Environmental Conditions Control Filamentation and Virulence in Candida albicans

描述(申请人提供)：白色念珠菌是世界范围内口腔念珠菌病的主要原因。从酵母(卵形的萌芽细胞)到菌丝(细长的细胞端到端连接)的可逆转变的能力是使白色念珠菌成为成功的机会致病菌的关键毒力特征。向细丝的转变与其他毒力因子共同调节，并发生在几种寄主环境条件下，包括370℃、血清、碳/氮饥饿和中性pH。暴露在这些生长条件下会激活许多信号转导途径，促进毒力相关基因的表达和菌丝生长，但对控制细丝特异转录因子的表达和活性的机制知之甚少。虽然可以使用抗真菌疗法来治疗念珠菌病，但许多抗真菌药物都有副作用，而且是抑菌的，而不是杀菌的。我们研究的长期目标是深入了解形态发生，以确定新的抗真菌药物靶点。我们的目标是了解白色念珠菌如何通过转录和翻译后机制对环境刺激做出反应。我们的研究将集中在Ume6、Nrg1和Rfg1，它们是调节菌丝特异基因表达和丝化的关键转录因子。我们假设环境细丝诱导条件对Ume6、Nrg1和Rfg1的转录调控和活性具有不同的控制作用。为了解释我们的假设，我们提出了以下特定目标：特定目标1：确定UME6和NRG1UME6和NRG1mRNA转录调控机制(S)对环境微丝诱导条件的响应。具体目标2：测定Ume6、Nrg1和Rfg1在几种环境条件下的转录活性。为了解决目标1，我们将使用启动子缺失分析与LacZ报告分析、凝胶迁移率改变分析和质谱仪来确定控制NRG1和UME6表达的转录调控因子，以响应特定的环境刺激。为了解决目标2，我们将使用一个带有LacZ报告基因的单杂交系统来研究Ume6、Nrg1和Rfg1对环境条件的转录活性。我们将使用RT-qPCR在这两个目的中量化LacZ的表达。在我们的研究完成后，我们希望对白色念珠菌形态发生的环境调节有更好的了解，这将有助于开发新的抗真菌疗法。 公共卫生相关性：白色念珠菌是全世界口腔真菌感染的主要原因，部分原因是全球艾滋病毒大流行。我们的研究将提供对一个关键的毒力因素的洞察，即在酵母和丝状之间切换的能力，这使这种生物成为一个成功的机会病原体。这一知识将有助于开发新的抗真菌疗法。

DESCRIPTION (provided by applicant): Candida albicans is the leading cause of oral candidiasis worldwide. The ability to undergo a reversible transition from yeast (ovoid, budding cells) to hyphal filaments (elongated cells attached end-to-end) is a key virulence trait that allows C. albicans to be a successful opportunistic pathogen. The transition to filaments is co-regulated with other virulence factors and occurs in response to several host environmental conditions including 370C, serum, carbon/nitrogen starvation and neutral pH. Exposure to these growth conditions activates numerous signal transduction pathways that promote virulence-related gene expression and hyphal growth, but little is known about the mechanisms that control the expression and activity of filament-specific transcription factors. Although antifungal therapies are available to treat candidiasis, many antifungal drugs have adverse side effects and are fungistatic, not fungicidal. The long term goal of our studies is to gain insight into morphogenesis to identify novel antifungal drug targets. Our objective is to understand how C. albicans regulators of filamentation respond to environmental stimuli via transcriptional and posttranslational mechanisms. Our studies will focus on Ume6, Nrg1 and Rfg1, key transcription factors that regulate hyphal- specific gene expression and filamentation. We hypothesize that environmental filament-inducing conditions differentially control the transcriptional regulation and activity of Ume6, Nrg1 and Rfg1. We propose the following Specific Aims to address our hypothesis: Specific Aim 1: Determine the mechanism(s) by which UME6 and NRG1 are transcriptionally regulated in response to environmental filament-inducing conditions. Specific Aim 2: Determine the transcriptional activity of Ume6, Nrg1 and Rfg1 in response to several environmental conditions. In order to address Aim 1, we will use a promoter deletion analysis with a lacZ reporter assay, electrophoretic mobility shift assays and mass spectrometry to identify the transcriptional regulators that control NRG1 and UME6 expression in response to specific environmental stimuli. To address Aim 2, we will use a one-hybrid system with a lacZ reporter gene to investigate the transcriptional activity of Ume6, Nrg1 and Rfg1 in response to environmental conditions. We will use RT-qPCR in both Aims to quantify lacZ expression. At the completion of our studies, we expect to have a better understanding of the environmental regulation of C. albicans morphogenesis, which will aid in the development of new antifungal therapies. PUBLIC HEALTH RELEVANCE: Candida albicans is the leading cause of oral fungal infections worldwide due, in part, to the global HIV pandemic. Our studies will provide insight into a key virulence factor, the ability to switch between yeast and filamentous forms, which makes this organism a successful opportunistic pathogen. This knowledge will aid in the development of new antifungal therapies.