A molecular toolbox for hypothesis testing in the dermatophytes

描述（由申请人提供）：皮肤真菌是霉菌，与曲霉和二态真菌密切相关。这些霉菌引起皮肤和指甲感染，包括脚癣，这是世界上最常见的真菌感染。在美国，仅脚气对医疗保健系统的经济影响估计每年就超过4亿美元。同样，头部真菌感染是美国和世界各地主要城市中心常见的儿科感染，在社会经济萧条的人群中尤为常见。在最近的一项研究中，美国日托中心22-50%的儿童表现出皮肤真菌性头皮感染的症状。对皮肤真菌的研究和资助仍然不足。最近对皮肤真菌的基因组测序和EST分析将使研究人员能够产生关于皮肤真菌的假设，最终目标是开发皮肤真菌感染的检测（诊断），治疗（药物）和预防（疫苗或预防）方法。检验假设的标准方法是改变真菌细胞的基因型，然后评估产生的表型。然而，用于改变基因型和评估表型的工具严重限制了皮肤癣菌。本提案的目的是开发一个分子工具箱，使研究人员能够测试有关皮肤真菌致病性的假设。本课题的具体目标如下：目标1 -开发利用分裂标记改变皮肤真菌基因型的技术，以最大限度地实现同源重组，以及利用农杆菌转化进行随机诱变和基因表达控制。目的2 -评估用于评估皮肤真菌表型的交替宿主病原体模型。人类皮肤“筏”将用于研究真菌与分化的角质形成细胞的相互作用。皮肤真菌感染也将在蜡蛾幼虫（Galleria mellonella）中进行测试，这种蜡蛾幼虫目前用于其他真菌感染。一旦完成，这些目标将提供必要的假设检验工具，与基因组序列和表达分析相结合，以解决致病性和毒性问题，最终目标是开发诊断、治疗和预防策略。公共卫生相关性：皮肤真菌是导致多种皮肤和指甲感染的致病真菌，包括脚癣和头癣，这是社会经济落后地区患者常见的头部真菌感染。据估计，皮肤癣菌是世界上最常见的真菌感染。这一提议将开发一套分子工具，可以与即将完成的基因组序列结合使用，以解决有关皮肤真菌预防、治疗和诊断的重要问题。

DESCRIPTION (provided by applicant): Dermatophytes are molds, closely related to Aspergillus and the dimorphic fungi. These molds cause infections within the skin and nails, including athlete's foot, the most common fungal infection worldwide. In the United States, the economic impact of athlete's foot alone on the health care system is estimated to exceed $400 million a year. Similarly, fungal infection of the head is a frequent pediatric infection in major urban centers in the U.S. and worldwide, and is especially common in socio-economically depressed populations. In a recent study, 22-50% of children in a U.S. daycare exhibited symptoms of dermatophytic scalp infection. The dermatophytes remain under-studied and under-funded. Recent genome sequencing and EST analysis of the dermatophytes will enable researchers to generate hypotheses concerning the dermatophytes with the ultimate goal to develop methods for the detection (diagnostics), treatment (drugs), and prevention (vaccines or prophylaxis) of dermatophyte infections. A standard method for testing hypotheses is to alter the genotype of the fungal cell and then assess the resulting phenotype. However, the tools for altering the genotype and assessing the phenotype are severely limited for dermatophytes. The objective of this proposal is to develop a molecular toolbox that will allow researchers to test hypotheses relating to dermatophyte pathogenicity. The specific aims of the proposal are as follows: Aim 1 - To develop techniques for altering the dermatophyte genotype, using split markers to maximize homologous recombination, and Agrobacterium tumefaciens transformation for random mutagenesis and gene expression control. Aim 2 - To evaluate alternate host pathogen models for assessing dermatophyte phenotypes. Human skin "rafts" will be used to study the interactions of the fungus with differentiated keratinocytes. Dermatophyte infections will also be tested in Galleria mellonella, a wax moth larvae which is currently in use for other fungal infections. When completed, these aims will provide hypothesis-testing tools that are essential, in combination with genome sequences and expression analysis, to address questions of pathogenicity and virulence, with the ultimate goal of the development of diagnostics, treatments and prevention strategies for the dermatophytes. PUBLIC HEALTH RELEVANCE: Dermatophytes are pathogenic fungi responsible for a variety of skin and nail infections, including athlete's foot and tinea capitis, a common fungal infection of the head in patients in socio- economically depressed areas. It is estimated that dermatophytes are the most common fungal infection worldwide. This proposal will develop a set of molecular tools that can be used in combination with the soon-to-be-completed genome sequences to address important questions about dermatophyte prevention, treatment and diagnosis.