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HISTOPLASMA CAPSULATUM MACROPHAGE--GENETIC INTERACTIONS

荚膜组织浆细胞巨噬细胞--遗传相互作用

基本信息

批准号：
2519573
负责人：
GEORGE S KOBAYASHI
金额：
$ 28.16万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-09-30 至 1999-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2519573
关键词：
alveolar macrophages artificial chromosomes clone cells complementary DNA fungal genetics gene expression gene targeting host organism interaction human tissue laboratory mouse messenger RNA northern blottings polymerase chain reaction protein purification protein sequence respiratory infections sequence tagged sites

项目摘要

Histoplasma capsulatum , a pathogenic dimorphic fungus that causes pulmonary and systemic disease in the normal and compromised host, poses a particular threat to patients who are immunologically defective, particularly those with the Acquired Immunodeficiency Syndrome (AIDS). In the AIDS patient progressive disseminated histoplasmosis is a significant cause of morbidity and mortality. Histoplasma capsulatum causes disease by parasitizing and surviving within non-activated macrophages. Little is known about the mechanisms by which the fungus survives and thrives within this environment. By understanding the genetic bases of how H. capsulatum subverts the normal killing mechanism of the macrophage and creates an hospitable intracellular niche, it may be possible to design more effective therapies and vaccines that can be used to treat the compromised patient and those at high risk to develop disease. Histoplasma capsulatum invades the human host through the lungs. The ability to survive within alveolar macrophages is therefore the first obstacle the organism must overcome to cause infection and a prime target for therapeutic intervention. Macrophages are highly phagocytic cells that kill microbes they have ingested by production of reactive oxygen and nitrogen metabolites, synthesis of bactericidal peptides, and formation of lysosomal acid hydrolases. Intracellular pathogens have developed diverse strategies to survive within macrophages, and in recent years we have uncovered a few of the tricks that used to avoid macrophage killing. However, the mechanisms by which H. capsulatum achieves the goal of intracellular survival is less well delineated. It neither escape from phagosomes nor inhibit phagosome-lysosome fusion; rather, these organisms survive within phagolysosomes, and appear to alter the intraphagosomal environment by moderating the pH within this organelle. We do not understand the mechanism by which this occurs, nor do we know whether the phagocytic event that mediates the internalization of these yeast is equivalent to the paradigms that have been established primarily using opsonized erythrocytes. There are 4 goals in our application. We plan to: (1) isolate and identify genes that are turned on and the products that are expressed during the process of attachment to- and survival within- macrophage; (2) clone and sequence the amplified mRNAs and perform comparative analysis of the peptide sequences they code for and select those that have low homology frequency with human sequences; (3) clone and characterize selected cDNA sequences from 2 cDNA libraries made from mRNA purified during attachment to macrophage infection and then perform gene knock-out experiments to identify the role these genes play in attachment and survival; and (4) locate the position of the cloned genes in our H. capsulatum yeast artificial chromosome (YAC) library.

荚膜组织胞浆菌，一种致病的二态真菌，可引起正常和受损宿主的肺部和全身疾病，姿势对免疫缺陷的患者来说是一个特别的威胁，尤其是患有获得性免疫缺陷综合症(艾滋病)的人。在……里面艾滋病患者进行性播散性组织胞浆菌病是一种重要的致病和死亡的原因。组织胞浆菌通过以下途径致病寄生并存活于未激活的巨噬细胞内。小才是已知真菌在体内存活和繁衍的机制这种环境。通过了解囊状血吸虫的遗传基础颠覆巨噬细胞的正常杀伤机制并创造一个热情好客的细胞内利基，可能会设计出更多可用于治疗受损患者的有效疗法和疫苗患者和高危人群罹患疾病。组织胞浆菌通过肺部侵入人体宿主。这个因此，在肺泡巨噬细胞内存活的能力是第一个生物体必须克服的引起感染的障碍和主要目标进行治疗干预。巨噬细胞是高度吞噬的细胞，通过产生活性氧和杀死它们摄入的微生物氮代谢产物，杀菌肽的合成和形成溶酶体酸性水解酶。细胞内的病原体已经发展出不同的在巨噬细胞内生存的策略，近年来我们已经揭示了一些过去用来避免杀死巨噬细胞的诀窍。然而，包裹体实现这一目标的机制对细胞内存活的描述较少。它既不能逃脱吞噬小体也不会抑制吞噬小体-溶酶体融合；相反，这些生物在吞噬小体内存活，并似乎改变吞噬小体内通过调节细胞器内的酸碱度来调节环境。我们不会了解这种情况发生的机制，我们也不知道介导这些酵母内化的吞噬事件是等同于主要使用已调理的红细胞。在我们的申请中有4个目标。我们计划：(1)分离和鉴定启动的基因和在过程中表达的产物巨噬细胞的附着和存活过程；(2)克隆和对扩增的mRNAs进行测序，并进行比较分析它们编码的多肽序列并选择那些同源性较低的序列频率与人类序列一致；(3)克隆和鉴定所选的基因从附着过程中纯化的mRNA中提取的两个cDNA文库中的序列巨噬细胞感染，然后进行基因敲除实验确定这些基因在依恋和生存中所起的作用；以及(4) 克隆的基因在我们胶囊酵母中的定位人工染色体(YAC)文库。