Contact-Dependent Expression of Met3 in Candida Biofilms

描述（由申请人提供）：真菌病原体白色念珠菌能够在惰性表面（如用于医疗器械的表面）和生物表面（如皮肤、指甲和粘膜）上形成强粘附的生物膜。这一特性代表了一个关键特征，它促进了这种机会主义的持续和传播，部分解释了它在免疫功能低下的个体、新生儿和老年人中越来越多地作为一种重要病原体出现。众所周知，与浮游生物相比，微生物群落以生物膜的形式生长，并表现出不同的表型（如获得性耐药性），但这些表型的分子基础仍不清楚。最近的研究表明，与非生物表面接触后，白色念珠菌特异性地、快速地上调硫同化和谷胱甘肽生物合成途径以及许多其他基因，其中许多基因的功能未知（103）。这些观察结果使我们想知道，在分子水平上，真核生物是如何感知和反应与外来（非生物或生物）表面的接触，然后将这些信息转化为基因表达和细胞行为的改变模式的。这个应用程序的直接目的是确定关键成分，其中调节接触依赖基因表达的白色念珠菌。为了实现这一点，我们将使用MET3基因，该基因编码ATP硫化酶，催化硫同化途径的第一步。MET3基因在细胞接触非生物表面50分钟内上调20倍以上，因此可以用来开发一个实验系统来阐明接触依赖基因表达。在Specific Aim 1中，我们将定义c/s作用区域，该区域调节MET3在表面接触时的差异表达。这一目标的完成将导致鉴定DNA中特定的序列元件，这是MET3接触依赖性表达所必需的，并将进一步产生白色念珠菌中MET3启动子的详细图谱，很少有这种水平的表征。在特异性目标2中，将鉴定接触依赖基因表达所需的反式作用元件。这一目标的完成将导致MET3浮游和粘附相关表达所需的调控元件的阐明，并定义转录因子，这些转录因子对接触刺激有独特的反应。最后，在Specific Aim 3中，我们将评估MET3和硫同化途径在发病机制中的作用。念珠菌的硫同化途径与其哺乳动物宿主的硫同化途径有质的不同，为开发新的治疗方法提供了有希望的靶点。了解接触传感的潜在机制将为干扰细胞粘附从而控制初始接触点的生物膜形成提供新的方法。

DESCRIPTION (provided by applicant): The fungal pathogen, Candida albicans is able to form strongly adherent biofilms on inert surfaces such as those used for medical devices and on biological surfaces such as the skin, nails and mucous membranes. This attribute represents a key trait, which facilitates the persistence and dissemination of this opportunist, accounting in part for its increasing emergence as an important pathogen in immunocompromised individuals, neonates and the elderly. While it is widely known that microbial communities grow as biofilms and display distinct phenotypes [e.g. acquired drug resistance] as compared with their planktonic counterparts, the molecular basis through which these phenotypes are manifest remains unknown. Recent work shows that upon contact with an abiotic surface, C. albicans specifically and rapidly up regulates pathways for sulfur assimilation and glutathione biosynthesis and a number of other genes, many of which are of unknown function (103). These observations led us to ask how, at the molecular level, does a eukaryotic organism sense and respond to contact with a foreign (abiotic or biotic) surface and then transduce this information into altered patterns of gene expression and cell behavior. The immediate objective of this application is to identify key components, which regulate contact-dependent gene expression in C. albicans. To accomplish this we will use the MET3 gene, which encodes an ATP sulfurylase catalyzing the first step of the sulfur assimilation pathway. The MET3 gene is up-regulated over 20-fold within 50 minutes of cell contact to the abiotic surface and thus can be used to develop an experimental system to elucidate contact-dependent gene expression. In Specific Aim 1, we will define the c/s-acting regions, which regulate the differential expression of MET3 upon surface contact. Completion of this Aim will result in the identification of specific sequence elements in the DNA, which are required for contact- dependent expression of MET3 and will further produce a detailed map of the MET3 promoter in C. albicans, for which few have been characterized to this level. In Specific Aim 2, trans-acting elements required for contact-dependent gene expression will be identified. Completion of this Aim will result in the elucidation of the regulatory elements required for both planktonic and adherence-associated expression of MET3 and define transcription factors, which are uniquely responsive to the contact stimulus. Finally, in Specific Aim 3 we will assess the role of MET3 and the sulfur assimilation pathway in pathogenesis. The Candida sulfur assimilation pathway is qualitatively different from that of its mammalian host, providing promising targets for the development of new therapeutics. Understanding the underlying mechanism of contact sensing should provide novel approaches for interfering with cell adherence and thus controlling biofilm formation at the point of initial contact.