Lipid-mediated fungal pathogenesis

摘要 本方案的长期目标是研究新生隐球菌(CN)的致病机制(S)。 重点研究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，当进入肺部并通过血液传播时，会导致生命- 对易患脑膜炎的患者造成威胁，导致高发病率和死亡率。目前对此的治疗方法 疾病可能会产生无法忍受和剂量限制的副作用。1因此，有必要制定新的治疗策略来更好地控制疾病。 与隐球菌病相关的高死亡率。 尽管疫苗被誉为过去一个世纪公共卫生领域最伟大的成就之一，但 针对隐球菌病和一般真菌感染的安全和有效疫苗的开发一直是主要的 这一障碍主要是由于缺乏对保护免疫的基础机制的了解。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CD4T细胞淋巴细胞减少症， 因为免疫缺陷患者最容易患上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(Sgl1)基因，得到的突变体(Dsgl1)高度浓缩 2在野生型(WT)CN细胞中不能检测到的甾醇糖苷(SGS)是存在于 真菌和其他微生物种类繁多，容易刺激宿主免疫。3-5在上一个供资周期中， 我们在小鼠模型中证明了CN Dsgl1突变体是不致病的，并且，在鼻腔给药时，突变细胞 在肺环境中被迅速消除。2非常有趣的是，接受CN Dsgl1突变的小鼠现在受到保护，当 用强毒CN WT攻击，即使当小鼠的CD4或CD8 T细胞耗尽时，这种保护也能实现， 模仿HIV宿主(图12和图2)。我们验证了Sgl1在第二真菌病原菌烟曲霉中的作用 (AF)。Af DsglA也是无毒的(图9)，并能够预防二次Af WT感染(图11)。重要的是 我们启动了高通量筛选，发现了在体外抑制Sgl1或Sgla活性的小分子，积累 真菌细胞中的SGS(图6)在低糖的最低浓度下对CN和Af具有体外抗真菌活性 浓度(图2和3)，并阻止CN向大脑扩散(图5)，模拟观察到的表型 与各自的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的缺失完全取消了CN Dsgl1.6的保护性表型 事实上，CN Dcap59/Dsgl1双重突变体的管理并不能预防二次感染(图8)。 基于这些观察，我们假设CN Sgl1是真菌毒力的主要调节因子，通过调节 SGS的水平，通过GXM刺激宿主对隐球菌病的免疫反应(图1)。至 为了验证这一假设，我们提出了以下目标： 目的1.确定CN-Sgl1和Af-SglA在真菌毒力中的作用。 目的2.研究宿主对CN蓄积SGS的免疫应答。 这一建议将为真菌SGS在CN中调节病理生物学的作用和机制提供新的见解。 重要的是，这些研究还可能导致开发新的抗真菌策略来预防和/或治疗 侵袭性真菌感染，如隐球菌病和曲霉病，在免疫受损的宿主中。

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.