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.

Potential effects of Trachyspermum copticum essential oil and propolis alcoholic extract on Mep3 gene expression of Microsporum canis isolates.

OBJECTIVE: The purpose of this study was to evaluate the effects of Trachyspermum copticum (T. copticum) essential oil and propolis alcoholic extract on growth and transcription of Mep3 gene of Microsporum canis (M. canis) strains. METHODS: The antifungal activity was assayed by broth macrodilution method. Fungal isolates were grown in soy peptone liquid medium and treated with T. copticum oil and propolis extract. Total RNAs of M. canis were subjected to reverse transcription-polymerase chain reaction (RT-PCR). Specific primers of Actin and Mep3 genes were used. RESULTS: The results revealed that MIC values of T. copticum oil against M. canis strains were ranged from 0.2-30.5 µg/mL, with 42.3% of the strains inhibited at 0.9 µg/mL. In addition, MIC values of propolis extract against M. canis strains were ranged from 0.2-488.2 µg/mL, with 34.6% of the strains inhibited at 0.9 µg/mL. RT-PCR analysis of Mep3 and Actin expression showed DNA fragments of 661 and 690 bp amplified in all isolates before treatments with T. copticum essential oil and propolis extract. Both T. copticum and propolis completely inhibited the expression of Mep3 gene. CONCLUSION: We reported for the first time that T. copticum and propolis inhibits the expression of Mep3 gene in M. canis strains in relation to a remarkable inhibition in protease production by the fungus.

Microsporum fulvum IBRL SD3: as novel isolate for chicken feathers degradation.

Keratinous wastes have increasingly become a problem and accumulate in the environment mainly in the form of feathers, generated mainly from a large number of poultry industries. As keratins are very difficult to degrade by general proteases, they pose a major environmental problem. Therefore, microorganisms which would effectively degrade keratins are needed for recycling such wastes. A geophilic dermatophyte, Microsporum fulvum IBRL SD3 which was isolated from a soil sample collected from a chicken feather dumping site using a baiting technique, was capable to produce keratinase significantly. The crude keratinase was able to degrade whole chicken feathers effectively. The end product of the degradation was protein that contained essential amino acids and may have potential application in animal feed production. Thus, M. fulvum could be a novel organism to produce keratinase for chicken feathers degradation.

Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Subtilisin Sub3 is involved in adherence of Microsporum canis to human and animal epidermis.

The aim of this study was to assess the role of the secreted keratinolytic subtilisin-like protease Sub3 in adherence of Microsporum canis to epidermis from various susceptible species, in addition to cat for which this role was recently demonstrated. Firstly, we showed by immunostaining that Sub3 is not expressed in arthroconidia from an M. canis SUB3 RNA-silenced strain but is present on the surface of arthroconidia from a SUB3 non-silenced parental strain. Secondly, comparative adherence assays using arthroconidia from both M. canis strains and skin explants from humans, dogs, horses, rabbits, guinea pigs, mice and cats revealed that only 8-16% of arthroconidia from the SUB3 silenced strain adhered to different types of epidermis when compared to the control strain. Attempts to restore fungal adherence by the addition of recombinant Sub3 failed in the tested conditions. Overall results show for the first time that Sub3 is necessary for the adherence of M. canis arthroconidia to epidermis from humans and other animal species than cat, supporting the idea that Sub3 plays a central role in colonization of keratinized host structures by M. canis, whatever the host.

Inhibition of the keratinolytic subtilisin protease Sub3 from Microsporum canis by its propeptide (proSub3) and evaluation of the capacity of proSub3 to inhibit fungal adherence to feline epidermis.

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats, dogs and humans. Proteolytic enzymes have been postulated to be key factors involved in the invasion of the stratum corneum and keratinized epidermal structures. Among these proteases, the secreted subtilisin protease Sub3 was found to be required for adherence of M. canis arthroconidia to feline epidermis. This protease is synthetized as a preproenzyme consisting of a signal peptide followed by the propeptide and the protease domain. In order to assess whether the enzymatic activity of Sub3 could be responsible for the role of the protease in the adherence process, we expressed and characterized the propeptide of Sub3 and demonstrated that this propeptide is a strong inhibitor of its mature enzyme. This propeptide acts as a noncompetitive inhibitor with dissociation constants, K(I) and [Formula: see text] of 170 and 130 nM respectively. When tested for its capacity to inhibit adherence of M. canis to feline epidermis using an ex vivo adherence model made of feline epidermis, the propeptide does not prevent adherence of M. canis arthroconidia because it loses its capacity to inhibit rSub3 following a direct contact with living arthroconidia, presumably through inactivation by fungal membrane-bound proteases.

Enzymatic activity of Microsporum canis and Trichophyton mentagrophytes from breeding rabbits with and without skin lesions.

Microsporum canis and Trichophyton mentagrophytes are zoophilic dermatophytes which can cause skin infections in animals and humans. The clinical expression of this infection strongly varies depending on host, fungal species as well as enzyme production. No comparative studies are available on the enzymatic activities of M. canis and T. mentagrophytes isolated from breeding rabbits. Thus, the aim of this work was to assess the capability of M. canis and T. mentagrophytes isolated from rabbits both with and without lesions in producing different enzymes. The relationship of dermatophyte enzymatic activities and presence/absence of skin lesions has also been investigated. A total of 260 isolates of T. mentagrophytes and 25 isolates of M. canis sampled both from healthy and lesioned skin of rabbits, as well as from air samples of positive farms were examined. The results showed that T. mentagrophytes and M. canis from rabbits produce different enzymes. However, only elastase and gelatinase were linked to the appearance of lesions in T. mentagrophytes infections, whereas lipase in those by M. canis.

Difference in keratinase activity of dermatophytes at different environmental conditions is an attribute of adaptation to parasitism.

Dermatophytes are a group of morphologically and physiologically related moulds, which cause well-defined infection called dermatophytosis. The enzymatic ability of fungi to decompose keratin has long been interpreted as a key innovation in the evolution of animal dermatology. In the present study, keratinase activity profile among Trichophyton mentagrophytes, Trichophyton rubrum, Microsporum canis and Microsporum gypseum isolated on keratin substrates such as human hair, human nail and chicken feather at variable environmental conditions of temperature, pH and metal ions was elucidated. All the above-mentioned fungal strains were isolated from soil using To-KA-Va baiting technique and keratinolytic activity was measured spectrophotometrically. In the temperature range of 30-40 °C and slightly alkaline pH (7.0-8.0), Trichophyton produced the highest activity of keratinase. It can be presumed that high enzyme production of Trichophyton species at normal body temperature range and pH could be an attribute for obligate anthropization in some dermatophytes.

Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH.

The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.

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.

Extracellular protease expression in Microsporum gypseum complex, its regulation and keratinolytic potential.

Two soil isolates of Microsporum gypseum were studied for the production of extracellular proteases. Both the strains secreted protease on glucose-gelatin medium. The enzyme activity peaked on day 15 at 28 °C. Asparagine repressed protease yield. Sugars caused catabolite repression of protease formation. Protease activities of both the isolates were significantly affected by incubation period, culture media and carbohydrates used. Both the strains grew on the skin bait and caused a gravimetrically measurable loss of the substrate. Despite less pronounced differences in the keratinase levels, great variations occurred in the amount of keratin degraded by two isolates. Keratinase production as well as loss in substrate mass was better in glucose-lacking flasks than those containing the sugar. Although the rate of keratin degradation was independent of enzyme production, statistically positive correlations were recorded between loss in substrate mass: yielded dry mycelial weight and substrate degradation: keratinase levels.

Secreted dipeptidyl peptidases as potential virulence factors for Microsporum canis.

Dermatophytoses caused by Microsporum canis are frequently encountered in cats and dogs; they are highly contagious and readily transmissible to humans. In this study, two single genes, respectively coding for dipeptidyl peptidases IV and V (DppIV and DppV), were isolated and characterized. Both proteins share homology with serine proteases of the S9 family, some of which display properties compatible with implication in pathogenic processes. Both genes are expressed in vivo in experimentally infected guinea-pigs and in naturally infected cats, and when the fungus is grown on extracellular matrix proteins as the sole nitrogen and carbon source. DppIV and V were produced as active recombinant proteases in the yeast Pichia pastoris; the apparent molecular weight of rDppV is 83 kDa, whereas rDppIV appears as a doublet of 95 and 98 kDa. Like other members of its enzymatic subfamily, rDppIV has an unusual ability to cleave Pro-X bonds. This activity does not enhance the solubilization of keratin by fungal secreted endoproteases, and the protease probably acts solely on small soluble peptides. RDppV showed no ability to induce delayed-type hypersensitivity (DTH) skin reactions in guinea-pigs, despite the known immunogenic properties of homologous proteins.

Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes.

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats and humans. The mechanisms involved in adherence of M. canis to epidermis have never been investigated. Here, a model was developed to study the adherence of M. canis to feline corneocytes through the use of a reconstructed interfollicular feline epidermis (RFE). In this model, adherence of arthroconidia to RFE was found to be time-dependent, starting at 2 h post-inoculation and still increasing at 6 h. Chymostatin, a serine protease inhibitor, inhibited M. canis adherence to RFE by 53%. Moreover, two mAbs against the keratinolytic protease subtilisin 3 (Sub3) inhibited M. canis adherence to RFE by 23%, suggesting that subtilisins, and Sub3 in particular, are involved in the adherence process.

Secreted proteases from dermatophytes.

Dermatophytes are highly specialized pathogenic fungi that exclusively infect the stratum corneum, nails or hair, and it is evident that secreted proteolytic activity is important for their virulence. Endo- and exoproteases-secreted by dermatophytes are similar to those of species of the genus Aspergillus. However, in contrast to Aspergillus spp., dermatophyte-secreted endoproteases are multiple and are members of two large protein families, the subtilisins (serine proteases) and the fungalysins (metalloproteases). In addition, dermatophytes excrete sulphite as a reducing agent. In the presence of sulphite, disulphide bounds of the keratin substrate are directly cleaved to cysteine and S-sulphocysteine, and reduced proteins become accessible for further digestion by various endo- and exoproteases secreted by the fungi. Sulphitolysis is likely to be an essential step in the digestion of compact keratinized tissues which precedes the action of all proteases.

[A study of the growth and enzymatic activity of Microsporum gypseum and Trichophyton ajelloi isolates from sewage sludge]. / Badania wzrostu i aktywnosci enzymatycznej szczepów Microsporum gypseum i Trichophyton ajelloi wyizolowanych z osadów sciekowych.

The study was to compare growth and enzymatic activity of Microsporum gypseum and Trichophyton ajelloi isolates from sewage sludge. Agar media and the API-ZYM test were used. The isolates showed weak gelatinase, catalase and urease activities and did not produce cellulase, pectate lyase and polygalacturonase. In some strains poor amylase and DNA-se activities were observed. No strain was able to hydrolyze casein. The strains were found to hydrolyze tributyrin, rapeseed oil and Biodiesel oil and to grow on Diesel oil medium. On the medium containing tributyrin and on the media with rapeseed oil and Biodiesel oil additions, inhibition and stimulation of fungal growth was observed, respectively. Diesel oil did not affect the growth of these fungi. The growth and enzymatic activity of M. gypseum was found to be better than the growth and activity of T. ajelloi. Higher enzymatic activity can be associated with the pathogenicity of M. gypseum.

RNA silencing in the dermatophyte Microsporum canis.

Dermatomycoses caused by Microsporum canis are frequent in domestic animals and easily transmissible to humans. Several proteases secreted by this fungus were identified as potential virulence factors, but the construction of deficient strains is required to investigate their role in the pathogenesis of the disease. Using target genes encoding two of these proteases, a first evaluation of the utility of RNA-mediated silencing as a reverse genetic tool in dermatophytes was carried out. SUB3 and DPPIV, respectively coding for a subtilisin and a dipeptidyl peptidase, were both down-regulated, by means of two plasmid constructs designed to express an RNA hairpin that corresponds to part of their respective sequence. The degree of attenuation was evaluated by enzymatic assay of the transformants culture supernatants, and by real-time reverse transcriptase-polymerase chain reaction. Enzymatic activities and expression levels varied from less than 5% to 100% of that of control transformants obtained with plasmid without hairpin inserts. Inhibition was globally more efficient for SUB3 than for DPPIV. These results show that RNA silencing can be used for functional genomics in M. canis, and particularly to circumvent the limits and technical difficulties of conventional disruption methods.

Extracellular proteolytic activity and molecular analysis of Microsporum canis strains isolated from symptomatic and asymptomatic cats.

Microsporum canis is the main zoophylic dermatophyte in dogs and cats, and it is also an important zoonotic agent. The literature showed that cats are asymptomatic carriers of M. canis. This is apparently due to host resistance and/or the presence of strains with lower virulence. This study was aimed to evaluate the keratinolytic, elastinolytic and collagenolytic activities of M. canis strains and their relationship with symptomatic and asymptomatic cats. In addition, these strains were analysed by RFLP. The strains isolated from cats with clinical dermatophytosis had higher keratinase and elastase activity than those isolated from asymptomatic animals (p minus than 0.05). There were not differences in RFLP patterns based on Hind III digestion.

In vitro antifungal and anti-elastase activity of some aliphatic aldehydes from Olea europaea L. fruit.

Olea europaea preparations are traditionally employed in a variety of troubles, including skin infections. Olive extracts and some of their pure compounds have shown antimicrobial activity in vitro. The present study deals with the antifungal activity of some aliphatic aldehydes from olive fruit [hexanal, nonanal, (E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal] against Tricophyton mentagrophytes (6 strains), Microsporum canis (1 strains) and Candida spp. (7 strains). The capability of these substances to inhibit elastase, a virulence factor essential for the dermatophytes colonization, and their cytotoxicity on cultures of reconstructed human epidermis, are also described. Aldehydes tested, inhibited the growth of T. mentagrophytes and M. canis in the range of concentration between <1.9 and 125 microg/ml; the unsaturated aldehydes showed the most broad spectrum of activity in that inhibited all strains tested. None of the aldehydes exhibited activity against Candida spp. strains. (E)-2-octenal and (E)-2-nonenal inhibited the elastase activity in a concentration-dependent manner; the anti-elastase activity suggests an additional target of the antimicrobial activity of these compounds. Aldehydes were devoid of cytotoxicity on cultures of human reconstructed epidermis. The antifungal activity of the aldehydes from olive fruit here reported, substantiates the use of olive and olive oil in skin diseases and suggests that these natural compounds could be useful agents in the topical treatment of fungal cutaneous infections.

[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.