Capsular-Independent Inhibition of Phagocytosis by Cryptococcus neoformans

新生隐球菌吞噬作用的荚膜独立抑制

基本信息

批准号：
8779933
负责人：
Christina Homer
金额：
$ 3.46万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-01 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8779933
关键词：
Acquired Immunodeficiency Syndrome Antibodies Binding Biological Assay Brain Cessation of life Characteristics Clinical Cryptococcus neoformans Custom Defect Diagnosis Disease Drug Targeting Family Family member Gene Family Genes Genetic Hand Human In Vitro Infection Injection of therapeutic agent Investigation Knock-out Laboratories Lung Mannose Masks Measures Mediating Methods Modeling Molecular Mus Mycoses PTPN6 gene Pattern Pattern Recognition Phagocytosis Phagocytosis Inhibition Pharmaceutical Preparations Phenotype Phosphoric Monoester Hydrolases Polysaccharides Protein Family Protein Tyrosine Kinase Proteins Publishing Recombinants Relative (related person)Role Signal Pathway Signal Transduction Spleen Surface Tail Testing Tyrosine Veins Virulence Yeasts base beta-Glucans capsule chromatin immunoprecipitation drug development fungus in vivo macrophage member mortality mutant novel overexpression pathogen prevent public health relevance receptor research study success trait transcription factor transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): Cryptococcus neoformans is an opportunistic yeast pathogen responsible for over 1 million infections and 600,000 deaths annually. In a published study, we identified a transcriptional regulator Gat201 that inhibits phagocytosis independently of the polysaccharide capsule. Previously, the major component of the polysaccharide capsule, glucoronoxylomannan (GXM), was believed to be the only mechanism by which the fungus could evade phagocytosis by macrophages. Our laboratory performed studies using custom microarrays to define which genes were controlled by Gat201 and screened each Gat201-regulated gene for its effect on phagocytosis. We determined that Blp1, a protein regulated by Gat201, inhibited C. neoformans phagocytosis by macrophages. I performed subsequent chromatin immunoprecipitation and sequencing (ChIP-Seq) experiments and discovered that two other proteins in the same family as Blp1 (Blp2 and Blp4) are targets of Gat201. Thus, my aims are as follows: (1) Determine whether Blp1, Blp2 and Blp4 contribute in a redundant manner to the GXM-independent phagocytosis inhibition controlled by Gat201 by constructing double and triple mutant strains and triply-misregulated Blp strains (Blp1 knockout, Blp2 and Blp4 overexpressed) in C. neoformans. Analysis of the phenotypes of these mutant strains will show whether Blp1, Blp2, and Blp4 function in the same virulence mechanism in vitro and in vivo. I anticipate this study will reveal some promising drug targets for treating cryptococcal infection. (2 & 3) Determine the molecular mechanism of Blp1 phagocytosis inhibition. Since Blp1 likely does not act through GXM, I will determine the novel mechanism by which this protein inhibits phagocytosis. I will test two competing hypotheses: Blp1 interacts with pathogen-associated molecular patterns (PAMPs) on C. neoformans so that macrophages do not recognize it as a pathogen or Blp1 engages inhibitory receptors on macrophages. I will test the first hypothesis by comparing phagocytosis levels of wildtype C. neoformans and the fungus lacking Blp proteins by wildtype macrophages and macrophages lacking the Syk activation factor in opsonized and unopsonized conditions. To test the second hypothesis, I will compare phagocytosis levels of wildtype and Blp-deficient C. neoformans by wildtype macrophages and macrophages lacking the SHP-1 inhibition factor in opsonized and unopsonized conditions. Together, these two aims will elucidate the mechanism by which C. neoformans protects itself from macrophages independently of its polysaccharide capsule.

描述（由申请人提供）：Neoformans是一种机会性的酵母病原体，每年可导致100万多个感染和600,000人死亡。在一项已发表的研究中，我们确定了一个转录调节剂GAT201，该调节剂与多糖胶囊独立抑制吞噬作用。以前，据信多糖胶囊（GXM）的主要成分被认为是唯一的机制，使真菌可以通过巨噬细胞逃避吞噬作用。我们的实验室使用自定义微阵列进行了研究，以定义哪些基因由GAT201控制，并筛选了每个GAT201调节的基因对吞噬作用的影响。我们确定BLP1是由GAT201调控的蛋白质，抑制了巨噬细胞的新生虫吞噬作用。我进行了随后进行的染色质免疫沉淀和测序（CHIP-SEQ）实验，并发现与BLP1同一家族中的另外两个蛋白质（BLP2和BLP4）是GAT201的靶标。 Thus, my aims are as follows: (1) Determine whether Blp1, Blp2 and Blp4 contribute in a redundant manner to the GXM-independent phagocytosis inhibition controlled by Gat201 by constructing double and triple mutant strains and triply-misregulated Blp strains (Blp1 knockout, Blp2 and Blp4 overexpressed) in C. neoformans.对这些突变菌株的表型的分析将显示BLP1，BLP2和BLP4在体外和体内是否在相同的毒力机理中功能。我预计这项研究将揭示一些有望治疗隐球菌感染的药物靶标。（2＆3）确定BLP1吞噬作用抑制的分子机制。由于BLP1可能不会通过GXM起作用，因此我将确定该蛋白质抑制吞噬作用的新机制。我将测试两个竞争的假设：BLP1与C. Neoformans上与病原体相关的分子模式（PAMP）相互作用，以使巨噬细胞不会将其识别为病原体或BLP1在巨噬细胞上抑制抑制受体。我将通过比较野生型C. Neoformans的吞噬作用水平和缺乏野生型巨噬细胞和巨噬细胞缺乏BLP蛋白的真菌来检验第一个假设，而真菌缺乏BLP蛋白，而巨噬细胞和巨噬细胞缺乏Syk激活因子，而在调查和无儿子中则缺乏SYK激活因子。为了检验第二个假设，我将通过野生型巨噬细胞和巨噬细胞和巨噬细胞中缺乏SHP-1抑制因子的吞噬症和BLP缺陷型新形构型吞噬作用水平。总之，这两个目标将阐明新生梭菌保护自身免受巨噬细胞免受多糖胶囊的侵害的机制。