Contact-Dependent Expression of Met3 in Candida Biofilms

描述(申请人提供)：真菌病原体，白色念珠菌能够在惰性表面形成强烈附着的生物膜，如用于医疗器械的表面，以及在皮肤、指甲和粘膜等生物表面上。这一属性代表了一个关键特征，有助于这种机会主义的持续和传播，部分原因是它越来越多地成为免疫功能低下的个人、新生儿和老年人的重要病原体。虽然众所周知，与浮游微生物群落相比，微生物群落以生物膜的形式生长，并表现出不同的表型[例如获得性抗药性]，但这些表型明显的分子基础仍不清楚。最近的工作表明，与非生物表面接触后，白色念珠菌特异性地和迅速地上调硫同化和谷胱甘肽生物合成的途径以及许多其他基因，其中许多基因的功能未知(103)。这些观察让我们想知道，在分子水平上，真核生物是如何感知并响应与外来(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。这项应用的直接目标是确定调控白色念珠菌接触依赖性基因表达的关键成分。为了实现这一点，我们将使用MET3基因，该基因编码一种催化硫同化途径第一步的ATP硫酰基酶。MET3基因在细胞接触非生物表面的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的基因表达。在特定的目标1中，我们将定义c/S作用区域，它调控MET3在表面接触时的差异表达。这一目标的完成将导致在DNA中鉴定特定的序列元件，这些元件是MET3接触依赖表达所必需的，并将进一步产生白色念珠菌中MET3启动子的详细图谱，其中很少有人达到这一水平。在具体目标2中，将确定接触性基因表达所需的反式作用元件。这一目标的完成将导致阐明MET3的浮游和附着相关表达所需的调控元件，并定义对接触刺激唯一反应的转录因子。最后，在特定的目标3中，我们将评估MET3和硫同化途径在发病机制中的作用。假丝酵母菌的硫同化途径与其宿主哺乳动物的硫同化途径有质的不同，为新疗法的开发提供了有希望的靶点。了解接触感应的潜在机制应该为干扰细胞黏附提供新的方法，从而控制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.