Pathogenesis of Dermatophytosis: Sensing the Host Tissue.

The genera Trichophyton, Microsporum, and Epidermophyton include filamentous fungi that cause dermatophytosis, a superficial infection of the skin, stratum corneum, nail beds, and hair follicles. The ability of dermatophytes to adhere to these substrates and adapt to the host environment is essential for the establishment of infection. Several fungal enzymes and proteins participate in this adaptive response to the environment and to keratin degradation. Transcription factors such as PacC and Hfs1, as well as heat shock proteins, are involved in sensing and adapting to the acidic pH of the skin in the early stages of fungal-host interaction. During dermatophyte growth, with keratin as the sole carbon source, the extracellular pH shifts from acidic to alkaline. This creates an environment in which most of the known keratinolytic proteases exhibit optimal activity. These events culminate in the establishment and maintenance of the infection, which can be chronic or acute depending on the dermatophyte species. This review focuses on these and other molecular aspects of the dermatophyte-host interaction.

Melanogenesis in dermatophyte species in vitro and during infection.

Dermatophytes are keratinophilic fungi that are the most common cause of fungal skin infections worldwide. Melanin has been isolated from several important human fungal pathogens, and the polymeric pigment is now recognized as an important virulence determinant. This study investigated whether dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum gypseum, produce melanin or melanin-like compounds in vitro and during infection. Digestion of the pigmented microconidia and macroconidia of dermatophytes with proteolytic enzymes, denaturant and hot concentrated acid yielded dark particles that retained the size and shape of the original fungal cells. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical signal, consistent with the pigments being a melanin. Immunofluorescence analysis demonstrated reactivity of a melanin-binding mAb with the pigmented conidia and hyphae, as well as the isolate particles. Laccase, an enzyme involved in melanization, was detected in the dermatophytes by an agar plate assay using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Skin scrapings from patients with dermatophytoses contained septate hyphae and arthrospores that were reactive with the melanin-binding mAb. These findings indicate that dermatophytes can produce melanin or melanin-like compounds in vitro and during infection. Based on what is known about the function of melanin as a virulence factor of other pathogenic fungi, this pigment may have a similar role in the pathogenesis of dermatophytic diseases.

[Enzyme activities of dermatophytes isolated from different clinical samples by ApiZYM method]. / Ce,itli klinik orneklerden izole edilen dermatofitlerin enzim aktivitelerinin ApiZYM yöntemiyle tayini.

Dermatophytes' enzymes may have a role in chronic superficial infections. In this study it was planned to investigate the possible relationship between the enzymatic activities of the dermatophytes and acute or chronic course of the infections. Dermatophytes were isolated from 58 (72%) out of 81 patients with superficial infections. The infections were divided into two groups as acute (< or = 6 weeks) or chronic (> 6 weeks). Isolated fungi were identified by the classical methods and ApiZYM method. ApiZYM exhibits 19 different enzymatic activities. From 11 acute cases; five Epidermophyton floccosum, three Trichophyton rubrum, two Microsporum canis and one Trichophyton mentagrophytes strains were isolated. Of the 47 chronic cases, 96% was due to T. rubrum and 4% due to T. mentagrophytes. Production of alkaline phosphatase, leucine arylamidase and beta-glucosidase enzymes were detected in all tested strains. There was no difference between the enzymatic activities of acute and chronic cases. Alpha-mannosidase activity was detected in all of the species isolated from acute cases whereas none of the chronic cases were positive (P < 0.001). The results of this study suggest that alpha-mannosidase activity may play a role in both cutaneous inflammatory response caused by dermatophytes and the chronicity of the lesions.

Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes.

BACKGROUND: We have focused on the DNA topoisomerase II genes of pathogenic fungi and have previously applied polymerase chain reaction (PCR)-based identification of several species including the some of the major dermatophyte species. OBJECTIVE: To identify the dermatophytes (18 species) to a species level by PCR and PCR-restriction fragment length polymorphism (RFLP) techniques, without determining the nucleotide sequence. METHODS: The genomic DNAs of the dermatophytes (ten species of Trichophyton, seven species of Microsporum, and Epidermaphyton floccosum) were amplified by PCR using a common primer set (dPsD1) for the dermatophytes, followed by nested PCR using other primer sets (dPsD2, PsT and PsME) that contained primers specific for the DNA topoisomerase II genes of the dermatophytes. PCRs using PsT and PsME were used for the species-identification of Trichophyton, Microsporum and E. floccosum. The PCR products generated by dPsD2 were digested with restriction enzymes (Hinc II, Hinf, Afl II and PflM I), and the restriction profiles were analyzed. RESULTS: Of the eighteen species of dermatophytes, five species (T. rubrum, T. violaceum, M. canis, M. gypseum and E. floccosum) were specifically identified by the PCR using PsT and PsME to the species level, and the remaining species were identified by the unique restriction profiles for each species in the PCR-RFLP analysis, except that the restriction profile of T. mentagrophytes var. interdigitale was identical to that of T. mentagrophytes var. quinckeanum. CONCLUSION: PCR and PCR-RFLP techniques targeting the DNA topoisomerase II gene are simple and rapid, and quite useful as tools for the identification of dermatophytes to the species level.

Molecular taxonomy of dermatophytes and related fungi by chitin synthase 1 (CHS1) gene sequences.

In the present study, the nucleotide sequences of the CHS1 gene from dermatophytes and related fungi in the genera Chrysosporium, Epidermophyton, Microsporum and Trichophyton were investigated using molecular methods. About 440-bp genomic DNA fragments of the CHS1 gene from 21 species were amplified by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these fungi showed more than 83% similarity. The molecular taxonomy of the CHS1 gene sequences revealed that Microsporum was genetically distinct from Chrysosporium and Trichophyton, as classified by morphological characteristics.

Inhibitors of the fungal cell wall. Synthesis of 4-aryl-4-N-arylamine-1-butenes and related compounds with inhibitory activities on beta(1-3) glucan and chitin synthases.

As part of our project devoted to the search for antifungal agents, which act via a selective mode of action, we synthesized a series of new 4-aryl- or 4-alkyl-N-arylamine-1-butenes and transformed some of them into 2-substituted 4-methyl-tetrahydroquinolines and quinolines by using a novel three-step synthesis. Results obtained in agar dilution assays have shown that 4-aryl homoallylamines not possessing halogen in their structures, tetrahydroquinolines and quinolines, display a range of antifungal properties in particular against Epidermophyton floccosum and Microsporum canis. Regarding the mode of action, all active compounds showed in vitro inhibitory activities against beta(1-3) glucan-synthase and mainly against chitin-synthase. These enzymes catalyze the synthesis of beta(1-3) glucan and chitin, respectively, major polymers of the fungal cell wall. Since fungal but not mammalian cells are encased in a cell wall, its inhibition may represent a useful mode of action for these antifungal compounds.

Phylogenetic relation of Epidermophyton floccosum to the species of Microsporum and Trichophyton in chitin synthase 1 (CHS1) gene sequences.

The Nucleotide sequence of the chitin synthase 1 (CHS1) gene of Epidermophyton floccosum, an anthrophophilic dermatophyte which is the type species of the genus Epidermophyton was analyzed to determine its phylogenetic relation to eight other dermatophyte species belonging to the genera Microsporum and Trichophyton, which were sequenced in our previous studies. A genomic DNA fragment about 620 bp in length of the CHS1 gene was amplified from E. floccosum by polymerase chain reaction (PCR) and was sequenced. The CHS1 nucleotide sequence showed more than 85% similarity with sequences derived from the other dermatophytes. Phylogenetic analyses of the sequences from E. floccosum revealed that the genus Epidermophyton may be genetically distinct from Microsporum and Trichophyton.

[Dermatophytes do not produce sialidase in vitro]. / Dermatophyten bilden in vitro keine Sialidase.

Sialidase (EC 3.2.1.18) is a pathogenicity factor of many microorganisms, and may also play a role in adhesion of dermatophytes to the epithelia of their hosts by the hydrolytical cleavage of terminal, negatively charged sialic acids of glycoconjugates on the cell surfaces, thus allowing fungal lectins to bind to the subterminal sugars. Therefore, 116 strains of seven species of dermatophytes were investigated for sialidase production. Two highly sensitive, quantitative sialidase assays were applied to cell homogenates and culture supernatants from seven different media of the fungi, but were always negative for sialidase activity. However, sialidase activity was always detected in Ophiostoma stenoceras used as a positive control cultivated in parallel; the enzyme was inducible by sialylated mucins. A sialidase-dependent pathomechanism for dermatophytes appears unlikely based on the results presented.

[Exoenzymes of dermatophytes isolated from acute and chronic tinea]. / Exoenzimas de dermatofitos aislados de tiñas agudas y crónicas.

The presence of five enzymes (deoxyribonuclease, elastase, lipase, caseinase and hemolysin) in 76 strains of dermatophytes 47 of Trichophyton rubrum, 10 of T. mentagrophytes, five of T. tonsurans, 10 of Microsporum canis and four of Epidermophyton floccosum) isolated from 30 cases of acute dermatophytosis and from 46 chronic ones was determined by a qualitative plate assay; in the same way, the presence of these five enzymes with the acute and chronic dermatophytosis was correlated. It was observed that three of the enzymes were produced by the strains with a meaningful frequency; deoxyribonuclease was produced by 84.2% of the strains; elastase by 82.9%; and lipase by 65.8%. In T. rubrum the DNase was produced in 100% of strains. DNase and elastase were related to fungi which caused acute or chronic dermatophytosis in 93.3/78.2% and 96.6/74% respectively. On the other hand, lipase was present in 76% of strains, the ones that caused the chronic infections.

Brief communication: extracellular enzymatic activities of dermatophytes.

Eighty dermatophyte strains belonging to the genera Trichophyton, Microsporum, Epidermophyton and Chrysosporium were screened for their ability to produce extracellular enzymes using a semiquantitative method. The results obtained vary significantly when the studied genera are observed separately, though the enzyme beta-galactosidase was detected in none of the four studied genera.

Lipolytic activity of some dermatophytes. II. Isolation and characterisation of the lipase of Epidermophyton floccosum.

The physicochemical properties, stability and localisation of lipase from the anthropophilic dermatophyte Epidermophyton floccosum have been investigated. The lipase was best secreted by older cultures at an optimum pH of 6.5-7 in the temperature range 40-45 degrees C. Neither the skin-surface lipids nor the body temperature of man significantly affected the activity of the lipase.

Elastase: production by ringworm fungi.

Isolants of nine species of Trichophyton, one of Epidermophyton, and four of Microsporum were assayed for elastase activity. The species or isolants with elastase activity were obtained from patients with inflammatory ring-worm infection. In Nannizzia fulva (M. fulvum), plus-mating-type strains were elastase-positive and minus-mating-type strains elastase-negative. A genetic study of mating type and elastase activity indicated a monogenic basis for both mating type and elastase activity.