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.

组织囊肿囊肿，一种致病的二态真菌，引起正常和受损的宿主中的肺和全身性疾病，姿势对免疫学上有缺陷的患者的特殊威胁，特别是患有获得免疫缺陷综合征（AIDS）的人。在艾滋病患者进行性散发性组织质症是重要的发病率和死亡率的原因。组织囊肿囊肿会引起疾病在未激活的巨噬细胞中寄生和存活。几乎没有知道真菌在内部生存和繁荣的机制已知这个环境。通过了解H. capsulatum的遗传基础颠覆巨噬细胞的正常杀戮机制，并创建一个热情好客的细胞内利基，可能有可能设计更多可用于治疗受损的有效疗法和疫苗患者和患有高风险的患者。组织囊肿通过肺侵袭了人类宿主。这因此，在肺泡巨噬细胞中生存的能力是第一个生物体必须克服引起感染和主要目标的障碍用于治疗干预。巨噬细胞是高度吞噬细胞杀死微生物，它们通过产生活性氧和氮代谢物，杀菌肽的合成和形成溶酶体酸水解酶。细胞内病原体已经发展出多种多样在巨噬细胞中生存的策略，近年来我们已经发现了一些用于避免巨噬细胞杀死的技巧。但是，H。capsulatum实现目标的机制细胞内存活率不太明确。它没有逃脱吞噬小体或抑制吞噬体 - 溶酶体融合；相反，这些生物在吞噬物体内生存，似乎改变了阴道内的环境通过调节该细胞器中的pH值。我们没有了解发生这种情况的机制，我们也不知道是否介导这些酵母内在化的吞噬事件是相当于主要使用的范例调子红细胞。我们的应用中有4个目标。我们计划：（1）隔离并识别打开的基因以及在巨噬细胞内的附着和生存过程；（2）克隆和序列放大的mRNA并进行比较分析他们为肽序列编码并选择那些同源性较低的肽序列人类序列的频率；（3）克隆并表征选定的cDNA 来自在附着过程中由mRNA制成的2个cDNA库的序列进行巨噬细胞感染，然后进行基因敲除实验确定这些基因在依恋和生存中的作用；（4）找到克隆基因在我们的H. capsulatum酵母中的位置人造染色体（YAC）图书馆。