Lipid-mediated fungal pathogenesis

脂质介导的真菌发病机制

基本信息

批准号：
10494244
负责人：
Maurizio Del Poeta
金额：
$ 65.99万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10494244
关键词：
Achievement Acquired Immunodeficiency Syndrome Antifungal Agents Antigens Aspergillosis Aspergillus fumigatus Biochemical Biological Assay Blood Circulation Brain CD4 Positive T Lymphocytes CD8-Positive T-Lymphocytes Cells Characteristics Cryptococcosis Cryptococcus neoformans Crystallization Data Defect Development Disease Dose-Limiting Environment Funding Fungal Antigens Fungal Vaccines Genes Glucose Glucuronic Acids Glycolipids Goals Growth HIV Immune response Immunity Immunocompromised Host Immunologic Deficiency Syndromes In Vitro Incubated Infection Intranasal Administration Knowledge Lead Libraries Life Lipids Lung Lymphopenia Mannose Mediating Meningoencephalitis Morbidity - disease rate Mus Mycoses Pathogenesis Pathogenicity Patients Phenotype Polysaccharides Primary Infection Public Health Role Sterols Structure T-Lymphocyte Testing Therapeutic Vaccines Virulence Virulent Xylose base blood glucose regulation capsule fungus glucosidase high throughput screening improved in vitro activity in vivo inhibitor insight microorganism mortality mouse model mutant novel pathogenic fungus prevent screening secondary infection side effect small molecule sterol glucoside treatment strategy

项目摘要

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.

抽象的该提议的长期目标是研究加密鸡（CN）的致病性机制关注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，进入肺并通过血液传播后，会导致生命 - 威胁易感患者的脑膜脑炎，导致高发病和死亡率。目前的疗法疾病可能具有无法忍受和限制剂量的副作用。1因此，有必要采取新的治疗策略以更好地控制与隐球菌病有关的高死亡率。尽管在过去一个世纪中，疫苗被誉为公共卫生中最大的成就之一，但开发安全有效的疫苗，以防止隐球菌病以及真菌感染，这是主要的障碍主要是由于缺乏对保护性免疫的机制的知识。另外，真菌疫苗还需要在免疫缺陷的疾病中有效，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入性真菌感染的影响。在先前的研究中，我们删除了固醇 - 葡萄糖苷酶1（SGL1）基因，所得突变体（DSGL1）高度富集在固醇糖苷（SGS）中，否则在野生型（WT）CN细胞中无法检测到的糖苷。2SGS是A中存在于A中多种真菌和其他微生物，容易刺激宿主免疫。3-5在上一个资金周期中，我们表明，在小鼠模型中，CN DSGL1突变体不是致病性的，并且在鼻内给药后，是突变细胞的 2非常有趣的是，接收CN DSGL1突变体的小鼠现在受到保护受到毒气CN WT的挑战，即使小鼠是CD4+或CD8+ T细胞耗尽的，也可以实现这种保护模仿艾滋病毒+宿主（图12和2）。我们验证了SGL1在第二个真菌病原体中的作用（AF）。 AF DSGLA也没有毒性（图9），能够防止继发性AF WT感染（图11）。重要的是，我们开始了高吞吐量筛选，并发现在体外抑制SGL1或SGLA活性的小分子积累真菌细胞中的SG（图6），在低葡萄糖的最小培养基中对CN和AF发挥体外抗真菌活性浓度（图2和3），并阻止CN向大脑的传播（图5），模仿观察到的表型具有各自的DSGL1或DSGLA突变体。此外，CN的关键特征是其多糖胶囊，主要由葡萄糖酸，木糖和 mannose（GXM）。有趣的是，我们发现GXM的缺失完全消除了CN DSGL1.6的保护表型实际上，CN DCAP59/DSGL1双突变体的给药不能预防继发感染（图8）。基于这些观察结果，我们假设CN SGL1是通过调节的真菌毒力的主要调节剂通过GXM通过GXM刺激宿主免疫反应对隐球菌病（图1）。到检验该假设，我们提出以下目的：目的1。确定CN SGL1和AF SGLA在真菌毒力中的作用。目的2。研究宿主免疫反应，以累积CN积累SG。该提案将提供有关真菌SG调节CN病理生物学的作用和机制的新见解。重要的是，这些研究还可能导致发展新型抗真菌策略，以预防和/或治疗免疫功能低下的宿主中的侵入性真菌感染，例如隐球菌病和曲霉病。