NRG1 Regulation of Candida albicans Virulence

描述（由申请人提供）：白色念珠菌是念珠菌病最常见的病原体，目前是医院感染的第四大原因。C.白色念珠菌能够在单细胞酵母样形式（芽生孢子）和丝状形式（根据丝状结构的轻微变化称为菌丝或假菌丝）之间进行可逆的形态转变。这些形态发生转换是其致病潜力的关键。这一观点是基于大量使用C.不能经历这种形态发生转换的白色念珠菌突变株（即Δ cph 1、Δ efg 1、Δ tup 1）。然而，所有这些菌株都被锁定在一种或另一种形式中，因此无法充分说明形态发生转变在毒力中的作用。此外，这些突变株已经使用URA-爆破技术构建，其固有的问题对亲本和破坏株之间的遗传同质性提出了严重的问题。为了克服这些问题，我们构建了一个C。白念珠菌菌株，其中我们已经将最近发现的NRG 1基因置于四环素可调控启动子的控制下。In C. Nrg 1 p是一种DNA结合蛋白，其功能是作为表达的负调节因子。实验证实了多西环素（DOX）在许多实验室条件下控制该菌株形态转变的能力。该基因表达系统的优点还在于，它可以用于动物宿主，其中通过简单地将DOX加入到饮用水中来调节目的基因的表达。该建议的具体目的是：i）在念珠菌病的鼠模型中使用该菌株，以评估NRG 1的作用和在C.白色念珠菌毒力和ii）在增殖和生物膜生长条件下鉴定Nrg 1 p的下游靶标。

DESCRIPTION (provided by applicant): Candida albicans is the most common etiological agent of candidiasis, now the fourth leading cause of nosocomial infections. C. albicans is able to undergo reversible morphological transitions between unicellular yeast-like forms (blastospore) and filamentous forms (called hypha or pseudohypha depending on slight variations in the structure of the filament). These morphogenetic conversions are pivotal to its pathogenic potential. This belief is based upon the results obtained from a large number of virulence studies using C. albicans mutant strains unable to undergo this morphogenetic switch (i.e. delta cph1, delta efg1, delta tup1). However, all these strains are locked in one or other form so conclusions on the role of the morphogenetic transitions in virulence cannot be adequately addressed. Also, these mutant strains have been constructed using the URA-blaster technique with inherent problems that place serious questions on the genetic homogeneity between parental and disrupted strains. To overcome these problems, we have constructed a C. albicans strain which in which we have placed the recently discovered NRG1 gene under the control of a tetracycline-regulatable promoter. In C. albicans, Nrg1p is a DNA-binding protein that functions as a negative regulator of filamentation. Experiments confirmed the ability of doxycycline (DOX) to control the morphological transitions in this strain under a number of laboratory conditions. This gene expression system has also the advantage that it can be used in an animal host where expression of the gene of interest is regulated by simply adding DOX to the drinking water. The specific aims of this proposal are: i) to use this strain in a murine model of candidiasis in order to assess the role of NRG1 and the morphogenetic conversions in C. albicans virulence and ii) to identify downstream targets of Nrg1p under both planktonic and biofilm growing conditions.