An immunoinhibitory cell wall glycoprotein (mannan) from Trichophyton rubrum.

Trichophyton rubrum causes 90% of chronic dermatophyte infections. Most patients with widespread chronic T. rubrum infection fail to express a delayed hypersensitivity reaction to intradermally injected trichophytin. We propose that cell-mediated immunity to T. rubrum may be suppressed in chronic infections by the mannan cell wall component of the fungus. The proposed suppressive effect of T. rubrum mannan on cell-mediated immunity was tested by measuring the ability of extracted mannan to inhibit lymphoproliferative responses of human mononuclear leukocytes to antigens, mitogens, and an anti-T-cell receptor antibody (anti-CD3) in vitro. Mannan was found to be highly antigenic in two of five donors and weakly antigenic in the other three. Despite its antigenic property, mannan exhibited a dose-related ability to inhibit lymphoproliferation stimulated by other agents including 1) antigens from Candida albicans, T. rubrum, and tetanus toxoid (ID50 = 250 micrograms/ml); 2) anti-CD3 antibody (ID50 = 250 micrograms/ml); and 3) Phaseolus limensis mitogenic lectin (ID50 = 64 micrograms/ml). Mannan added to cultures later than 24 h after initiation had no inhibitory influence, but culture of cells with mannan for a period of 24 h prior to the addition of stimulus enhanced the inhibitory effect of the glycoprotein. Lymphoproliferation in response to recombinant interleukin-2 (IL-2) was not inhibited. The influence of time of addition of mannan and the failure of mannan to inhibit IL-2-stimulated lymphoproliferation demonstrate that the suppressive effect of mannan must be pharmacologic rather than cytotoxic. The observed ability of T. rubrum cell wall mannan to suppress cell-mediated immune function in vitro may provide an important clue to a mechanism enabling the fungus to avoid elimination in chronically infected patients.

[Determination of guanine and cytosine contents of Trichophyton rubrum nuclear DNA with the thermal denaturation technique].

We report here on the G + C mol% content of T. rubrum DNA (including T. rubrum types I-V and an isolate from a patient with tinea pedis) as determined by a thermal denaturation technique. The results are as follows: 45.5, 57.37, 44.22, 45.38, 45.54, and 49.41 mol% for T. rubrum types I-V and isolate respectively. The results are similar to those in the literature, except that the value of T. rubrum type II is slightly higher. Our observations indicate that the morphological heterogeneity of different types of T. rubrum do not seem to be reflected in their G + C mol% values. The authors suggest that the method used here for determining G + C mol% is easy and reproducible.

Purified trichophytin: lymphocyte reactivity in vitro and trichophytin-induced suppression.

Three trichophytin preparations from different strains of Trichophyton mentagrophytes were produced according to the ethylene glycol method and assessed for their lymphocyte stimulation activity in vitro (LST). These trichophytin preparations showed significant variations in their effects on cord lymphocytes reactivity. The preparations also varied in their stimulation of lymphocytes from patients with dermatophytosis. Conclusions could be drawn about the immunological specificity, sensitivity and lymphocyte toxicity, which is important for the standardization of antigens. Furthermore, it was demonstrated that trichophytin-preincubated lymphocytes mediated a suppression of lymphocyte reactivity to this antigen.

Lipid composition of the mycelial and spore forms of Trichophyton verrucosum.

Neutral lipid composition and that of phospholipids of mycelial and spore forms of Trichophyton verrucosum were examined. It was found that arthrospores had more than twice as high content of lipids (99.3 mg/g) than the corresponding mycelial form (44.7 mg/g). Differences were also found in the qualitative composition: almost two times more neutral lipids (58.5 mg/g) and three times more phospholipids (40.8 mg/g) occurred in the spores than in the mycelial form 30.6 and 14.1 mg/g, respectively). Analysis of the neutral lipid fraction composition of both examined forms of T. verrucosum showed that the basic component were triglycerides, constituting about 70% in the spores and 44% in the mycelium of all the lipids. In the case of phospholipids no significant differences were observed between the spore and mycelial forms, phosphatidylcholine and phosphatidylethanolamine were predominating in both forms. A much higher content of lipids in the infectious form of the fungus, the arthrospores, suggest a possibility of participation of this fraction in the pathogenicity of the fungus.

New diagnostic methods in dermatological mycology.

In dermatological mycology, direct microscopy and culture are the main diagnostic procedures. Microscopic examination of a wet KOH preparation is usually satisfactory for the detection of the infecting organism based on the morphological characteristics of the fungal elements. However, examination of the KOH mount requires skill and experience. In order to help the unskilled personnel to recognize fungal elements by microscopic examination, a number of differential strains have been described. Great strides have been made to improve the sensitivity and specificity of direct microscopy for the detection of fungi also on chemical properties of the fungal cell wall. Thus fluorescence microscopy using fluorescent brighteners, e.g. Blancophor BA holds great promise for its wide application in dermatologic mycology. Culture media enjoy considerable application in the isolation and identification of dermatophytes and Candida species from clinical specimens. Some novel media have been formulated recently to assist in the early detection and identification of some fastidious dermatophytes e.g. Trichophyton verrucosum. In dermatological mycology the predictive diagnostic value of the diagnostic tests has to be delineated for each disease state. As an example, direct microscopy is a useful diagnostic procedure for the detection of dermatophytes and Pityrosporum-yeasts in clinical materials, but less valuable for the detection of Candida. Furthermore, considerable attention needs to be focused on the utilization of new and old diagnostic tests from a series of viewpoints including necessity, usefulness, clinical relevance and cost-benefit analysis.

Studies in the differentiation between Microsporum ferrugineum Ota and Trichophyton soudanense Joyeux.

A study, conducted with 20 isolates of Microsporum ferrugineum and 12 isolates of Trichophyton soudanense, revealed that some of the discrepancies in the literature regarding their characteristics and differentiation were due to methodology, strain variation and the use of an insufficient number of isolates. We found all isolates of T. soudanense to be urease negative and gelatinase positive (usually by the first week); to produce brown to black colonies on Lowenstein-Jensen medium; to rapidly decompose casein and more slowly tyrosine; to grow well or better at 37 degrees C as compared to room temperature; to produce reflexive branching on cornmeal Tween agar and abundant microconidia on casero medium and to exhibit no sexual reaction with either mating type of arthroderma simii. All but one isolate demonstrated restricted growth on lactose agar and only three isolates perforated hair. In contrast, we found 18 of 20 isolates of M. ferrugineum to be urease positive in urea broth (most isolates were negative on urea agar); all produced light-colored colonies on Lowenstein-Jensen medium; spreading colonies on lactose agar and failed to perforate hair in vitro or to produce reflexive branching. Most isolates manifested poorer to no growth at 37 degrees C compared to room temperature and all but one failed to decompose casein and tyrosine. A few strains produced macroconidia and/or microconidia on casero medium and some reacted sexually with A. simii (a) (-) mating type. Gelatin hydrolysis was variable.(ABSTRACT TRUNCATED AT 250 WORDS)

Analytical isoelectric focusing of secreted dermatophyte proteins applied to taxonomic differentiation of Microsporum and Trichophyton species (preliminary studies).

Culture filtrates were prepared from dermatophytes under standard conditions and adapted for analytical isoelectric focusing in thin layer polyacrylamide gels over the pH range 3.5-9.5. Dermatophytes grown in trypticase soy broth secreted a large number of proteins displaying a wide range of isoelectric points (pIs). Trichophyton megninii extracts contained a triplet of proteins focusing in the pH 8.0-8.5 range that were absent in taxonomically related T. kuryangei isolates. Single ascospore isolates and standard tester strains of Nannizzia otae (+) mating type were differentiated from the (-) mating type by proteins focusing at pH 6.5 and 8.4. These were markedly reduced in the (+) type. The isofocused pattern of Microsporum canis conformed closely to the (-) mating type of N. otae. The protein patterns of T. megninii and T. kuryangei were distinct from those obtained with M. canis and M. equinum because of an intense-staining broad protein band, pI 7.2, and three periodic acid-Schiff-positive glycoproteins focusing in the acidic range which were absent in the Microsporum species. A characteristic protein or doublet (pI 8.7) was present in the Microsporum species and absent in the Trichophyton species. Analytical isoelectric focusing is a potentially useful method to distinguish inter- and intra-species differences in the pattern of secreted dermatophyte proteins present in culture filtrates and in trichophytins. The information derived may be useful in the classification of species.

"Nonfibrillar" chitin associated with walls and septa of Trichophyton mentagrophytes arthrospores.

Two morphologically distinct forms of chitin were found in the arthrospore walls and septa of Trichophyton mentagrophytes. Two-thirds of the total wall chitin was the microfibrillar and chitinase-sensitive form. The remaining chitin existed in a previously uncharacterized "nonfibrillar" form and was insensitive to the action of Streptomyces chitinase. Exhaustive digestion of the arthrospore walls and septa with beta (1 leads to 3)-glucanase and chitinase followed by extraction with NaOH (1 N, 100 degrees C, 3 h) resulted in a fraction which retained the original wall shape. This fraction consisted of 85% N-acetylglucosamine, 2.0% galactosamine, 2.5% glucose, and 0.4% amino acids, 74% of which were lysine. Both its infrared spectrum and its X-ray diffraction pattern were almost identical to those of authentic chitin. There was no evidence of the presence of muramic acid, hexuronic acid, phosphate, or sulfate in this fraction. Its resistance to chitinase was due neither to the presence of protective wall layers or melanin nor to its close or covalent association with beta-glucan. Aside from its nonfibrillarity, this hexosamine polymer differed from authentic chitin in that it was soluble in 6 N HCl and 7.5 N NaOH. The development of this nonfibrillar chitin layer in the cell wall during arthrosporogenesis of T. mentagrophytes may be related to the arthrospores being resistant to a variety of antifungal agents.

Effect of undecanoic acid on lipid composition of Trichophyton rubrum.

Phospholipid and neutral lipid composition of undecanoic acid (UDA) resistant mutant of Trichophyton rubrum were compared with those of the UDA sensitive parent strain of this dermatophyte. When grown in glucose-peptone broth under identical conditions, contents of neutral and phospholipids in UDA resistant mutant were nearly double of those in the UDA sensitive parent strain. Glyceride, sterol, sterol ester and phospholipid contents of mycelium increased when UDA sensitive strain was grown in presence of low concentration of UDA, sufficient to cause partial growth inhibition of this strain. UDA in growth medium did not change the lipid composition of the UDA resistant mutant.

Fatty acids of dermatophytes.

The cellular fatty acids (C11-C20) from 18 species and strains within the genera Microsporum, Trichophyton, and Epidermophyton were determined by gas liquid chromatography. In addition, the effects of incubation time and temperature on fatty acid composition were investigated in selected species. The dermatophytes investigated represented anthropophilic, geophilic, and zoophilic species. Linoleic (18:2), oleic (18:1), and palmitic acid (16:0), accounted for 83.6-94.5% of the fatty acids of dermatophytes. Fatty acid composition and degree of unsaturation did not show any correlations with taxonomic status or ecological group. Incubation time influenced fatty acid composition slightly, but tendencies towards unsaturation and chain elongation were not observed. Elevated incubation temperature (37 degrees C) tended to increase the degree of total fatty acid unsaturation.

Isolation and partial characterization of immunologically reactive fractions from chitinase digested cell wall of Trichophyton mentagrophytes.

Cell wall preparations of Trichophyton mentagrophytes were digested with chitinase following which various fractions were isolated by ultrafiltration and Sephadex gel filtration. All fractions isolated contained both polysaccharide and peptide material. A correlation was seen between those fractions capable of eliciting immediate and delayed skin reactions in sensitized guinea pigs and those capable of stimulating the in vitro proliferation of lymphocytes taken from sensitized guinea pigs. These immunologically active fractions also developed precipitin lines with antiserum taken from sensitized animals. A low molecular weight fraction was found to be completely reactive immunologically (UM2(a)), and appeared to have a molecular weight in the range of 2,000--4,000 as assessed by ultrafiltration and gel filtration studies.

Characterization of immunologically active peptides from the cell wall of T. mentagrophytes.

A low molecular weight fraction from chitinase digested cell walls of T. mentagrophytes containing both polysaccharide and peptide moieties was found to have immunological reactivity at both the cellular and humoral level. This fraction (UM2(a)) was further degraded by treatment with either a combination of pronase and carboxypeptidase A or with trypsin. Peptides were separated from the carbohydrate-rich fraction by ultrafiltration. The carbohydrate-rich fraction retained the ability to induce both immediate and delayed skin reactions in sensitized guinea pigs and to stimulate the proliferation of sensitized lymphocytes in vitro. The peptide moieties retained reactivity in that they caused delayed reactions and lymphocyte proliferation but were unable to induce immediate or Arthus reactions in sensitized animals. Tryptic peptides from UM2(a) were purified by ion exchange chromatography. A high proportion of these peptides demonstrated immunological activity at both the cellular and humoral level since they were capable of inducing delayed reactions and/or lymphocyte transformation, as well as being capable of blocking the complement fixation reaction between UM2(a) and specific antiserum.

Immunological cross-reactivity between a glycoprotein isolated from Trichophyton mentagrophytes and human isoantigen A.

A glycoprotein isolated from the cell wall of Trichophyton mentagrophytes was assessed for its cross-reaction with human blood group isoantigens. Rabbit antiglycoprotein antibodies agglutinated human erythrocytes of blood groups A1 and A2, and precipitated Blood Group Substance A in agarose gels. Erythrocytes of blood group B were only slightly agglutinated, and O(Rho+) and O(Rho-) erythrocytes were not. Additionally, the glycoprotein was shown to specifically inhibit isoagglutination of erythrocytes of group A. Partial identity between the glycoprotein and a crude extract of the fungus was demonstrated by immunodiffusion. Analyses revealed the glycoprotein to be composed of approximately 17% protein and 80% carbohydrate. The glycoprotein was found by indirect immunofluorescence to be located in the mycelial cell wall. The possibility that cross-reacting antigens may lead to a chronic, spreading infection is discussed.

Carbohydrate composition and taxonomy of Geotrichum, Trichosporon and allied genera.

The yeast-like genera Geotrichum and Trichosporon are heterogeneous and are related with anamorphs of both ascomycetous and basidiomycetous fungi. A rearrangement can be obtained using carbohydrate composition of intact cells, studied with the aid of gas-liquid chromatography. The genus Geotrichum is restricted to ascomycetous species with a dominance of galactomannans, whereas Trichosporon is reserved for basidiomycete-like, xylose-containing species. Consequently, some new combinations are introduced in both genera. Representatives of related genera are included for comparison: e.g. Dipodascus, Hyphophichia, Cryptococcus and Filobasidium.

Architecture and chemistry of microconidial walls of Trichophyton mentagrophytes.

The ultrastructure and chemical composition of the walls of Trichophyton mentagrophytes microconidia were investigated with particular emphasis on the localization of the major structural components within the walls. The walls consisted of carbohydrate (56.1% neutral polysaccharide, and 16.0% chitin), protein (22.6%), lipid (6.5%), ash (1.7%), and trace amounts of melanin (0.2%) and phosphorus (0.2%). in thin sections, three distince layers were recognized. The electron-transparent pellicle (15 to 20 nm thick) covering the outermost surface of the wall consisted of a glycoprotein-lipid complex and was mostly extracted by sodium phosphate buffer (0.1 M, pH 6.5) containing 8 M urea, 1% (vol/vol) mercaptoethanol, and 1% (wt/vol) sodium dodecyl sulfate. The middle electron-dense layer (30 to 50 nm thick) represented the proteinaceous rodlet layer embedded in polysaccharides and could be completely solubilized by hot alkali extraction (1 N NaOH, 100 DEGREES C, 1 h). The thick inner layer (200 to 300 nm thick) was relatively resistant to the above treatments and was found to consist of amorphous glucans and microfibrillar chitin. Approximately half of the inner wall glucans was susceptible to (1 leads to 3)-beta-glucanase.