NRG1 Regulation of Candida albicans Virulence

描述(申请人提供)：白色念珠菌是最常见的念珠菌病病原体，现在是医院感染的第四大原因。白念珠菌能够经历单细胞酵母样形态(胚芽孢子)和丝状形态(根据细丝结构的微小变化而称为菌丝或假菌丝)之间的可逆形态转变。这些形态发生的转换是其致病潜力的关键。这一信念是基于对无法经历这种形态发生转换的白色念珠菌突变株(即Delta cph1、Delta efg1、Delta Tup1)进行的大量毒力研究的结果。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得到充分的解决。此外，这些突变菌株是使用URA-Blaster技术构建的，存在固有问题，这些问题严重影响了亲本菌株和破坏菌株之间的遗传同质性。为了克服这些问题，我们构建了一株白色念珠菌，其中我们将最近发现的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，起着丝状化负调节的作用。实验证实，在一些实验室条件下，多西环素(DOX)能够控制该菌株的形态转变。该基因表达系统还具有的优点是，它可以用于动物宿主中，在动物宿主中，只需向饮用水中添加DOX就可以调节目的基因的表达。这项建议的具体目的是：i)在白念珠菌病小鼠模型中使用该菌株，以评估NRG1在白念珠菌毒力中的作用和形态发生转化；ii)确定Nrg1p在浮游和生物膜生长条件下的下游靶标。

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.