Lipid-mediated fungal pathogenesis

摘要 本项目的长期目标是研究新型隐球菌（Cn）的致病机制 专注于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，一旦进入肺部并通过血液传播，就会导致生命- 在易感患者中威胁脑膜脑炎，导致高发病率和死亡率。目前的治疗方法 这种疾病可能会产生难以忍受且剂量有限的副作用。1因此，需要新的治疗策略来更好地控制 隐球菌病的高死亡率 尽管疫苗被誉为过去世纪公共卫生领域最伟大的成就之一， 开发安全有效的疫苗来对抗隐球菌病和一般的真菌感染， 这一障碍主要是由于缺乏对保护性免疫机制的了解。此外，真菌 疫苗需要在免疫缺陷的情况下也是有效的，例如AIDS患者中的CD 4 + T细胞淋巴细胞减少症， 因为免疫缺陷的患者最容易感染隐球菌病和其他侵袭性真菌感染。 在以前的研究中，我们删除了甾醇葡萄糖苷酶1（Sgl 1）基因，得到的突变体（Dsgl 1）高度富集 在甾醇葡糖苷（SG）中，其在野生型（WT）Cn细胞中不可检测。2 SG是存在于 各种真菌和其他微生物，并易于刺激宿主免疫力。3 -5在上一个供资周期， 我们发现Cn Dsgl 1突变体在小鼠模型中不是致病性的，并且在鼻内给药后， 非常有趣的是，接受Cn Dsgl 1突变体的小鼠现在受到保护， 用毒性Cn WT攻击并且即使当小鼠是CD 4+或CD 8 + T细胞耗尽时也实现这种保护， 模拟HIV+宿主（图12和2）。我们验证了Sgl 1在第二种真菌病原体烟曲霉中的作用， （Af）. Af DsglA也是无毒的（图9）并且能够保护免受继发性Af WT感染（图11）。重要的是， 我们启动了高通量筛选，发现在体外抑制Sgl 1或SglA活性的小分子， 真菌细胞中的SG（图6）在低葡萄糖的基本培养基中对Cn和Af发挥体外抗真菌活性 浓度（图2和3），并阻断Cn向大脑的传播（图5），模拟观察到的表型 与相应的Dsgl 1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、木糖和葡萄糖醛酸组成。 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的保护性表型 事实上，给予Cn Dcap 59/Dsgl 1双突变体不能防止继发感染（图8）。 基于这些观察，我们假设Cn Sgl 1是真菌毒力的主要调节因子，通过调节 通过GXM刺激宿主对隐球菌病的免疫应答的SGs水平（图1）。到 为了检验这一假设，我们提出以下目标： 目标1。研究Cn Sgl 1和Af SglA在真菌致病力中的作用。 目标2.目的：研究宿主对Cn积累的SGs的免疫应答。 这一建议将提供新的见解的作用和机制，真菌SG调节Cn的病理生物学。 重要的是，这些研究还可能导致新的抗真菌策略的发展，以预防和/或治疗 侵袭性真菌感染，如隐球菌病和曲霉病，免疫功能低下的宿主。

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.