Lipid-mediated fungal pathogenesis

摘要 该提案的长期目标是研究新型隐球菌（Cn）的致病机制 重点关注 Cn 糖脂如何调节真菌毒力和宿主免疫反应。 Cn 是一种真菌病原体，进入肺部并通过血流传播后，会导致生命- 对易感患者来说威胁脑膜脑炎，导致高发病率和死亡率。目前针对此的治疗方法 该疾病可能会产生无法忍受的剂量限制性副作用。1因此，需要新的治疗策略来更好地控制 与隐球菌病相关的高死亡率。 尽管疫苗被誉为上个世纪公共卫生领域最伟大的成就之一， 开发针对隐球菌病和一般真菌感染的安全有效的疫苗一直是一个主要任务 障碍主要是由于缺乏对支持保护性免疫的机制的了解。此外，真菌 疫苗也需要在免疫缺陷的情况下有效，例如艾滋病患者的 CD4 T 细胞淋巴细胞减少症， 因为免疫缺陷患者最容易受到隐球菌病和其他侵袭性真菌感染。 在之前的研究中，我们删除了甾醇葡萄糖苷酶1（Sgl1）基因，所得突变体（Dsgl1）高度富集 存在于甾醇糖苷 (SG) 中，否则在野生型 (WT) Cn 细胞中无法检测到。2 SG 是存在于 多种真菌和其他微生物，容易刺激宿主免疫力。3-5 在上一个资助周期中， 我们证明 Cn Dsgl1 突变体在小鼠模型中不具有致病性，并且在鼻内给药后，突变细胞 在肺部环境中被迅速消除。2非常有趣的是，接受 Cn Dsgl1 突变体的小鼠现在受到保护 受到有毒 Cn WT 的攻击，即使小鼠 CD4 或 CD8 T 细胞耗尽，也能实现这种保护， 模仿 HIV 宿主（图 12 和 2）。我们验证了 Sgl1 在第二种真菌病原体烟曲霉中的作用 （AF）。 Af DsglA 也没有毒力（图9）并且能够防止继发性Af WT 感染（图11）。重要的是， 我们发起了高通量筛选，发现了体外抑制 Sgl1 或 SglA 活性的小分子， 真菌细胞中的 SG（图 6）在低葡萄糖基本培养基中对 Cn 和 Af 发挥体外抗真菌活性 浓度（图2和3），并阻止Cn传播到大脑（图5），模仿观察到的表型 与相应的 Dsgl1 或 DsglA 突变体。 此外，Cn的一个关键特征是其多糖胶囊，主要由葡萄糖醛酸、木糖和 甘露糖（GXM）。非常有趣的是，我们发现 GXM 的缺失完全消除了 Cn Dsgl1.6 的保护表型 事实上，施用 Cn Dcap59/Dsgl1 双突变体并不能防止继发感染（图 8）。 基于这些观察，我们假设 Cn Sgl1 通过调节真菌毒力，是真菌毒力的主要调节因子。 SG 的水平，通过 GXM 刺激宿主针对隐球菌病的免疫反应（图 1）。至 检验这个假设，我们提出以下目标： 目标 1. 确定 Cn Sgl1 和 Af SglA 对真菌毒力的作用。 目标 2. 研究宿主对 Cn 积累的 SG 的免疫反应。 该提案将为真菌 SG 调节 Cn 病理学的作用和机制提供新的见解。 重要的是，这些研究还将有可能导致开发新的抗真菌策略来预防和/或治疗 免疫功能低下宿主的侵袭性真菌感染，例如隐球菌病和曲霉病。

ABSTRACT The long-term goal of this proposal is to study mechanism(s) of pathogenicity of Cryptococcus neoformans (Cn) focusing on how Cn glycolipids regulate fungal virulence and the host immune response. Cn is a fungal pathogen that, upon entering the lung and disseminating through the bloodstream, causes a life- threatening meningo-encephalitis in susceptible patients, leading to high morbidity and mortality. Current therapies for this disease can have intolerable and dose-limiting side effects.1 Thus, new treatment strategies are warranted to better control the high mortality associated with cryptococcosis. Although vaccines have been hailed as one of the greatest achievements in public health during the past century, the development of safe and efficacious vaccines against cryptococcosis, and fungal infections in general, has been a major hurdle mainly due to the lack of knowledge about the mechanisms that underpin protective immunity. Additionally, fungal vaccines need to be effective also in conditions of immunodeficiency, such as CD4+ T cell lymphopenia in AIDS patients, because immunodeficient patients are the most susceptible to cryptococcosis and other invasive fungal infections. In previous studies, we deleted the sterol-glucosidase 1 (Sgl1) gene and the resulting mutant (Dsgl1) is highly enriched in sterol glucosides (SGs), which are otherwise not detectable in wild-type (WT) Cn cells.2 SGs are glycolipids present in a variety of fungi and other microorganisms and are prone to stimulate host immunity.3-5 During the previous funding cycle, we showed that Cn Dsgl1 mutant is not pathogenic in a mouse model and, upon intranasal administration, the mutant cells are rapidly eliminated in the lung environment.2 Very interestingly, mice receiving Cn Dsgl1 mutant are now protected when challenged with virulent Cn WT and this protection is achieved even when mice are CD4+ or CD8+ T cell depleted, mimicking the HIV+ host (Fig.12 and2). We validated the role of Sgl1 in second fungal pathogen, Aspergillus fumigatus (Af). The Af DsglA is also not virulent (Fig. 9) and able to protect against a secondary Af WT infection (Fig. 11). Importantly, we initiated a high throughput screening and found small molecules that inhibit Sgl1 or SglA activity in vitro, accumulates SGs in fungal cells (Fig. 6), exert in vitro antifungal activity against Cn and Af in minimum media with low glucose concentrations (Figs. 2 and 3), and block the dissemination of Cn to the brain (Fig. 5), mimicking the phenotypes observed with the respective Dsgl1 or DsglA mutants. In addition, a key characteristic of Cn is its polysaccharide capsule, mainly made of glucuronic acid, xylose and mannose (GXM). Very interestingly, we found that deletion of GXM totally abrogates the protective phenotype of Cn Dsgl1.6 In fact, the administration of the Cn Dcap59/Dsgl1 double mutant does not protect against a secondary infection (Fig. 8). Based on these observations, we hypothesize that Cn Sgl1 is a major regulator of fungal virulence by modulating the level of SGs, which, through GXM, stimulate the host immune response against cryptococcosis (Fig. 1). To test this hypothesis, we propose the following aims: Aim 1. To determine the role of Cn Sgl1 and Af SglA on fungal virulence. Aim 2. To study the host immune response against Cn accumulating SGs. This proposal will provide novel insights on the role and mechanisms by which fungal SGs regulate the pathobiology in Cn. Importantly, these studies will also potentially lead to the development of novel antifungal strategies to prevent and/or treat invasive fungal infections, such as cryptococcosis and aspergillosis, in immunocompromised hosts.