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）上形成强粘附性生物膜。这一属性代表了一个关键特征，这有利于这种机会主义者的持续存在和传播，部分原因是它作为免疫功能低下的个体、新生儿和老年人中的重要病原体越来越多地出现。虽然众所周知，微生物群落作为生物膜生长，并显示出与它们的共生对应物相比不同的表型[例如获得性耐药性]，但这些表型表现的分子基础仍然未知。最近的研究表明，当接触非生物表面时，C。白念珠菌特异性地快速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尚不清楚（103）。这些观察使我们想知道，在分子水平上，真核生物如何感知和响应与外来（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。本申请的直接目标是鉴定调节C.白色念珠菌为了实现这一点，我们将使用MET 3基因，该基因编码催化硫同化途径第一步的ATP硫酸化酶。MET 3基因在细胞与非生物表面接触的50分钟内上调超过20倍，因此可用于开发实验系统以阐明接触依赖性基因表达。在具体目标1中，我们将定义c/s作用区域，其调节表面接触后MET 3的差异表达。该目标的完成将导致DNA中特异性序列元件的鉴定，这些序列元件是MET 3的接触依赖性表达所需的，并且将进一步产生C中MET 3启动子的详细图谱。白色念珠菌，很少有人被描述到这个水平。在具体目标2中，将确定接触依赖性基因表达所需的反式作用元件。该目标的完成将导致阐明MET 3浮游和粘附相关表达所需的调节元件，并定义对接触刺激具有独特反应的转录因子。最后，在具体目标3中，我们将评估MET 3和硫同化途径在发病机制中的作用。假丝酵母硫同化途径与其哺乳动物宿主的硫同化途径在性质上不同，为开发新的治疗方法提供了有希望的靶点。了解接触感应的潜在机制应该提供新的方法来干扰细胞粘附，从而控制在初始接触点的生物膜形成。

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.