Molecular study of calcium channels in fungal pathogens.

真菌病原体中钙通道的分子研究。

• 批准号: 6941780
• 负责人: ANGIE GELLI
• 金额: $ 33.4万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6941780
• 关键词: Cryptococcus neoformans; acidity /alkalinity; biological signal transduction; calcium channel; calmodulin; cell membrane; drug resistance; fungal genetics; fungal proteins; gene environment interaction; genetic regulation; host organism interaction; intermolecular interaction; laboratory mouse; meningitis; molecular pathology; mycosis; osmotic pressure; protein localization; protein structure function; temperature; virulence; voltage /patch clamp

DESCRIPTION (provided by applicant): In order to proliferate within host cells and subsequently promote disease, fungal pathogens require an active calcium-calmodulin-dependent signaling cascade. The molecular mechanisms that determine how the calcium response is initiated and propagated in fungal pathogens remain largely unknown. A possible working model would state that calcineurin is activated by an increase in cytosolic calcium levels and calmodulin in response to signals specific to the host environment. Activated calcineurin would then subsequently dephosphorylate specific proteins required for fungal pathogenesis. We propose that calcium channels in pathogenic fungal cells initiate calcium signaling by responding to particular stimuli that are specific to the host environment (i.e. alkaline pH, 5% CO2, iron levels etc). Signal-specificity is achieved by the association of the calcium channel with key signaling proteins that could be recruited by calmodulin's interaction with the C-terminus of the calcium channel. The overall aim of the proposed research is to characterize the cellular and molecular mechanism by which pathogenic fungi use calcium channels to couple host-specific signals to a calcium/calmodulin-mediated signaling cascade that is required for colonization of the host environment. In order to elucidate the molecular mechanism of calcium channel function and regulation, structure-function studies using conventional patch clamp techniques in conditions that mimic the host environment will be performed. The calcium channel mutants generated for the structure-function studies, will be tested for virulence in an animal model of cryptococcal meningitis. Channel activation and regulation will be examined in cells that lack key signaling molecules in order to determine whether these signaling proteins regulate channel function as a means to impart signal specificity. A detailed study of calcium channel function and regulation is imperative not only for a clear understanding of the mechanism(s) underlying the signal-response coupling in the pathogenic fungal-host relationship but also for the potential development of small molecules that could function to prevent fungal proliferation in the host. For example, occlusion of the channel pore, or a change in channel voltage-sensitivity or the prevention of regulatory proteins from interacting with the channel could represent viable means by which small molecules may function to perturb channel activity, inhibit fungal cell proliferation within the host and ultimately prevent disease.

描述（由申请人提供）：为了在宿主细胞内增殖并随后促进疾病，真菌病原体需要活跃的钙 - 钙调蛋白依赖性信号传导级联。确定如何在真菌病原体中启动和传播钙反应的分子机制仍然在很大程度上未知。一个可能的工作模型将指出，钙调神经蛋白会通过胞质钙水平的增加和钙调蛋白的增加而激活，这是对特定于宿主环境的信号的响应。然后，活化的钙调蛋白随后将去磷酸化的真菌发病机理所需的特定蛋白质。我们提出，致病真菌细胞中的钙通道通过响应特定于宿主环境的特定刺激（即碱性pH，5％CO2，铁水平等）来启动钙信号传导。信号特异性是通过钙通道与关键信号蛋白的关联来实现的，这些信号蛋白可以通过钙调蛋白与钙通道的C末端的相互作用来募集。拟议研究的总体目的是表征细胞和分子机制，通过这种机制，致病真菌使用钙通道将特定于宿主特异性信号与钙/钙调蛋白介导的信号级联级联相结合，这是宿主环境定植所需的。为了阐明钙通道功能和调控的分子机制，在模仿宿主环境的条件下，使用常规贴片夹技术进行结构功能研究。用于结构功能研究的钙通道突变体将在隐球菌脑膜炎的动物模型中测试毒力。通道激活和调节将在缺乏关键信号分子的细胞中检查，以确定这些信号蛋白是否调节通道功能作为赋予信号特异性的一种手段。对钙通道功能和调控的详细研究不仅对于对病原真菌宿主关系中信号反应偶联的机制的清晰了解至关重要，而且对于可能起作用的小分子的潜在发展，这些分子的潜在发展可以防止宿主中的真菌增殖。 例如，通道孔的阻塞，或通道电压敏感性的变化或预防调节蛋白与通道相互作用的预防可以代表可行的手段，而小分子可以通过这种方式来抑制宿主内部的真菌细胞在扰动通道活性，并最终预防疾病。