7 of the animals and Trichophyton verrucosum from one. The isolation of the latter species was epidemically important as a potential source of infection.
Arthroderma cuniculi, which seems to be associated with wild fox colonization, has been reported for the first time from Japan.

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Fifty-five strains of Candida albicans from various sources were compared in vitro with respect to their cell surface hydrophobicity and their adhesive capability to human buccal epithelial cells and a polystyrene surface. The capability of yeast to adhere to both surfaces varied markedly from strain to strain. However, there was no discernible difference in the mean number of yeast cell adhesivity to the epithelial cells between the isolates from pathological specimens and those from healthy individuals, whereas the average number of cells from the laboratory strains was significantly smaller than that of the fresh isolates. In contrast, similar adhesive capabilities to the plastic surface were obtained from the various isolates and laboratory strains. C. albicans cells, irrespective of their origin, showed low cell surface hydrophobicity with two exceptions. Yeast cell surface hydrophobicity was not closely correlated with the adhesion to epithelial cells. Further, some of the hydrophilic yeast cells adhered in considerable numbers to a polystyrene surface.

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The in vitro antifungal activity of SS717, a new topical imidazole, was studied in comparison with bifonazole (BFZ) using an agar dilution technique.
The SS717 activity toward several yeasts and molds was the highest in neutral medium, but it was lowered by an increase of inoculum size, prolongation of the incubation period and the addition of calf serum to the medium. Composition of the culture medium also significantly altered the activity of SS717. It exhibited a greater activity on casitone agar (CA) and brain heart infusion agar than on Sabouraud dextrose agar (SDA). Such a difference in SS717 activity between the two media was clearly observed in yeast-like fungi: the MIC values against clinical isolates of Candida albicans were from 10 to >20 (μg/ml) on SDA and from 0.63 to 10 (μg/ml) on CA. SS717 exhibited antifungal activity toward a wide range of pathogenic fungi, including those causing dermatophytoses and other superficial mycoses to a similar or greater extent than did BFZ.

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In order to characterize the mechanism of antifungal action of a new imidazole antimycotic, SS717, the effects of this drug on growth, viability, ergosterol biosynthesis, cell membrane permeability, and several other metabolic parameters were studied using a wild-type strain of Candida albicans (TIMM 0144) and two mutant strains therefrom which were resistant to vibunazole and had a reduced ability to synthesize ergosterol.
SS717 exhibited a similarly potent fungicidal activity at a concentration of 80μg/ml or above toward both the wild-type strain and the two mutant strains. Whereas IC₉₉ values of SS717 for the two mutant strains increased only 7 to 8 fold, their IC₅₀ values increased 82 to 129 fold over corresponding values of the wild-type strain. Biosynthesis of total lipids and alkali-insoluble glucan of the wild-type strain was inhibited by this drug to a greater extent than that of other macromolecules at concentrations below 40μg/ml. However, ergosterol biosynthesis was much more sensitive to SS717, which caused a potent inhibition even at concentrations as low as 0.08μg/ml. Apart from this effect, SS717 at concentrations above 20μg/ml induced a rapid release of intracellular K⁺ and PO₄^3- from the wild-type strain, as well as a rapid elevation of pH of an ambient medium, probably the result of K⁺ release. The extent of K⁺ release induced at drug concentrations above 20μg/ml from cells of the mutant strains was equal to or greater than that from the wild-type strain cells.
On the basis of all these results, it is suggested that SS717 at relatively low concentrations exerts fungistatic action mainly through inhibition of ergosterol biosynthesis, while at higher concentrations the drug achieves fungicidal action mainly by directly damaging the cell membrane. It is further likely that a change of the lipid composition of the cell membrane produced by the former action secondarily amplifies the latter action.

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A 43-year-old man with folliculitis barbae candidomycetica who is a methamphetamine abuser is reported. After an operation of gastric perforation and the administration of several antibiotics, many pustules emerged primarily in his bearded and whiskered area. KOH-mount examination revealed many fungal elements in the extracted beards, and histopathological study revealed dense PMN infiltration and abscess formation around a destroyed hair follicle. PAS stain showed many fungal elements of spores and pseudohyphae in inner root sheath and hair cortex. In addition, endothrix Candida-growth was verified by PAP method using monoclonal and polyclonal anti-Candida antibodies. Candida albicans type A was isolated and identified from the beard. Clotrimazole application and griseofulvin medication were both ineffective, but the skin lesions were finally successfully treated with itraconazole medication.

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New active antifungal agents with 1-(2-phenyl-1-alkenyl)-1H-imidazole structure were prepared and their structure-activity relationships were examined.
The most potential activity among these derivatives was observed in Z-2-(2, 4-dichlorophenyl)-3-methyl-1-pentenyl-1H-imidazole (coded as GBR-14206), which displayed a marked activity against not only Candida but Trichophyton. It was of particular interest to note that the antifungal profiles of these styrylimidazole derivatives were dependent on the geometry in the styryl portion; the Z-isomers were found to be more potent against Candida than the corresponding E-isomers, whereas the E-isomers were more effective for Trichophyton.
Thus, the geometry in the styryl portion of 1-(2-phenyl-1-alkenyl)-1H-imidazole derivatives is crucial for their differential antifungal potency.

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The scalps of 40 adults and 10 children without tinea capitis, which included 16 patients with tinea corporis due to Microsporum canis, and 34 family members were examined using the hairbrush method. In addition, 8 patients with tinea capitis and 21 pet cats which were sources of infection in the same homes were also investigated. The results were as follows: 1) M. canis was isolated in 32 of the 50 cases (64%) who had no scalp lesions. 2) Positive rates and number of colonies correlated with the source of infection within homes, i. e., many cats, one cat or a tinea capitis patient, in that order. The dermatophyte was not detected in families only with a patient with tinea corporis and without cats. 3) when the source of infection was treated simultaneously, scalp lesions did not develop in any of the adults. However, one case of tinea capitis developed in a three-year-old boy. 4) Positive rate and number of colonies were shown to decrease after hairwash. 5) Positive rate and number of colonies correlated with the results of the hairbrush method on the source of infection.
From the above findings we can infer that in most cases the dermatophyte exists only as a saprophyte. Moreover, if the source of infection is being addressed, we believe it is unnecessary to treat adult cases without scalp lesions. Prophylactic treatment in children may, however be necessary or advisable.

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Trichophyton rubrum a major pathogenic fungus in dermatophytosis, produced a new type of extracellular proteinase, in addition to those previously reported, when cultivated in liquid medium containing bovine serum albumin or in fetal calf serum. This enzyme had an optimal pH 4.5 for azocoll and the activity was strongly inhibited by PMSF and chymostatin. The related enzyme profiles suggested that this proteinase was one of the serine proteinases, and it was found in isolates from both tinea superficialis and granuloma trichophyticum. The proteolytic activity of isolates from granuloma trichophyticum was higher than that from tinea superficialis in both acid and alkaline proteinases, but the isolate growth from tinea superficialis was better than that from granuloma trichophyticum.

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