RESUMEN
BACKGROUND: The T indotineae population shows a high amount of terbinafine resistant isolates based on different point mutations of squalene epoxidase erg1 (ergosterol) gene. A significant proportion of these isolates also show azole resistance. OBJECTIVES: Elucidation of the molecular mechanism for azole resistance, especially the identification of mutations in the sterol 14-α demethylase Erg11 genes, which encode for enzymes interacting with azoles. METHODS: Sequencing of putative Erg11 genes and analysis of phenotypic resistance pattern using a microplate-laser-nephelometry-based growth assay. RESULTS: Four different types of Erg11B mutants were detected; two double mutants with Ala230Thr/Asp441Gly, respectively, Ala230/Tyr444His and single mutants with Gly443Glu, Tyr444Cys and Tyr444His. All isolates featured the wild type genotype of Erg11A. All strains demonstrated different combinations of Erg1 and Erg11 genotypes. CONCLUSION: Resistance against terbinafine and azoles developed several times independently within the T indotineae population. The challenge for fungal treatment is, therefore, that species identification is not enough for prediction of therapeutic efficacy of antifungals. In the future, it will also become important to analyse genes involved in resistance mechanisms.
Asunto(s)
Farmacorresistencia Fúngica , Mutación Puntual , Escualeno-Monooxigenasa , Esterol 14-Desmetilasa , Trichophyton/genética , Antifúngicos/farmacología , Azoles/farmacología , Farmacorresistencia Fúngica/genética , Evolución Molecular , Proteínas Fúngicas/genética , Pruebas de Sensibilidad Microbiana , Escualeno-Monooxigenasa/genética , Esterol 14-Desmetilasa/genética , Terbinafina/farmacología , Trichophyton/enzimologíaRESUMEN
In recent years, cases involving terbinafine-resistant Trichophyton isolates have been reported increasingly, particularly in India. We present 14 cases of terbinafine treatment failure in Trichophyton-infected Danish patients due to acquired resistance. Patients infected with Trichophyton rubrum (n = 12) or Trichophyton interdigitale (n = 2) with elevated terbinafine MICs during 2013-2018 were included. Antifungal susceptibility testing (AFST) was performed following a modified EUCAST E.Def 9.3.1 method (5 days of incubation) with or without cycloheximide and chloramphenicol (CC) supplementation of the growth medium. The squalene epoxidase (SE) target gene was sequenced, and 3-dimensional enzyme homology modeling was performed. Most patients (12/14 [86%]) were male. The mean age was 53.5 years (range, 11 to 77 years). The mean duration of infections was 4.8 years at the time of resistance detection. Prior systemic terbinafine treatment was documented for all patients, and topical therapy for 62% (information was missing in one case). Overall, nine isolates (64%) displayed high terbinafine resistance (MICs, 4 to >8 mg/liter), while two (14%) displayed moderate (MICs, 1 to 2 mg/liter) and three (21%) displayed low (MICs, 0.125 to 0.25 mg/liter) terbinafine resistance compared with control isolates. MICs generated with or without CC supplementation were similar, but CC prevented contamination. Known and novel SE amino acid substitutions (F397L, L393F, L393S, F415S, H440Y F484Y, and I121M V237I) were detected in resistant but not control isolates. Three-dimensional homology modeling suggested a role of the novel I121M and V237I alterations. Terbinafine resistance has been detected in Denmark using a modified EUCAST method, which facilitated susceptibility testing of dermatophytes. Action is needed for this emerging public health problem.
Asunto(s)
Antifúngicos/farmacología , Terbinafina/farmacología , Trichophyton/efectos de los fármacos , Trichophyton/patogenicidad , Adolescente , Adulto , Anciano , Antifúngicos/uso terapéutico , Niño , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mutación/genética , Escualeno-Monooxigenasa/genética , Escualeno-Monooxigenasa/metabolismo , Terbinafina/uso terapéutico , Trichophyton/enzimología , Adulto JovenRESUMEN
Dermatophytes are fungi that have an ability to invade keratinised structures. Enzymes secreted by dermatophytes can underlie fungal survival on the host and development of infection. It is possible that the range of activity of keratinases from various dermatophytes is limited to specific species of animals and groups of people. The aim of this study was to carry out phenotypic analysis of the degree of keratinolytic activity of Trichophyton verrucosum strains using hairs of humans and various animal species as substrates. Our results indicated that the activity of keratinases is substrate-induced. The host range of T. verrucosum can be defined as wide. The highest activity of keratinases was recorded in media containing keratin from cow (Bos taurus) and sheep (Ovis aries) hairs in comparison with that from other tested species. The production of keratin-degrading enzymes is a function of time, with the peak of their activity occurring on day 15 of incubation. The role of keratin-degrading enzymes in the pathogenesis of dermatophytosis is becoming increasingly clearer. Given the conceptual understanding that keratin breakdown may require more than just one enzyme, the use of phenotypic methods is an optimal approach to in vitro study of the decomposition of species-specific keratin.
Asunto(s)
Cabello/metabolismo , Especificidad del Huésped , Queratinas/metabolismo , Trichophyton/enzimología , Trichophyton/metabolismo , Animales , Bovinos , Humanos , Hidrólisis , OvinosRESUMEN
Dermatophytosis, the commonest superficial fungal infection, has gained recent attention due to its change of epidemiology and treatment failures. Despite the availability of several agents effective against dermatophytes, the incidences of chronic infection, reinfection, and treatment failures are on the rise. Trichophyton rubrum and Trichophyton interdigitale are the two species most frequently identified among clinical isolates in India. Consecutive patients (n = 195) with suspected dermatophytosis during the second half of 2014 were included in this study. Patients were categorized into relapse and new cases according to standard definitions. Antifungal susceptibility testing of the isolated Trichophyton species (n = 127) was carried out with 12 antifungal agents: fluconazole, voriconazole, itraconazole, ketoconazole, sertaconazole, clotrimazole, terbinafine, naftifine, amorolfine, ciclopirox olamine, griseofulvin, and luliconazole. The squalene epoxidase gene was evaluated for mutation (if any) in 15 T. interdigitale and 5 T. rubrum isolates exhibiting high MICs for terbinafine. A T1189C mutation was observed in four T. interdigitale and two T. rubrum isolates. This transition leads to the change of phenylalanine to leucine in the 397th position of the squalene epoxidase enzyme. In homology modeling the mutant residue was smaller than the wild type and positioned in the dominant site of squalene epoxidase during drug interaction, which may lead to a failure to block the ergosterol biosynthesis pathway by the antifungal drug.
Asunto(s)
Alilamina/farmacología , Antifúngicos/farmacología , Escualeno-Monooxigenasa/metabolismo , Trichophyton/efectos de los fármacos , Trichophyton/enzimología , Alilamina/análogos & derivados , Arthrodermataceae/efectos de los fármacos , Ciclopirox/farmacología , Farmacorresistencia Fúngica/genética , Fluconazol/farmacología , Itraconazol/farmacología , Pruebas de Sensibilidad Microbiana , Morfolinas/farmacología , Escualeno-Monooxigenasa/genética , Terbinafina/farmacología , Trichophyton/genética , Voriconazol/farmacologíaRESUMEN
Recalcitrant dermatophytoses are on the rise in India. High MICs of terbinafine (TRB) and squalene epoxidase (SQLE) gene mutations conferring resistance in Trichophyton spp. have been recently documented. However, studies correlating laboratory data with clinical response to TRB in tinea corporis/cruris are lacking. For this study, we investigated the clinicomycological profile of 85 tinea corporis/cruris patients and performed antifungal susceptibility testing by CLSI microbroth dilution and SQLE mutation analysis of the isolates obtained and correlated these with the responses to TRB. Patients confirmed by potassium hydroxide (KOH) mounting of skin scrapings were started on TRB at 250 mg once a day (OD). If >50% clinical clearance was achieved by 3 weeks, the same dose was continued (group 1). If response was <50%, the dose was increased to 250 mg twice a day (BD) (group 2). If the response still remained below 50% after 3 weeks of BD, the patients were treated with itraconazole (ITR; group 3). Overall, skin scrapings from 64 (75.3%) patients yielded growth on culture. Strikingly, all isolates were confirmed to be Trichophyton interdigitale isolates by internal transcribed spacer (ITS) sequencing. Thirty-nine (61%) of the isolates had TRB MICs of ≥1 µg/ml. Complete follow-up data were available for 30 culture-positive patients. A highly significant difference in modal MICs to TRB among the three treatment response groups was noted (P = 0.009). Interestingly, 8 of the 9 patients in group 3 harbored isolates exhibiting elevated TRB MICs (8 to 32 µg/ml) and SQLE mutations. The odds of achieving cure with TRB MIC < 1 µg/ml strains were 2.5 times the odds of achieving cure with the strain exhibiting MIC ≥1 µg/ml.
Asunto(s)
Antifúngicos/farmacología , Proteínas Fúngicas/genética , Escualeno-Monooxigenasa/genética , Terbinafina/farmacología , Tiña/tratamiento farmacológico , Trichophyton/efectos de los fármacos , Administración Oral , Adolescente , Adulto , Anciano , ADN de Hongos/genética , ADN Intergénico/genética , Esquema de Medicación , Farmacorresistencia Fúngica/genética , Femenino , Proteínas Fúngicas/metabolismo , Expresión Génica , Humanos , Itraconazol/farmacología , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mutación , Técnicas de Tipificación Micológica , Escualeno-Monooxigenasa/metabolismo , Tiña/microbiología , Tiña/patología , Trichophyton/enzimología , Trichophyton/genética , Trichophyton/aislamiento & purificaciónRESUMEN
Resistance to antifungals is a leading concern in the treatment of human mycoses. We demonstrate that the salA gene, encoding salicylate 1-monooxygenase, is involved in resistance of the dermatophyte Trichophyton rubrum to terbinafine, one of the most effective antifungal drugs against dermatophytes. A strain with multiple copies of salA was constructed and exhibited elevated expression of salA and increased terbinafine resistance. This reflects a mechanism not yet reported in a pathogenic fungus.
Asunto(s)
Farmacorresistencia Fúngica/genética , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Terbinafina/farmacología , Trichophyton/efectos de los fármacos , Trichophyton/genética , Antifúngicos/farmacología , Farmacorresistencia Fúngica/efectos de los fármacos , Genes Fúngicos/genética , Pruebas de Sensibilidad Microbiana , Transcripción Genética , Transformación Genética , Trichophyton/enzimología , Regulación hacia Arriba/genéticaRESUMEN
Dermatophyte research has renewed interest because of changing human floras with changing socioeconomic conditions, and because of severe chronic infections in patients with congenital immune disorders. Main taxonomic traits at the generic level have changed considerably, and now fine-tuning at the species level with state-of-the-art technology has become urgent. Research on virulence factors focuses on secreted proteases now has support in genome data. It is speculated that most protease families are used for degrading hard keratin during nitrogen recycling in the environment, while others, such as Sub6 may have emerged as a result of ancestral gene duplication, and are likely to have specific roles during infection. Virulence may differ between mating partners of the same species and concepts of zoo- and anthropophily may require revision in some recently redefined species. Many of these questions benefit from international cooperation and exchange of materials. The aim of the ISHAM Working Group Dermatophytes aims to stimulate and coordinate international networking on these fungi.
Asunto(s)
Dermatomicosis , Hongos , Animales , Arthrodermataceae/clasificación , Arthrodermataceae/enzimología , Arthrodermataceae/inmunología , Arthrodermataceae/patogenicidad , Biodiversidad , Dermatomicosis/inmunología , Dermatomicosis/microbiología , Dermatomicosis/transmisión , Hongos/clasificación , Hongos/enzimología , Hongos/inmunología , Hongos/patogenicidad , Humanos , Investigación/tendencias , Trichophyton/clasificación , Trichophyton/enzimología , Trichophyton/inmunología , Trichophyton/patogenicidadRESUMEN
Signaling pathways are highly diverse in filamentous fungi, allowing the cells to receive and process ambient information. Interaction of components from different pathways results in signaling networks. The mitogen-activated protein kinase (MAPK) pathway is dependent on phosphorylation that is accomplished by kinase proteins. Thus, the STE/PAK protein kinase family plays essential roles in MAPK signal transduction, regulating several cellular functions. The STE/PAK protein displays an autoinhibitory (Cdc42/Rac interactive binding-CRIB) domain on its N-terminal portion, which interacts with the C-terminal catalytic kinase domain. Based on current knowledge, for the STE/PAK kinase to be activated, molecular signals (e.g., interaction with the activated form of Rac1 and Cdc42 proteins) or proteolytic cleavage by caspase 3 is necessary. Both mechanisms release the kinase domain from the CRIB interaction. Here, we hypothesize a novel molecular mechanism for the activation of STE20/PAKA kinase in Trichophyton rubrum based on an alternative pre-mRNA splicing process. Our data suggest that, because of the retention of intron 1 of this gene, it is theoretically possible that the translation of STE20/PAKA kinase will be free of its autoinhibitory CRIB domain. These findings indicate a rapid response system to environmental changes. Furthermore, STE20/PAKA may be a potential T. rubrum virulence factor and an interesting target for new drugs against dermatophytes.
Asunto(s)
Empalme Alternativo/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas Quinasas/química , Proteínas Quinasas/genética , Precursores del ARN/genética , Trichophyton/enzimología , Trichophyton/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Intrones/genética , Dominios Proteicos , Proteínas Quinasas/metabolismo , Precursores del ARN/metabolismo , Transcripción GenéticaRESUMEN
Pseudoaspidinol is a phloroglucinol derivative with Antifungal activity and is a major active component of Dryopteris fragrans. In our previous work, we studied the total synthesis of pseudoaspidinol belonging to a phloroglucinol derivative and investigated its antifungal activity as well as its intermediates. However, the results showed these compounds have low antifungal activity. In this study, in order to increase antifungal activities of phloroglucinol derivatives, we introduced antifungal pharmacophore allylamine into the methylphloroglucinol. Meanwhile, we remained C1â»C4 acyl group in C-6 position of methylphloroglucinol using pseudoaspidinol as the lead compound to obtain novel phloroglucinol derivatives, synthesized 17 compounds, and evaluated antifungal activities on Trichophyton rubrum and Trichophyton mentagrophytes in vitro. Molecular docking verified their ability to combine the protein binding site. The results indicated that most of the compounds had strong antifungal activity, in which compound 17 were found to be the most active on Trichophyton rubrum with Minimum Inhibitory Concentration (MIC) of 3.05 µg/mL and of Trichophyton mentagrophytes with MIC of 5.13 µg/mL. Docking results showed that compounds had a nice combination with the protein binding site. These researches could lay the foundation for developing antifungal agents of clinical value.
Asunto(s)
Antifúngicos , Proteínas Fúngicas , Simulación del Acoplamiento Molecular , Escualeno-Monooxigenasa , Trichophyton/enzimología , Alilamina/química , Antifúngicos/síntesis química , Antifúngicos/química , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Floroglucinol/química , Escualeno-Monooxigenasa/antagonistas & inhibidores , Escualeno-Monooxigenasa/químicaRESUMEN
Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.
Asunto(s)
Antifúngicos/farmacología , Naftalenos/farmacología , Mutación Puntual/genética , Escualeno-Monooxigenasa/genética , Trichophyton/efectos de los fármacos , Trichophyton/genética , Arthrodermataceae/efectos de los fármacos , Arthrodermataceae/enzimología , Arthrodermataceae/genética , Farmacorresistencia Fúngica/genética , Pruebas de Sensibilidad Microbiana , Terbinafina , Trichophyton/enzimologíaRESUMEN
Hedgehogs have increased in popularity as pets in Spain but there are no data of infection rates of this exotic animal with dermatophytes in our country. During the period of 2008-2011 a total of 20 pet hedgehogs (19 African pygmy hedgehogs and 1 Egyptian long-eared hedgehog) suspected of having dermatophytoses were studied. This is the first survey of the occurrence of T. erinacei in household hedgehogs in Spain. The T. erinacei infection rate was 50% (9 out of 19 African pygmy hedgehogs, and the one Egyptian long-eared hedgehog surveyed). Morphological identification of the isolates was confirmed by molecular analysis. All the strains had the same ITS sequence and showed 100% sequence similarity to T. erinacei type strain CBS 511.73 (AB 105793). The Spanish isolates were confirmed as T. erinacei urease positive. On the basis of ITS sequences, T. erinacei is a species close to but separate from the taxa included in the A. benhamiae complex. Review of the current literature on DNA-based methods for identification of species included in this complex has highlighted the urgent need to reach a consensus in species circumscription and classification system accepted by all mycologists.
Asunto(s)
Erizos/microbiología , Mascotas/microbiología , Tiña/veterinaria , Trichophyton/clasificación , Trichophyton/aislamiento & purificación , Animales , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Femenino , Genotipo , Masculino , Técnicas Microbiológicas , Microscopía , Filogenia , Prevalencia , Análisis de Secuencia de ADN , España/epidemiología , Tiña/epidemiología , Tiña/microbiología , Trichophyton/enzimología , Trichophyton/genética , Ureasa/análisisRESUMEN
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.
Asunto(s)
Epidermophyton/patogenicidad , Interacciones Huésped-Patógeno , Microsporum/patogenicidad , Tiña/microbiología , Tiña/patología , Trichophyton/patogenicidad , Animales , Epidermophyton/enzimología , Humanos , Hidrólisis , Queratinas/metabolismo , Microsporum/enzimología , Trichophyton/enzimologíaRESUMEN
OBJECTIVE: The aim of this study was to evaluate the efficiency of pyocyanin pigment as a novel compound active against tyrosinase with its depigmentation efficiency for combating Trichophyton rubrum which could be a major causative agent of tinea corporis. METHODS: Fifty swabs of fungal tinea corporis infections were collected and identified. Five MDRPA isolates were tested for their levels of pyocyanin production. The purified extracted pyocyanin was characterized by UV spectrum and FT-IR analysis. Pyocyanin activity against tyrosinase was determined by dopachrome micro-plate. In addition, the antidermatophytic activity of pyocyanin against T. rubrum was detected by radial growth technique. In vivo novel trial was conducted to evaluate the efficiency and safety of pyocyanin as an alternative natural therapeutic compound against T. rubrum causing tinea corporis. RESULTS: Purified pyocyanin showed highly significant inhibitory activity against tyrosinase and T. rubrum. In vivo topical treatments with pyocyanin ointment revealed the efficiency of pyocyanin (MIC 2000 µg/ml) to cure tinea corporis compared to fluconazole, which showed a partial curing at a higher concentration (MIC 3500 µg/ml) after two weeks of treatment. In addition, the results revealed complete healing and disappear of hyperpigmentation by testing the safety of pyocyanin ointment and its histopathological efficiency in the skin treatment without any significant toxic effect. CONCLUSION: Pyocyanin pigment could be a promising anti-tyrosinase and a new active compound against T. rubrum, which could be a major causative agent of tinea corporis. In fact, if pyocyanin secondary metabolite is going to be used in practical medication, it will support the continuous demand of novel antimycotic natural agents against troublesome fungal infections.
Asunto(s)
Antifúngicos/metabolismo , Monofenol Monooxigenasa/antagonistas & inhibidores , Piocianina/metabolismo , Piocianina/uso terapéutico , Tiña/tratamiento farmacológico , Trichophyton/enzimología , Administración Tópica , Animales , Modelos Animales de Enfermedad , Femenino , Pruebas de Sensibilidad Microbiana , Pomadas/administración & dosificación , Piocianina/administración & dosificación , Conejos , Resultado del Tratamiento , Trichophyton/efectos de los fármacosRESUMEN
BACKGROUND: Dermatophytosis is the most important superficial fungal infection which affects nearly 20% of human population worldwide. Recurrence of disease and emerging resistance of Trichophyton rubrum to synthetic antifungals are the main problems in control of dermatophytosis. The purpose of this study was to evaluate the effect of cold atmospheric plasma (CAP) on T. rubrum growth, ergosterol biosynthesis and keratinase activity. METHODS: A CAP system, comprised of helium 98% - oxygen 2% (He/O2), was used. Trichophyton rubrum conidia suspensions were treated with CAP in time periods of 90, 120, 150 and 180 s in 96-well microplates. Fungal growth was evaluated by counting the colony forming unit (CFU). Fungal dry weight, ergosterol biosynthesis and keratinase activity were evaluated in CAP-treated T. rubrum and untreated controls. RESULTS: T. rubrum growth was significantly inhibited by 62%-91%. CAP strongly suppressed fungal ergosterol biosynthesis by 27%-54%. The keratinase activity was increased by 7.30%-21.88% up to 120 s CAP exposure. CONCLUSION: Our results demonstrated for the first time that CAP inhibits T. rubrum growth, suppresses ergosterol biosynthesis and increases moderately keratinase activity in a dose-dependent manner. Overall, CAP exposure could be a potentially useful method for treatment of clinical cases of human and animal dermatophytoses.
Asunto(s)
Ergosterol/biosíntesis , Péptido Hidrolasas/química , Gases em Plasma/farmacología , Trichophyton/enzimología , Animales , Antifúngicos/farmacología , Atmósfera , Frío , Helio/química , Humanos , Oxígeno/química , Tiña/tratamiento farmacológicoRESUMEN
Trichophyton mentagrophytes is a keratinophilic pathogenic fungus that infects both humans and animals. Subtilisins are important for T. mentagrophytes virulence, particularly when invading the epidermal barrier of the host. Subtilisin gene SUB6 belongs to a seven-member gene family (SUB1-SUB7) encoding the subtilisin serine proteases. Additionally, the SUB6 gene product Sub6, which is thought to be the major allergen Tri r2 in Trichophyton rubrum, elicits both immediate- and delayed-type hypersensitivity (DTH) reactions in humans. To assess its gene function, SUB6 was disrupted using the Agrobacterium tumefaciens-mediated transformation method. Polymerase chain reaction and Southern blot analyses were used to confirm the disruption. In vitro virulence analyses comparing the mutant with the wild-type strain showed that proteolytic activity was significantly increased in the SUB6 gene disruption strain (SUB6::hph), which corresponded to the significantly increase in MEP4 (metalloprotease gene) and SUB3 expression of SUB6::hph. The SUB6::hph -infected animals showed attenuated clinical symptoms and pathological changes, and because of the persistently high level of immunosuppressive cytokine IL-10, the increase in DTH-related cytokines IFN-γ, TNF-α and IL-12 was delayed and lower than that in animals infected with the wild-type strain. These results suggested that SUB6::hph had attenuated virulence in vivo, and that a genetically-linked regulatory effect may account for the increase in proteolytic activity and the residual pathogenicity of the mutant strain.
Asunto(s)
Proteínas Fúngicas/metabolismo , Subtilisina/metabolismo , Trichophyton/enzimología , Factores de Virulencia/metabolismo , Agrobacterium tumefaciens/genética , Animales , Modelos Animales de Enfermedad , Proteínas Fúngicas/genética , Técnicas de Inactivación de Genes , Cobayas , Subtilisina/genética , Tiña/microbiología , Tiña/patología , Transformación Genética , Trichophyton/genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
Metalloproteases (Mep) of the M36 family are important virulence factors for the host invasion by the dermatophyte Trichophyton mentagrophytes. Dermatophytes secrete keratinase to degrade human and animal keratin and invade the skin. In previous studies, primers designed from the MEP gene sequences of Aspergillus fumigatus and A. oryzae were used to amplify the MEP genes from T. mentagrophytes, and the five MEP genes (MEP1-MEP5) were expressed. Differences in the expression of these five MEP genes in different dermatophytes were observed in an in vitro protein induction study, indicating their different functions and proteolytic abilities. However, specific pathogenic functions and mechanisms of each of the metalloproteases, as well as differences in their proteolytic activities, remain uncertain. In the current study, Agrobacterium tumefaciens-mediated transformation (ATMT) was used to successfully transform five MEP genes, resulting in five MEP mutant strains. MEP3 showed strongest proteolytic activity, hair biodegradation ability, and animal pathogenicity among the mutant strains. The MEP4 and MEP5 mutants were the least pathogenic through the above tests. Therefore, we hypothesize that the MEP4 and MEP5 genes are most likely to significantly affect the pathogenicity of T. mentagrophytes.
Asunto(s)
Metaloproteasas/metabolismo , Trichophyton/enzimología , Trichophyton/patogenicidad , Factores de Virulencia/metabolismo , Agrobacterium tumefaciens/genética , Animales , Femenino , Vectores Genéticos , Cobayas , Masculino , Metaloproteasas/genética , Piel/microbiología , Piel/patología , Transformación Genética , Trichophyton/genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
Cysteine dioxygenase (CDO) is involved in regulation of intracellular cysteine levels by catabolising the cysteine to sulphite and sulphate. In keratinolytic fungi, sulphite is actively excreted to reduce disulphide bridges in keratin before its enzymatic degradation. The pathogenicity role of CDO was confirmed in cysteine-hypersensitive and growth-defective ΔCdo mutant of Arthroderma benhamiae on hair and nails. We analysed the CDO expression regulation in T. mentagrophytes (anamorph of A. benhamiae) mycelia by determining the Cdo mRNA and CDO protein levels and by analysing the proportion of two molecular forms of CDO in response to l-cystine exposure. Cdo mRNA levels in mycelia lysates were detected by reverse-transcription real-time polymerase chain reaction and CDO protein by western blot using mouse CDO-specific hyperimmune serum. The Cdo mRNA level increased gradually 2.5-4.5 h after exposure of the mycelium to l-cystine. The CDO protein, detected as two bands of different mobility, appeared earlier in comparison to mRNA (1 h) and culminated after 24 h. More mobile form prevailed after 4.5 h. The comparison of the dynamics in the Cdo mRNA and CDO protein levels indicates that T. mentagrophytes responds to l-cystine by increased transcription and apparently decreased degradation of the CDO and by changing towards higher mobility molecular form, similar to previous reports describing mammalian analogue.
Asunto(s)
Cisteína-Dioxigenasa/genética , Cistina/metabolismo , Proteínas Fúngicas/genética , Regulación Enzimológica de la Expresión Génica , Tiña/microbiología , Trichophyton/enzimología , Animales , Cisteína-Dioxigenasa/metabolismo , Femenino , Proteínas Fúngicas/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Micelio/enzimología , Micelio/genética , Micelio/crecimiento & desarrollo , Micelio/metabolismo , Trichophyton/genética , Trichophyton/crecimiento & desarrollo , Trichophyton/fisiología , Regulación hacia ArribaRESUMEN
We report a case of a 34-year-old Polish Caucasian male who was diagnosed with tinea manuum caused by Trichophyton rubrum var. raubitschekii. It would be the first described case of a dermatophytosis caused by this fungus in Poland and one of a few cases in Central Europe described so far. Admittedly, it would be the first case in Central Europe with no evidence pointing to African origin. The clinical condition improved after administering itraconazole (daily dose 100 mg orally) supplemented with a topical treatment, while the patient was totally cured after 2 months. The histopathological examination turned out to be highly useful in the diagnostic process. The genetic analysis of the urease gene pointed to a urease-positive T. rubrum rather than T. rubrum var. raubitschekii.
Asunto(s)
Tiña/diagnóstico , Tiña/microbiología , Trichophyton/clasificación , Trichophyton/aislamiento & purificación , Ureasa/metabolismo , Adulto , Antifúngicos/uso terapéutico , ADN de Hongos/química , ADN de Hongos/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/química , ADN Espaciador Ribosómico/genética , Genes de ARNr , Mano/patología , Humanos , Itraconazol/uso terapéutico , Masculino , Polonia , ARN de Hongos/genética , ARN Ribosómico 28S/genética , Análisis de Secuencia de ADN , Tiña/tratamiento farmacológico , Tiña/patología , Resultado del Tratamiento , Trichophyton/enzimología , Ureasa/genéticaRESUMEN
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.
Asunto(s)
Microbiología del Aire , Dermatomicosis/microbiología , Microsporum/enzimología , Conejos/microbiología , Trichophyton/enzimología , Animales , Dermatomicosis/veterinaria , Activación Enzimática , Gelatinasas/metabolismo , Lipasa/metabolismo , Microsporum/aislamiento & purificación , Microsporum/metabolismo , Elastasa Pancreática/metabolismo , Trichophyton/aislamiento & purificación , Trichophyton/metabolismoRESUMEN
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.