A molecular toolbox for hypothesis testing in the dermatophytes

性状（由申请人提供）：皮肤癣菌是霉菌，与曲霉菌和二型真菌密切相关。这些霉菌会导致皮肤和指甲内的感染，包括脚癣，这是世界上最常见的真菌感染。在美国，仅足癣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，在社会经济状况不佳的人群中尤其常见。在最近的一项研究中，22-50%的儿童在美国日托表现出头皮真菌感染的症状。皮肤癣菌仍然研究不足，资金不足。最近的基因组测序和EST分析的皮肤癣菌将使研究人员产生关于皮肤癣菌的假设，最终目标是开发方法的检测（诊断），治疗（药物），和预防（疫苗或预防）的皮肤癣菌感染。检验假设的标准方法是改变真菌细胞的基因型，然后评估所得的表型。然而，用于改变基因型和评估表型的工具对于皮肤癣菌来说是非常有限的。这项建议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。该提案的具体目标如下：目标1 -开发用于改变皮肤真菌基因型的技术，使用分裂标记以最大化同源重组，以及用于随机诱变和基因表达控制的根癌农杆菌转化。目的2 -评估皮肤癣菌表型的替代宿主病原体模型。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大蜡螟（Galleria mellonella）中进行测试，这是一种目前用于其他真菌感染的蜡螟幼虫。完成后，这些目标将提供必要的假设测试工具，结合基因组序列和表达分析，以解决致病性和毒力问题，最终目标是开发皮肤癣菌的诊断，治疗和预防策略。公共卫生相关性：皮肤癣菌是导致各种皮肤和指甲感染的病原性真菌，包括脚癣和头癣，这是社会经济不景气地区患者常见的头部真菌感染。据估计，皮肤癣菌是全世界最常见的真菌感染。该提案将开发一套分子工具，可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.