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，目前是医院感染的第四大原因。白色念珠菌能够在单细胞酵母样形态（囊胚）和丝状形态（根据丝状结构的细微变化称为菌丝或假菌丝）之间进行可逆的形态转变。这些形态发生的转变对其致病潜力至关重要。这一观点是基于大量的毒力研究得出的结果，这些研究使用的白色念珠菌突变株不能经历这种形态发生开关（即δ cph1， δ efg1, δ tup1）。然而，所有这些菌株都以一种或另一种形式锁定，因此关于形态发生转变在毒力中的作用的结论不能充分解决。此外，这些突变株是使用URA-blaster技术构建的，其固有的问题是亲本菌株和破坏菌株之间的遗传同质性存在严重问题。为了克服这些问题，我们构建了一个白色念珠菌菌株，我们将最近发现的NRG1基因置于四环素可调节启动子的控制下。在白色念珠菌中，Nrg1p是一种dna结合蛋白，作为丝化的负调节因子。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。这种基因表达系统的另一个优点是，它可以在动物宿主中使用，在动物宿主中，只需在饮用水中添加DOX即可调节目标基因的表达。该提案的具体目的是：i)在小鼠念珠菌病模型中使用该菌株，以评估NRG1在白色念珠菌毒力中的作用和形态发生转化；ii)在浮游和生物膜生长条件下确定Nrg1p的下游靶点。

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.