Genetics and visualization of innate host response to C. albicans infection in vi

说明(申请人提供)：念珠菌感染是第四位最常见的医院感染，并导致不成比例的死亡率。白色念珠菌是一种无处不在的共生菌，也是人类真菌病最常见的原因。弥漫性血念珠菌血症可归因于30%的死亡率，与化疗引起的中性粒细胞减少症、器官移植、慢性肉芽肿性疾病等原发遗传缺陷以及提供直接进入血流的静脉插管等医疗程序有关。人类流行病学和小鼠实验表明，先天免疫功能是抵御播散性疾病的主要屏障，但巨噬细胞在反应和清除疾病中的作用尚不清楚。不幸的是，技术限制限制了我们测试巨噬细胞在其自然感染环境中的作用的能力。在没有基质和可溶线索的情况下，分离的巨噬细胞摧毁白色念珠菌的能力相对较弱，这导致了这样的想法，即它们在限制感染方面起到的作用微乎其微，但可能会促进疾病在组织中的传播。然而，我们最近的非侵入性成像，使用了一种新的播散性透明的念珠菌-斑马鱼感染模型，表明巨噬细胞在其自然环境中具有增强的限制增殖的能力。 和白色念珠菌的萌发。我们假设巨噬细胞限制了白念珠菌的丝状生长，但促进了感染在全身的传播。我们将利用斑马鱼感染模型，通过活体实时成像、基因敲除、靶向细胞消融和真菌突变体来确定巨噬细胞在限制真菌增殖和促进传播方面的作用。带有标记巨噬细胞的转基因鱼将被用来跟踪单个真菌-吞噬细胞在体内的相互作用，我们将消融和扰动巨噬细胞，以确定它们在调节真菌形态发生转换、增殖和传播中的作用。拟议的实验将通过利用透明斑马鱼模型的优势来测试在哺乳动物模型中一直难以解决的先天免疫的基本问题。这些实验的结果将使我们对白念珠菌先天免疫的机制有一个更全面的基本了解，并为有针对性地筛选调节这些巨噬细胞活性的新宿主和病原体途径奠定基础。发现真菌感染期间巨噬细胞活动的潜在机制有可能导致新的治疗干预措施。 公共卫生相关性：传染病影响世界各地的人类，并在美国造成重大死亡率和发病率。目前控制真菌病的方法是不够的，特别是在免疫功能低下的患者群体中，因此研究替代治疗方法至关重要。这项研究的长期目标包括利用关于宿主-病原体相互作用的基本信息来开发治疗真菌疾病的疗法。

DESCRIPTION (provided by applicant): Candidemia is the 4th most common nosocomial infection, and leads to disproportionate mortality. Candida albicans is a ubiquitous commensal and the most frequent cause of fungal disease in humans. Disseminated bloodstream candidemia carries a >30% attributable mortality and is associated with chemotherapy-induced neutropenia, organ transplantation, primary genetic defects such as chronic granulomatous disease, and medical procedures such as intravenous catheterization that provide direct access to the bloodstream. Human epidemiology and mouse experiments have demonstrated that innate immune function is the primary barrier against disseminated disease, yet the role of macrophages in response and clearance of disease is still unclear. Unfortunately, technical constraints have limited our ability to test the roles of macrophages in their natural context of infection. The relatively weak capacity of isolated macrophages to destroy C. albicans in the absence of matrix and soluble cues has led to the idea that they play a marginal role limiting infection but could promote dissemination of disease among tissues. However, our recent non-invasive imaging, using a new transparent model of disseminated Candida-zebrafish infection, indicates that macrophages in their natural context have an enhanced ability to limit proliferation and germination of C. albicans. We hypothesize that macrophages limit filamentous growth of C. albicans but promote dissemination of infection throughout the body. We will exploit the zebrafish infection model to determine the role of macrophages in limiting fungal proliferation and promotion of dissemination using intravital real-time imaging, gene knockdown, targeted cell ablation, and fungal mutants. Transgenic fish with marked macrophages will be used to follow individual fungal-phagocyte interactions in vivo, and we will ablate and perturb macrophages to determine their role in regulating fungal morphogenetic switching, proliferation and dissemination. The proposed experiments will test basic questions of innate immunity that have been intractable in mammalian models by exploiting the advantages of the transparent zebrafish model. The results of the proposed experiments will yield a more complete basic understanding of the mechanisms underlying innate immunity to candidemia and set the stage for targeted screening for novel host and pathogen pathways regulating these macrophage activities. The discovery of mechanisms underlying macrophage activity during fungal infection has the potential to lead to new therapeutic interventions. PUBLIC HEALTH RELEVANCE: Infectious diseases affect humans worldwide and cause significant mortality and morbidity in the United States. Current methods to control fungal disease are inadequate, especially in the immunocompromised patient population, so it is critical to investigate alternative treatments. A long-term goal of this investigation involves leveraging basic information about host-pathogen interaction to develop therapeutics to treat fungal disease.