组氨酸激酶在马尔尼菲青霉菌中的功能研究
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摘要
在环境中存在的超过100,000种不同种类的真菌中,存在二态性的致病真菌:皮炎芽生菌(Blastomyces dermatitidis),粗球孢子菌(Coccidioides immitis),组织胞浆菌(Histoplasma capsulatum),巴西副球霉菌(Paracoccidioidesbrasilliensis),申克氏胞丝菌(Sporothrix schenckii)和马尔尼菲青霉菌(Penicilliummarneffei)。一旦这种二态性致病真菌的孢子侵入宿主体内,其形态就会由在外界环境的菌丝态转换为致病性的酵母形态。马尔尼菲青霉菌是青霉菌种中唯一具有温度控制的二态性致病菌。马尔尼菲青霉菌广泛分布于中国南部及东南亚地区,能引起呼吸道、皮肤以及系统性霉菌病,特别是在免疫功能低下和艾滋病病毒感染的患者中。
为更好的探讨马尔尼菲青霉菌的致病机制及其潜在的致病基因的功能,有必要对马尔尼菲青霉菌中一些与致病相关的重要基因进行深入的功能研究。本实验研究的重点是双组分调控系统-组氨酸激酶,它是二态性真菌感知和适应环境的的主要信号通路。最近的研究表明组氨酸激酶能够调控不同的过程,包括二次代谢产物的生成,并与植物和动物的病原菌的致病性有关。
本研究在马尔尼菲青霉菌的cDNA文库中获得一个新型的组氨酸激酶基因PmHHK1,构建了针对PmHHK1的干扰载体,通过RNA干扰技术,利用根癌农杆菌介导的转化方法来对其进行功能研究。干扰载体以木糖诱导的xylp为启动子。PmHHK1的基因干扰后马尔尼菲青霉菌生长受到抑制,孢子生成数量减少,细胞壁成分发生变化,一些致病因子及相关的调控基因出现下调。实验表明,PmHHK1基因对于调控马尔尼菲青霉菌的生长发育,致病性方面均有调控作用。双组分调控系统广泛存在于真核生物中,而在哺乳动物中确未被发现,因此可作为抗真菌病药物的潜在靶点进行进一步的研究。
Among the more than 100,000 different species of environmental fungi are six phylogenetically related ascomycetes called the dimorphic fungi:Blastomyces dermatitidis,Coccidioides immitis,Histoplasma capsulatum,Paracoccidioides brasiliensis,Sporothrix schenkii,and Penicillium marneffei.These fungi change morphology once spores are inhaled into the lungs of a mammalian host from hyphal molds in the environment to pathogenic yeast forms.Penicillium marneffei is the only known species of its genus that is temperature-dependent dimorphic.Now,it has emerged as one of the most common opportunistic pathogen causing respiratory,skin and systemic mycosis among immunocompromised and AIDS patients,endemic in South-east Asia and Southern China.
To obtain a better understanding of the key elements involved in Penicillium marneffei virulence and to identify possible drug targets,it is necessary to be able to study the important genes which are related to virulence of P.marneffei.In our study, we are interested in the histidine kinase.It is a major signal transduction pathway by which fungus sense and adapt to their environment.Recent work has revealed that the HK gene can regulate diverse processes,including differentiation,chemotaxis, secondary metabolite production,and virulence in plant and animal pathogens.
We cloned a novel Penicillium marneffei histidine kinase-PmHHK1 gene from cDNA library and investigated the biological functions using dsRNAi mediated by A. tumefaciens.The activity of RNA interference is under control of promoter xy1P, which can be induced by xylose.The reduction of PmHHK1 activity displays the morphogenesis changes including that sporulation and cell wall composition. Furthermore,several lines of evidence strongly indicate a central regulating role of PmHHK1 in virulence factor.Taken together,we propose that PmHHK1 gene might be involved in regulation of morphogenesis and pathogenicity.Two-component signaling systems are widespread in the eukaryotes outside the animal kingdom, therefore having potential implication for treating fungal diseases.
为更好的探讨马尔尼菲青霉菌的致病机制及其潜在的致病基因的功能,有必要对马尔尼菲青霉菌中一些与致病相关的重要基因进行深入的功能研究。本实验研究的重点是双组分调控系统-组氨酸激酶,它是二态性真菌感知和适应环境的的主要信号通路。最近的研究表明组氨酸激酶能够调控不同的过程,包括二次代谢产物的生成,并与植物和动物的病原菌的致病性有关。
本研究在马尔尼菲青霉菌的cDNA文库中获得一个新型的组氨酸激酶基因PmHHK1,构建了针对PmHHK1的干扰载体,通过RNA干扰技术,利用根癌农杆菌介导的转化方法来对其进行功能研究。干扰载体以木糖诱导的xylp为启动子。PmHHK1的基因干扰后马尔尼菲青霉菌生长受到抑制,孢子生成数量减少,细胞壁成分发生变化,一些致病因子及相关的调控基因出现下调。实验表明,PmHHK1基因对于调控马尔尼菲青霉菌的生长发育,致病性方面均有调控作用。双组分调控系统广泛存在于真核生物中,而在哺乳动物中确未被发现,因此可作为抗真菌病药物的潜在靶点进行进一步的研究。
Among the more than 100,000 different species of environmental fungi are six phylogenetically related ascomycetes called the dimorphic fungi:Blastomyces dermatitidis,Coccidioides immitis,Histoplasma capsulatum,Paracoccidioides brasiliensis,Sporothrix schenkii,and Penicillium marneffei.These fungi change morphology once spores are inhaled into the lungs of a mammalian host from hyphal molds in the environment to pathogenic yeast forms.Penicillium marneffei is the only known species of its genus that is temperature-dependent dimorphic.Now,it has emerged as one of the most common opportunistic pathogen causing respiratory,skin and systemic mycosis among immunocompromised and AIDS patients,endemic in South-east Asia and Southern China.
To obtain a better understanding of the key elements involved in Penicillium marneffei virulence and to identify possible drug targets,it is necessary to be able to study the important genes which are related to virulence of P.marneffei.In our study, we are interested in the histidine kinase.It is a major signal transduction pathway by which fungus sense and adapt to their environment.Recent work has revealed that the HK gene can regulate diverse processes,including differentiation,chemotaxis, secondary metabolite production,and virulence in plant and animal pathogens.
We cloned a novel Penicillium marneffei histidine kinase-PmHHK1 gene from cDNA library and investigated the biological functions using dsRNAi mediated by A. tumefaciens.The activity of RNA interference is under control of promoter xy1P, which can be induced by xylose.The reduction of PmHHK1 activity displays the morphogenesis changes including that sporulation and cell wall composition. Furthermore,several lines of evidence strongly indicate a central regulating role of PmHHK1 in virulence factor.Taken together,we propose that PmHHK1 gene might be involved in regulation of morphogenesis and pathogenicity.Two-component signaling systems are widespread in the eukaryotes outside the animal kingdom, therefore having potential implication for treating fungal diseases.
引文
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