Objective: To investigate the fungistatic activity and synergistic effects of natural products and their constituents, including traditional oriental medicines(TOMs).Methods: Fungistatic activities of TOMs prepared by...Objective: To investigate the fungistatic activity and synergistic effects of natural products and their constituents, including traditional oriental medicines(TOMs).Methods: Fungistatic activities of TOMs prepared by hot-water(115 ℃) or ethanol(70%; 40 ℃) extraction were determined by their minimum inhibitory concentration.To assess possible synergistic effects, minimum inhibitory concentrations of various combinations were evaluated.Results: By evaluating antifungal susceptibility of Trichophyton rubrum, which is a major causative fungus for several types of dermatophytosis, we confirmed that ethanol extracts were more active than hot-water extracts in 25 of the 36 TOMs, suggesting that the constituents with high hydrophobicity tend to contribute significantly to fungistatic activity.We selected four TOMs with high fungistatic activity, including Aucklandiae radix, Gentianae macrophyllae radix, Scutellariae radix, and Galla rhois, and their synergistic effects were investigated through the combination studies between TOMs or TOM-conventional drug terbinafine.In combinations between four TOMs, partial synergistic effects were observed in Aucklandiae radix–Galla rhois and Gentianae macrophyllae radix–Galla rhois combinations, as supported by the lowest fractional inhibitory concentration index value of 0.66 for both combinations.Furthermore, Galla rhois showed the strongest synergistic effect on growth inhibition of Trichophyton rubrumwith a fractional inhibitory concentration index value of 0.50 in combination with terbinafine.Conclusions: Our findings indicate that the combination of TOMs and TOM-terbinafine may be effective on treatment for chronic and recurrent dermatophytosis by improving fungistatic activity and led to decrease systemic toxicity in clinical practice.展开更多
BACKGROUND Facial cosmetic procedures become popular for people with a desire to have a younger appearance,and cosmetic technology has developed rapidly over the past several decades.However,increasing complications r...BACKGROUND Facial cosmetic procedures become popular for people with a desire to have a younger appearance,and cosmetic technology has developed rapidly over the past several decades.However,increasing complications related to cosmetic injections have been reported,and infection is one of the most serious problems and can cause anxiety and facial injury.We here report a case of Majocchi’s granuloma(MG)caused by Trichophyton rubrum after facial injection of hyaluronic acid.CASE SUMMARY A 37-year-old woman presented to our hospital with a history of red papules,nodules,and abscesses on her left zygomatic arch for 2 mo.She had received a cosmetic injection of hyaluronic acid on the left side of her face prior to the appearance of the lesions.MG caused by Trichophyton rubrum after facial injection of hyaluronic acid was diagnosed based on morphology and molecular biological identification.In vitro antifungal susceptibility testing was conducted according to the Clinical and Laboratory Standards Institute M38-A2 method.Minimal inhibitory concentrations were used to evaluate the antifungal susceptibility.The antifungal agents and their minimal inhibitory concentrations for the strain were terbinafine(<0.5μg/mL),itraconazole(0.06μg/mL),amphotericin B(0.25μg/mL),fluconazole(32μg/mL),voriconazole(0.125μg/mL),posaconazole(0.125μg/mL),and isavuconazole(0.06μg/mL).We initially administered 250 mg/d oral terbinafine for 2 mo,but the patient still had painful papules,nodules and abscesses on her face.Then,we adjusted the treatment to itraconazole 400 mg/d for 8 wk based on the in vitro antifungal susceptibility testing results.The skin lesions improved significantly,and there was no recurrence during follow-up.CONCLUSION This case revealed that facial injection of hyaluronic acid may cause serious MG.Antifungal susceptibility testing should be considered in the treatment of MG caused by Trichophyton rubrum.展开更多
Background: Trichophyton rubrum represents the most common infectious fungus responsible for dermatophytosis in human, but the mechanism involved is still not completely understood. An appropriate model constructed t...Background: Trichophyton rubrum represents the most common infectious fungus responsible for dermatophytosis in human, but the mechanism involved is still not completely understood. An appropriate model constructed to simulate host infection is the prerequisite to study the pathogenesis of dernlatophytosis caused by T.. rubrum. In this study, we intended to develop a new T. rubrum infection model in vitro, using the three-dimensional reconstructed epidermis - EpiSkin, and to pave the way for further investigation of the mechanisms involved in T. rubrum infection. Methods: The reconstructed human epidermis (RHE) was infected by inoculating low-dose (400 conidia) and high-dose (4000 conidia) T. rubrum conidia to optimize the infection dose. During the various periods after infection, the samples were processed for pathological examination and scanning electron microscopy (SEM) observation. Results: The histological analysis of RHE revealed a fully differentiated epidermis with a functional stratum corneum, which was analogous to the normal human epidermis. The results of hematoxylin and eosin staining and the periodic acid-Schiff staining showed that the infection dose of 400 conidia was in accord with the pathological characteristics of host dermatophytosis caused by T. rubrum. SEM observations further exhibited the process of 77 ruhrum infection in an intuitionistic way, Conclusions: We established the T. rubrum infection model on RHE in vitro successfully. It is a promising model fbr further investigation of the mechanisms involved in T. rubrum infection.展开更多
Trichophyton rubrum (T. rubrum) is the most common of the superficial fungi. In an effort to better understand the genetic and biochemical makeup of T. rubrum, we generated cDNA libraries from 3 growth stages and used...Trichophyton rubrum (T. rubrum) is the most common of the superficial fungi. In an effort to better understand the genetic and biochemical makeup of T. rubrum, we generated cDNA libraries from 3 growth stages and used these to isolate 4002 unique expressed sequence tags (ESTs). Sequence comparisons with the Genbank database allowed 1226 of the ESTs to be assigned putative functions or matched with homologs from other organisms. Of the remaining ESTs, 989 were only weakly similar to known sequences and 1787 had no identifiable functions, suggesting that they represent novel genes. We further analyzed the presence of several im-portant genes involved in the growth, metabolism, signal transduction, pathogenesis and drug resistance in T. rubrum. This information was used to newly elucidate important metabolic path-ways in T. rubrum. Taken together, our results should form the molecular basis for continued re-search on the physiological processes and pathogenic mechanisms of T. rubrum, and may lead to a better understanding of fungal drug resistance and identification of new drug targets.展开更多
Objectives Aspidin BB, a typical phloroglucinol derivative from Dryopteris fragrans, possesses significant antifungal property. This study aimed to investigate potential mechanism of antifungal activity of Aspidin BB ...Objectives Aspidin BB, a typical phloroglucinol derivative from Dryopteris fragrans, possesses significant antifungal property. This study aimed to investigate potential mechanism of antifungal activity of Aspidin BB against Trichophyton rubrum which is the most common pathogens responsible for chronic dermatophytosis. Methods The minimum inhibitory concentration (MIC) ofAspidin BB against strains was determined by broth microdilution. The effects of Aspidin BB on ergosterol biosynthesis were investigated by content determination based on UPLC method. Besides, the effects of drugs on squalene epoxidase (SE) in T. rubrum cell membrane were analyzed. Results MIC value of Aspidin BB against T. rubrum was 25.0 IJg/mL. Aspidin BB reduced ergosterol content significantly, but no notable effect on squalene epoxidase activity. Conclusion The results suggested that Aspidin BB inhibited ergosterol biosynthesis. However, it was not squalene epoxidase but other components may sever as possible targets in ergosterol biosynthesis pathway.展开更多
Background The prevalence of dermatophytoses and the development of new antifungal agents has focused interest on susceptibility tests of dermatophytes. The method used universally for susceptibility tests of dermatop...Background The prevalence of dermatophytoses and the development of new antifungal agents has focused interest on susceptibility tests of dermatophytes. The method used universally for susceptibility tests of dermatophytes was published as document (M38-A) in 2002 by the Clinical and Laboratory Standards Institute (CLSI), dealing with the standardization of susceptibility tests in filamentous fungi, though not including dermatophytes especially. However, it is not a very practical method for the clinical laboratory in routine susceptibility testing. In this test, we developed a novel rapid susceptibility assay --glucose consumption method (GCM) for dermatophytes. Methods In this study, we investigated the antifungal susceptibilities of dermatophytes to itraconazole (ITC), voriconazole (VOC), econazole nitrate (ECN) and terbinafine (TBF) by glucose consumption method (GCM), in comparison to the Clinical and Laboratory Standards Institute (CLSI) M38-A method. Twenty-eight dermatophyte isolates, including Trichophyton rubrum (T. rubrum) (n=-14) and Trichophyton mentagrophytes (T. mentagrophytes) (n=-14), were tested. In the GCM, the minimum inhibitory concentrations (MICs) were determined spectrophotometrically at 490 nm after addition of enzyme substrate color mix. For the CLSI method, the MICs were determined visually. Results Comparison revealed best agreement for TBF against T. mentagrophytes and T. rubrum, since MIC range, MIC50, and MIC90 were identical from two methods. However, for ITC and VOC, GCM showed wider MIC ranges and higher MICs than CLSI methods in most isolates. For ECN against T. rubrum, high MICs were tested by GCM (0.125-16 pg/ml) but not M38-A method (0.5-1 IJg/ml). The overall agreements for all isolates between the two methods within one dilution and two dilutions for ITC, VOC, ECN and TBF was 53.6% and 75.0%, 57.1% and 75.0%, 82.1% and 89.3%, and 85.7 and 85.7%, respectively. Conclusion Measurement of glucose uptake can predict the susceptibility of T. rubrum and T. mentagrophytes to ECN and TBF.展开更多
Background Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested tha...Background Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively. Methods HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (l×106 and 1.5×105 colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment. Results HaCaT cells constitutively expressed mRNA of membrane-bound TLR1,2, 4 and 6, Dectinl and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-I. Conclusion The cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.展开更多
Trichophyton rubrum is a dominating superficial dermatophyte, whose conidial germination is corre- lated to pathopoiesis and a highly important developmental process. To investigate the changes of physiology, biochemi...Trichophyton rubrum is a dominating superficial dermatophyte, whose conidial germination is corre- lated to pathopoiesis and a highly important developmental process. To investigate the changes of physiology, biochemistry and cytology during the germination, we selected 3364 function identified ESTs from T. rubrum cDNA library to construct cDNA microarrays, and compared the gene expression levels of conidia and germinating phase. Data analysis indicated that 335 genes were up-regulated during the germination, which mainly encoded translated, modified proteins and structural proteins. The constituents of cell wall and cell membrane were synthetized abundantly, suggesting that they are the foundation of cell morphogenesis. The ingredients of the two-component signal transduction sys- tem were up-regulated, presuming that they were important for the conidial germination. Genes of various metabolic pathways were expressed prosperously, especially the genes that participated in glycolysis and oxidative phosphorylation were up-regulated on the whole, demonstrating that in the environment with sufficient oxygen and glucose, conidia obtained energy through aerobic respiration. This paper provides important clues which are helpful to understanding the changes in gene expres- sion, signal conduction and metabolism characteristics during T. rubrum conidial germination, and possess significant meaning to the study of other superficial dermatophytes.展开更多
Trichophyton rubrum (T.rubrum) is one of the most common human fungal pathogens that cause chronic infections of the skin and nails. To identify antifungal responsive genes, cDNA microarray analysis was performed for ...Trichophyton rubrum (T.rubrum) is one of the most common human fungal pathogens that cause chronic infections of the skin and nails. To identify antifungal responsive genes, cDNA microarray analysis was performed for T. rubrum to reveal global transcriptional profiles of drug-specific responses to 5-Flucytosine (5-FC). cDNA microarray was constructed from the T. rubrum expressed sequence tag (ESTs) database, the minimum inhibitory concentration (MIC) of 5-FC was determined, and microarray hybridization and data analysis were applied. The expression pattern of 7 genes observed by microarray was confirmed by the quantitative real-time reverser transcription polymerase chain reaction (RT-PCR). Data analysis indicated that a total of 474 genes were found differentially expressed, 196 showed an increase in expression and 278 showed a decrease in expression. Marked down-regulation of genes involved in nucleotide metabolism (such as CDC21), transcription (such as E2F1), and RNA processing (such as SGN1, RIM4 and NOP1) was observed. Other genes involved in signal transduction, chaperones, inorganic ion transport, secondary metabolite biosynthesis, amino acid transport, lipid transport and potential drug resistance mechanism were also affected by 5-FC. Quantitative real-time RT-PCR of the selected genes confirmed the reliability of the microarray results. This is the first analysis of transcriptional profiles in response to 5-FC for T. rubrum. The findings may be valuable for the identification of genes involved in mechanisms of action and mechanisms of antifungal drug resistance of 5-FC.展开更多
Trichophyton rubrum (T.rubrum) is a common superficial fungus.Molecular and genetic studies of T.rubrum are still limited.In this paper,we report the global analysis of gene expression profiles at different growth pha...Trichophyton rubrum (T.rubrum) is a common superficial fungus.Molecular and genetic studies of T.rubrum are still limited.In this paper,we report the global analysis of gene expression profiles at different growth phases using cDNA microarray technology.A total of 2044 differentially expressed genes were obtained and clustered into three expression patterns.Our data confirmed previous results that many mRNAs were pre-stored in the conidia of T.rubrum.Transcriptional profiling and function analysis showed that some glycolytic enzymes share similar expression patterns and may be coregulated during the transition of growth phases.Some genes involved in small GTPase signaling pathways,and in cAMP-dependent and MAPK regulation pathways were induced in response to the growth dynamics of T.rubrum.Although the detailed biological roles of these T.rubrum genes are still unknown,our results suggest that these genes may be involved in regulation mechanisms in the life cycle of the fungus.展开更多
红色毛癣菌(Trichophyton rubrum,T.rubrum)是皮肤癣最主要病原;侧柏叶(Cacumen Platycladi)是侧柏[Platycladus orientali s(L.)Franco]的干燥嫩枝梢及叶,被《苗族医学》收录用于治疗皮肤癣。本研究旨在探讨侧柏叶抗T.rubrum的主要活...红色毛癣菌(Trichophyton rubrum,T.rubrum)是皮肤癣最主要病原;侧柏叶(Cacumen Platycladi)是侧柏[Platycladus orientali s(L.)Franco]的干燥嫩枝梢及叶,被《苗族医学》收录用于治疗皮肤癣。本研究旨在探讨侧柏叶抗T.rubrum的主要活性物质及其抗真菌机制,为抗皮肤癣新药研发提供帮助。本研究通过测定最小抑菌浓度和孢子萌发率分析了侧柏叶活性物质对T.rubrum的抑菌活性,使用电镜观察T.rubrum菌丝形态和孢子超微结构,并检测侧柏叶活性物质对T.rubrum细胞膜通透性、完整性和麦角甾醇含量的影响,从而阐述侧柏叶活性物质对T.rubrum抑菌机理,最后采用GC-MS和qRT-PCR方法找到其抗T.rubrum的作用靶点。结果显示,侧柏叶石油醚部位的α-蒎烯为主要抗菌活性成分,MIC为32μg·mL^(-1),极显著抑制孢子萌发(P<0.01);α-蒎烯可致细胞膜发生明显损伤和内容物流出、通透性极显著增加(P<0.01)以及24 h内细胞膜中麦角甾醇和24(28)脱氢麦角甾醇含量显著下降(P<0.05);引起脂质色谱图中ERG3活性抑制相关的“麦角甾烷-7,24-二烯-3β-醇”色谱峰出现;可极显著降低ERG3的相对表达量(P<0.01)。结果表明,α-蒎烯是侧柏叶中发挥抗T.rubrum活性最主要成分且以ERG3为其抗真菌靶点。展开更多
目的研究红色毛癣菌对人角质形成细胞模式识别受体TLR-2,TLR-4,Dectin-1表达及细胞因子分泌的影响,探讨角质形成细胞对红色毛癣菌的免疫应答及其机制。方法红色毛癣菌孢子与人永生化表皮细胞株HaCaT细胞共培养,采用Real time PCR检测共...目的研究红色毛癣菌对人角质形成细胞模式识别受体TLR-2,TLR-4,Dectin-1表达及细胞因子分泌的影响,探讨角质形成细胞对红色毛癣菌的免疫应答及其机制。方法红色毛癣菌孢子与人永生化表皮细胞株HaCaT细胞共培养,采用Real time PCR检测共培养后HaCaT细胞TLR-2,TLR-4,Dectin-1mRNA的表达情况;采用流式细胞技术检测共培养后不同时间段HaCaT细胞TLR-2,TLR-4及Dectin-1平均荧光强度;采用蛋白芯片抗体阵列检测共培养上清液中36种不同的细胞因子,趋化因子和急性时相蛋白的表达情况。结果共培养6 h后,TLR-2,TLR-4,Dectin-1mRNA表达上调;共培养量明24显h增后加TL。R结-2,论T L人R角-4,质D形ec成tin细-1胞平对均红荧色光毛强癣度菌增的高免,细疫胞识因别子和I应L-答8,,I在-30一9,定IF程N-度γ,上IL可-6通,IL过-1上3调在红Ha色Ca毛T癣细菌胞刺中激模后式分识泌别受体TLR-2,TLR-4及Dectin-1后分泌多种细胞因子来实现。展开更多
基金supported by the Grant K17281 from Korea Institute of Oriental Medicine(KIOM),provided by the Ministry of Science,ICT and Future Planning(MISP),Republic of Korea
文摘Objective: To investigate the fungistatic activity and synergistic effects of natural products and their constituents, including traditional oriental medicines(TOMs).Methods: Fungistatic activities of TOMs prepared by hot-water(115 ℃) or ethanol(70%; 40 ℃) extraction were determined by their minimum inhibitory concentration.To assess possible synergistic effects, minimum inhibitory concentrations of various combinations were evaluated.Results: By evaluating antifungal susceptibility of Trichophyton rubrum, which is a major causative fungus for several types of dermatophytosis, we confirmed that ethanol extracts were more active than hot-water extracts in 25 of the 36 TOMs, suggesting that the constituents with high hydrophobicity tend to contribute significantly to fungistatic activity.We selected four TOMs with high fungistatic activity, including Aucklandiae radix, Gentianae macrophyllae radix, Scutellariae radix, and Galla rhois, and their synergistic effects were investigated through the combination studies between TOMs or TOM-conventional drug terbinafine.In combinations between four TOMs, partial synergistic effects were observed in Aucklandiae radix–Galla rhois and Gentianae macrophyllae radix–Galla rhois combinations, as supported by the lowest fractional inhibitory concentration index value of 0.66 for both combinations.Furthermore, Galla rhois showed the strongest synergistic effect on growth inhibition of Trichophyton rubrumwith a fractional inhibitory concentration index value of 0.50 in combination with terbinafine.Conclusions: Our findings indicate that the combination of TOMs and TOM-terbinafine may be effective on treatment for chronic and recurrent dermatophytosis by improving fungistatic activity and led to decrease systemic toxicity in clinical practice.
基金the Scientific Research Project of Peking University Shenzhen Hospital,No. JCYJ2018011the"San-ming"Project of Medicine in Shenzhen,No. SZSM201812059。
文摘BACKGROUND Facial cosmetic procedures become popular for people with a desire to have a younger appearance,and cosmetic technology has developed rapidly over the past several decades.However,increasing complications related to cosmetic injections have been reported,and infection is one of the most serious problems and can cause anxiety and facial injury.We here report a case of Majocchi’s granuloma(MG)caused by Trichophyton rubrum after facial injection of hyaluronic acid.CASE SUMMARY A 37-year-old woman presented to our hospital with a history of red papules,nodules,and abscesses on her left zygomatic arch for 2 mo.She had received a cosmetic injection of hyaluronic acid on the left side of her face prior to the appearance of the lesions.MG caused by Trichophyton rubrum after facial injection of hyaluronic acid was diagnosed based on morphology and molecular biological identification.In vitro antifungal susceptibility testing was conducted according to the Clinical and Laboratory Standards Institute M38-A2 method.Minimal inhibitory concentrations were used to evaluate the antifungal susceptibility.The antifungal agents and their minimal inhibitory concentrations for the strain were terbinafine(<0.5μg/mL),itraconazole(0.06μg/mL),amphotericin B(0.25μg/mL),fluconazole(32μg/mL),voriconazole(0.125μg/mL),posaconazole(0.125μg/mL),and isavuconazole(0.06μg/mL).We initially administered 250 mg/d oral terbinafine for 2 mo,but the patient still had painful papules,nodules and abscesses on her face.Then,we adjusted the treatment to itraconazole 400 mg/d for 8 wk based on the in vitro antifungal susceptibility testing results.The skin lesions improved significantly,and there was no recurrence during follow-up.CONCLUSION This case revealed that facial injection of hyaluronic acid may cause serious MG.Antifungal susceptibility testing should be considered in the treatment of MG caused by Trichophyton rubrum.
基金This study was supported by the grants from Guangdong Natural Science Foundation,the Special Fund for Young Scientists of Third Affiliated Hospital of Sun Yat-sen University
文摘Background: Trichophyton rubrum represents the most common infectious fungus responsible for dermatophytosis in human, but the mechanism involved is still not completely understood. An appropriate model constructed to simulate host infection is the prerequisite to study the pathogenesis of dernlatophytosis caused by T.. rubrum. In this study, we intended to develop a new T. rubrum infection model in vitro, using the three-dimensional reconstructed epidermis - EpiSkin, and to pave the way for further investigation of the mechanisms involved in T. rubrum infection. Methods: The reconstructed human epidermis (RHE) was infected by inoculating low-dose (400 conidia) and high-dose (4000 conidia) T. rubrum conidia to optimize the infection dose. During the various periods after infection, the samples were processed for pathological examination and scanning electron microscopy (SEM) observation. Results: The histological analysis of RHE revealed a fully differentiated epidermis with a functional stratum corneum, which was analogous to the normal human epidermis. The results of hematoxylin and eosin staining and the periodic acid-Schiff staining showed that the infection dose of 400 conidia was in accord with the pathological characteristics of host dermatophytosis caused by T. rubrum. SEM observations further exhibited the process of 77 ruhrum infection in an intuitionistic way, Conclusions: We established the T. rubrum infection model on RHE in vitro successfully. It is a promising model fbr further investigation of the mechanisms involved in T. rubrum infection.
文摘Trichophyton rubrum (T. rubrum) is the most common of the superficial fungi. In an effort to better understand the genetic and biochemical makeup of T. rubrum, we generated cDNA libraries from 3 growth stages and used these to isolate 4002 unique expressed sequence tags (ESTs). Sequence comparisons with the Genbank database allowed 1226 of the ESTs to be assigned putative functions or matched with homologs from other organisms. Of the remaining ESTs, 989 were only weakly similar to known sequences and 1787 had no identifiable functions, suggesting that they represent novel genes. We further analyzed the presence of several im-portant genes involved in the growth, metabolism, signal transduction, pathogenesis and drug resistance in T. rubrum. This information was used to newly elucidate important metabolic path-ways in T. rubrum. Taken together, our results should form the molecular basis for continued re-search on the physiological processes and pathogenic mechanisms of T. rubrum, and may lead to a better understanding of fungal drug resistance and identification of new drug targets.
基金Application-oriented Research Project of Guangdong Provincial Department of Science and Technology(2015B020234009)Traditional Chinese Medicine Industry Research Project of State Administration of Traditional Chinese Medicine of People’s Republic of China(201507004)
文摘Objectives Aspidin BB, a typical phloroglucinol derivative from Dryopteris fragrans, possesses significant antifungal property. This study aimed to investigate potential mechanism of antifungal activity of Aspidin BB against Trichophyton rubrum which is the most common pathogens responsible for chronic dermatophytosis. Methods The minimum inhibitory concentration (MIC) ofAspidin BB against strains was determined by broth microdilution. The effects of Aspidin BB on ergosterol biosynthesis were investigated by content determination based on UPLC method. Besides, the effects of drugs on squalene epoxidase (SE) in T. rubrum cell membrane were analyzed. Results MIC value of Aspidin BB against T. rubrum was 25.0 IJg/mL. Aspidin BB reduced ergosterol content significantly, but no notable effect on squalene epoxidase activity. Conclusion The results suggested that Aspidin BB inhibited ergosterol biosynthesis. However, it was not squalene epoxidase but other components may sever as possible targets in ergosterol biosynthesis pathway.
基金This study was supported by a grant from the Guangdong Natural Science Foundation Committee (No. 06300760).Acknowledgement: We thank Dr. XIE Zhi from Department of Dermatology and Venereology, The Second Affiliated Hospital of Sun Yat-Sen University, for his critical review of the manuscript.
文摘Background The prevalence of dermatophytoses and the development of new antifungal agents has focused interest on susceptibility tests of dermatophytes. The method used universally for susceptibility tests of dermatophytes was published as document (M38-A) in 2002 by the Clinical and Laboratory Standards Institute (CLSI), dealing with the standardization of susceptibility tests in filamentous fungi, though not including dermatophytes especially. However, it is not a very practical method for the clinical laboratory in routine susceptibility testing. In this test, we developed a novel rapid susceptibility assay --glucose consumption method (GCM) for dermatophytes. Methods In this study, we investigated the antifungal susceptibilities of dermatophytes to itraconazole (ITC), voriconazole (VOC), econazole nitrate (ECN) and terbinafine (TBF) by glucose consumption method (GCM), in comparison to the Clinical and Laboratory Standards Institute (CLSI) M38-A method. Twenty-eight dermatophyte isolates, including Trichophyton rubrum (T. rubrum) (n=-14) and Trichophyton mentagrophytes (T. mentagrophytes) (n=-14), were tested. In the GCM, the minimum inhibitory concentrations (MICs) were determined spectrophotometrically at 490 nm after addition of enzyme substrate color mix. For the CLSI method, the MICs were determined visually. Results Comparison revealed best agreement for TBF against T. mentagrophytes and T. rubrum, since MIC range, MIC50, and MIC90 were identical from two methods. However, for ITC and VOC, GCM showed wider MIC ranges and higher MICs than CLSI methods in most isolates. For ECN against T. rubrum, high MICs were tested by GCM (0.125-16 pg/ml) but not M38-A method (0.5-1 IJg/ml). The overall agreements for all isolates between the two methods within one dilution and two dilutions for ITC, VOC, ECN and TBF was 53.6% and 75.0%, 57.1% and 75.0%, 82.1% and 89.3%, and 85.7 and 85.7%, respectively. Conclusion Measurement of glucose uptake can predict the susceptibility of T. rubrum and T. mentagrophytes to ECN and TBF.
基金This Work was supported by the grants from theFundamental Research Funds for the Central Universities (No. 10ykpy04) and the National Natural Science Foundation of China (No. 30600028).
文摘Background Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively. Methods HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (l×106 and 1.5×105 colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment. Results HaCaT cells constitutively expressed mRNA of membrane-bound TLR1,2, 4 and 6, Dectinl and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-I. Conclusion The cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.
基金the National High Technology Research and Development Program of China (Grant No. 2001AA223021) National Key Technologies R&D Programme (Grant No. 2002BA711A14)
文摘Trichophyton rubrum is a dominating superficial dermatophyte, whose conidial germination is corre- lated to pathopoiesis and a highly important developmental process. To investigate the changes of physiology, biochemistry and cytology during the germination, we selected 3364 function identified ESTs from T. rubrum cDNA library to construct cDNA microarrays, and compared the gene expression levels of conidia and germinating phase. Data analysis indicated that 335 genes were up-regulated during the germination, which mainly encoded translated, modified proteins and structural proteins. The constituents of cell wall and cell membrane were synthetized abundantly, suggesting that they are the foundation of cell morphogenesis. The ingredients of the two-component signal transduction sys- tem were up-regulated, presuming that they were important for the conidial germination. Genes of various metabolic pathways were expressed prosperously, especially the genes that participated in glycolysis and oxidative phosphorylation were up-regulated on the whole, demonstrating that in the environment with sufficient oxygen and glucose, conidia obtained energy through aerobic respiration. This paper provides important clues which are helpful to understanding the changes in gene expres- sion, signal conduction and metabolism characteristics during T. rubrum conidial germination, and possess significant meaning to the study of other superficial dermatophytes.
基金Supported by the National High Technology Research and Development Program of China (Grant No. 2006AA020504)
文摘Trichophyton rubrum (T.rubrum) is one of the most common human fungal pathogens that cause chronic infections of the skin and nails. To identify antifungal responsive genes, cDNA microarray analysis was performed for T. rubrum to reveal global transcriptional profiles of drug-specific responses to 5-Flucytosine (5-FC). cDNA microarray was constructed from the T. rubrum expressed sequence tag (ESTs) database, the minimum inhibitory concentration (MIC) of 5-FC was determined, and microarray hybridization and data analysis were applied. The expression pattern of 7 genes observed by microarray was confirmed by the quantitative real-time reverser transcription polymerase chain reaction (RT-PCR). Data analysis indicated that a total of 474 genes were found differentially expressed, 196 showed an increase in expression and 278 showed a decrease in expression. Marked down-regulation of genes involved in nucleotide metabolism (such as CDC21), transcription (such as E2F1), and RNA processing (such as SGN1, RIM4 and NOP1) was observed. Other genes involved in signal transduction, chaperones, inorganic ion transport, secondary metabolite biosynthesis, amino acid transport, lipid transport and potential drug resistance mechanism were also affected by 5-FC. Quantitative real-time RT-PCR of the selected genes confirmed the reliability of the microarray results. This is the first analysis of transcriptional profiles in response to 5-FC for T. rubrum. The findings may be valuable for the identification of genes involved in mechanisms of action and mechanisms of antifungal drug resistance of 5-FC.
基金supported by the National Natural Science Foundation of China (Grant No. 30870104)the Eleven-Fifth Mega-Scientific Project on Infectious Diseases,China (Grant Nos. 2008ZX10401-3 and 2009ZX10004-303)an intramural grant from the Institute of Pathogen Biology,Chinese Academy of Medical Sciences (Grant No. 2006IPB008)
文摘Trichophyton rubrum (T.rubrum) is a common superficial fungus.Molecular and genetic studies of T.rubrum are still limited.In this paper,we report the global analysis of gene expression profiles at different growth phases using cDNA microarray technology.A total of 2044 differentially expressed genes were obtained and clustered into three expression patterns.Our data confirmed previous results that many mRNAs were pre-stored in the conidia of T.rubrum.Transcriptional profiling and function analysis showed that some glycolytic enzymes share similar expression patterns and may be coregulated during the transition of growth phases.Some genes involved in small GTPase signaling pathways,and in cAMP-dependent and MAPK regulation pathways were induced in response to the growth dynamics of T.rubrum.Although the detailed biological roles of these T.rubrum genes are still unknown,our results suggest that these genes may be involved in regulation mechanisms in the life cycle of the fungus.
文摘目的研究红色毛癣菌对人角质形成细胞模式识别受体TLR-2,TLR-4,Dectin-1表达及细胞因子分泌的影响,探讨角质形成细胞对红色毛癣菌的免疫应答及其机制。方法红色毛癣菌孢子与人永生化表皮细胞株HaCaT细胞共培养,采用Real time PCR检测共培养后HaCaT细胞TLR-2,TLR-4,Dectin-1mRNA的表达情况;采用流式细胞技术检测共培养后不同时间段HaCaT细胞TLR-2,TLR-4及Dectin-1平均荧光强度;采用蛋白芯片抗体阵列检测共培养上清液中36种不同的细胞因子,趋化因子和急性时相蛋白的表达情况。结果共培养6 h后,TLR-2,TLR-4,Dectin-1mRNA表达上调;共培养量明24显h增后加TL。R结-2,论T L人R角-4,质D形ec成tin细-1胞平对均红荧色光毛强癣度菌增的高免,细疫胞识因别子和I应L-答8,,I在-30一9,定IF程N-度γ,上IL可-6通,IL过-1上3调在红Ha色Ca毛T癣细菌胞刺中激模后式分识泌别受体TLR-2,TLR-4及Dectin-1后分泌多种细胞因子来实现。