Ultrastructure of protoplasts from mycelium and microconidia of Trichophyton mentagrophytes

Protoplast formation from mycelium and microconidia of Trichophyton mentagrophytes was achieved with Novozym 234. Pretreatment procedures with dithiothreitol or urea mercaptoethanol sodium lauryl sulphate before digestion with Novozym 234 greatly reduced protoplast yield from mycelium. Snail gut enzyme did not protoplasts in good yield. Scanning electron microscopy of mycelium protoplasts showed the acquired spherical shape. The plasma membrane appeared finely granular although remnants of cell wall could sometimes be observed. Transmission electron microscopy showed the cell interior of these protoplasts was plasmolysed. Microconidia treated with Novozym 234 displayed a range of cell wall digestion, with intact protoplasts showing distinct cytoplasmic organelles.     

Characterization of the cyclophilin of Trichophyton mentagrophytes

A genetic approach to cyclophilins in a dermatophyte, Trichophyton mentagrophytes, was carried out. The nucleotide and deduced amino acid sequences of the cyclophilin of T. mentagrophytes shared about 70% sequence similarity with those of Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans. However, the first 21 amino acid and the C-terminal amino acid regions of 188 to 226 of the T. mentagrophytes cyclophilin were distinct from those of the other fungal cyclophilins. The recombinant glutathione S-transferase (GST)-T. mentagrophytes cyclophilin fusion protein produced by Escherichia coli was purified. The protease digest of the fusion protein had a molecular weight of about 13 kDa and peptidyl-prolyl cis-trans isomerase (PPI) activity. This digest protein from T. mentagrophytes was confirmed to be cyclophilin by proving PPI activity.     

The role of surface carbohydrates on the interaction of microconidia of Trichophyton mentagrophytes with epithelial cells

The presence of carbohydrate-binding adhesins on the microconidia of Trichophyton mentagrophytes surface and their role on cellular interactions were investigated. Flow cytometry showed that this fungus recognizes the sugars mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 degrees C than 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature of the microconidia adhesin. The interaction of the fungus to Chinese hamster ovary epithelial cells and its glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, which express mannose and galactose, respectively, as the terminal carbohydrate on the cell surface. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside to the interaction medium, pretreatment of Lec1 and Lec2 cells with lectins Concanavalina A and Arachis hypogaea and pretreatment with sodium periodate decreased the adhesion and the endocytic index. Examination of thin section by transmission electron microscopy showed that after fungal ingestion by Lec2 cells the fungi are enclosed in a 'loose'-type vacuole while the other cells are found within a 'tight'-type membrane-bound cytoplasmic vacuole. Our results suggest the occurrence of carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. This may have a role in the adhesion process during the infectious process of dermatophytosis.     

Dermatophytosis of pigs by Trichophyton mentagrophytes

We describe the isolation of Trichophyton mentagrophytes in two family-operated farms where the animals were suffering skin ailments characterized by a swelling and a reddening of the back and flanks.This condition affected 2 and 5% respectively of the animals on the farms, the younger ones being more frequently affected.     

Trichophyton mentagrophytes infection in a raccoon

Visible ringworm infection did not appear on a male raccoon captured in Bexar County, Texas. Shaving the abdomen of the animal caused the appearance of typical ringworm infection within a few months.Trichophyton mentagrophytes was isolated from the infected area. This is the first report on theTrichophyton infection in this animal.     

Repeated Ubiquitin Genes in Trichophyton mentagrophytes

Using Trichophyton mentagrophytes cDNA as a template, we performed PCR amplification with the UB1S and UB1R primers for isolation of a partial fragment of T. mentagrophytes ubiquitin gene. We screened the T. mentagrophytes cDNA library prepared with Uni-ZAP phage vector by hybridization with the T. mentagrophytes ubiquitin fragment, UB1S-UB1R. The nucleotide and deduced amino acid sequences of T. mentagrophytes ubiquitin gene encoded two ubiquitin repeats in 162 bp to 624 bp. The deduced amino acid sequences of ubiquitin from T. mentagrophytes shared about 98% similarity with those of Saccharomyces cerevisiae, Neurospora crassa, and Cryptococcus neoformans. Received: 28 April 1999 / Accepted: 22 June 1999     

Amino acid- or protein-dependent growth of Trichophyton mentagrophytes and Trichophyton rubrum

Culture conditions were examined for Trichophyton mentagrophytes and Trichophyton rubrum, which are major pathogens involved in dermatophytosis. They grew well in Sabouraud's dextrose broth or RPMI 1640. Growth in phosphate-buffered yeast nitrogen base supplemented with glucose was very slow, although growth improved significantly with the addition of amino acids or proteins to the medium. The fungi could also grow using human nail fragments as the only source of nutrition. Examination of proteases by substrate gel electrophoresis indicated that distinct sets of proteases are secreted from the dermatophytes in two different media, Sabouraud's dextrose broth and nail fragments. A protease inhibitor, phenylmethanesulfonyl fluoride, inhibited the growth of the fungi on nail fragments, but it did not inhibit their growth in Sabouraud's dextrose broth.     

Mating Genes of the Trichophyton mentagrophytes Complex

The mating type (-)-specific gene of the alpha-box and the mating type (+)-specific gene of the high-mobility group (HMG) DNA-binding domain were confirmed in zoophilic dematophytes of Arthroderma simii and A. vanbreuseghemii. The sequence of the alpha-box gene was 1,375 bp, containing 2 exons (from 172 to 463 bp and from 513 to 1,375 bp) in the A. simii (-) mating type strain and 1,380 bp, containing 2 exons (from 177 to 468 bp and from 518 to 1,380 bp) in the A. vanbreuseghemii (-) mating type strain. The sequence of the HMG gene was 1,871 bp, containing 2 exons (from 181 to 362 bp and from 426 to 1,440 bp, coding a protein of 398 amino acids) in the A. simii (+) mating type strain and 1,811 bp containing 2 exons (from 158 to 339 bp and from 403 to 1,381 bp, coding a protein of 386 amino acids) in the A. vanbreuseghemii (+) mating type strain. Of 15 animal isolates and 72 human isolates examined, the alpha-box gene was detected in five of the animal isolates and in none of the human isolates, while the HMG gene was detected in the other 10 of the animal isolates and in all of the human isolates. Phylogenetic analysis of the alpha-box and HMG genes of Trichophyton mentagrophytes complex strains and the Microsporum gypseum strain revealed that these strains were divided into 4 clusters; the first cluster consisting of A. vanbreuseghemii and the isolates from animals and humans, the second cluster consisting of A. simii, the third cluster consisting of A. benhamiae and the fourth cluster consisting of M. gypseum. These results indicate that anthropophilic T. mentagrophytes evolved from the A. vanbreuseghemii (+) mating strain.     

Morphological response of Trichophyton mentagrophytes to keratin

Summary The protein keratin can induce a reversible change in morphology of some granular strains ofTrichophyton mentagrophytes leading to a gross appearance which is indistinguishable from the pleomorphic mutation. The only other chemical which induced the same morphological change was a low concentration of sodium acetate. Pleomorphic cultures ofT. mentagrophytes were unaffected.