TWO-COMPONENT HISTIDINE KINASES OF CANDIDA ALBICANS

白色念珠菌是美国真菌感染的主要原因。目前在引起医院感染的所有病原体中排名第四。这种生物的毒力可能是多因素和组织特异性的，因为白色念珠菌能够感染许多不同的环境部位(例如，酸性阴道通道与血液和组织的中性pH)。二组分组氨酸激酶(HK)是原核生物和低等真核生物的环境感受器和磷酸传递蛋白，其活性导致与应激反应相关的基因转录激活。在致病真菌的HK基因中，我们最近分离到了一个白色念珠菌杂交HK(CaHK1)，它是目前所描述的所有HK基因中结构和功能上独一无二的。构建了cahk1缺失株，并与亲本株和基因重组株进行了体内外表型比较。在体外，空菌株的菌丝在pH 7.5时形成絮状，表明菌丝细胞表面的变化与caHK1的突变有关。与亲本菌株和基因重组菌株相比，空菌株在系统小鼠模型中也是无毒的，但在大鼠阴道粘膜模型中是有毒的。虽然Northern分析表明CaHK1在酸性和中性pH介质中都有表达，但我们推测Cahk1p的活性可能取决于其磷酸化状态。这项建议有三个具体目标。1.环境因素(温度、pH、渗透压)对CaHK1表达的影响将通过Northern分析进行体外检测；Cahk1p的翻译后修饰将被确定。2.我们将在口腔和阴道模型中评估CaHK1株的毒力，以扩大我们对Cahk1p在毒力中的作用的认识。3.Cahk1p与包括下游效应蛋白在内的其他蛋白的相互作用有待鉴定。综上所述，这些研究将部分确定Cahk1p的磷酸传递途径和CaHK1在念珠菌病发病机制中的作用。

Candida albicans is the leading cause of fungal infections in the US and, Candida spp. now rank fourth among all pathogens in causing nosocomial infections. Virulence in this organism is probably multi-factorial and tissue-specific, since C. albicans is capable of infecting a number of diverse environmental sites (for example, the acid vaginal canal vs. the neutral pH of blood and tissues). Two-component, histidine kinases (HK) are environmental sensor, phosphorelay proteins of prokaryotes and lower eukaryotes whose activity results in transcriptional activation of genes associated with an adaptive response to stress. Among the HK genes of pathogenic fungi, recently, we isolated a C. albicans hybrid HK (CaHK1), which is unique structurally and functionally among all HK genes thus far described. A cahk1 null strain has been constructed and compared phenotypically in vitro and in vivo to parental and gene-reconstituted strains. In vitro, hyphae of null strains flocculate at pH 7.5, indicating that cell surface changes in hyphae are associated with a mutation in caHK1 The null is also avirulent in a systemic murine model but virulent in a rat vaginal mucosal model when compared to parental and gene- reconstituted strains. While Northern analyses indicate that CaHK1 is expressed in both acid and neutral pH media, we hypothesize that activity of Cahk1p may depend upon its state of phosphorylation. There are 3 specific aims in this proposal. 1. The influence of environmental factors (temp, pH, osmolarity) on the expression of CaHK1 will be measured in vitro using Northern analyses; post-translational modifications of Cahk1p will be determined. 2. We will evaluate the virulence of CaHK1 strain constructs in oral and vaginal models in order to extend our knowledge on the role of Cahk1p in virulence. 3. Cahk1p interactions with other proteins including down-stream effector proteins are to be identified. In summary, these studies will partially define the Cahk1p phosphorelay pathway and function of CaHK1 in the pathogenesis of candidiasis.