HISTOPLASMA CAPSULATUM MACROPHAGE--GENETIC INTERACTIONS

荚膜组织浆细胞巨噬细胞--遗传相互作用

• 批准号: 2234580
• 负责人: GEORGE S KOBAYASHI
• 金额: $ 25.87万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2234580
• 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

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，一种致病性二型真菌， 正常和受损宿主的肺部和全身性疾病， 对免疫缺陷的病人来说是一个特别的威胁， 特别是那些患有获得性免疫缺陷综合症（艾滋病）的人。在 艾滋病患者进行性播散性组织胞浆菌病是一种有意义 发病和死亡的原因。荚膜组织胞浆菌通过以下方式引起疾病： 寄生并存活在未活化的巨噬细胞内。之甚少 了解真菌生存和繁荣的机制 这种环境。通过了解H.浆菌 破坏了巨噬细胞的正常杀伤机制， 好客的细胞内生态位，它可能是有可能设计更多的 有效的治疗方法和疫苗，可用于治疗受损的 患者和高风险人群发生疾病。 荚膜组织胞浆菌通过肺部侵入人类宿主。的 因此，在肺泡巨噬细胞内存活的能力是第一个 生物体必须克服的障碍才能引起感染， 进行治疗干预。巨噬细胞是高度吞噬细胞， 通过产生活性氧杀死它们摄入的微生物， 氮代谢产物，杀菌肽的合成， 溶酶体酸水解酶。细胞内病原体已经发展出多种多样的 在巨噬细胞内生存的策略，近年来， 发现了一些用来避免巨噬细胞死亡的技巧。 然而，H. capsulatum实现了 胞内存活的描述不太清楚。它既不能逃脱 吞噬体也不抑制吞噬体-溶酶体融合;相反，这些生物体 在吞噬溶酶体内存活，并似乎改变了吞噬小体内的 通过调节这个细胞器内的pH值来调节环境。我们不 我们不了解这种情况发生的机制，也不知道是否 介导这些酵母内化的吞噬事件是 相当于已经建立的主要使用 调理红细胞 在我们的应用程序中有四个目标。我们计划：（1）分离和鉴定 基因的开启和表达的产物， 附着于巨噬细胞并在巨噬细胞内存活的过程;（2）克隆和 测序扩增的mRNA，并进行比较分析， 它们编码的肽序列并选择那些具有低同源性的肽序列 频率与人类序列;（3）克隆和表征所选cDNA 来自2个cDNA文库的序列，所述cDNA文库由在附着期间纯化的mRNA制备 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;以及（4） 定位克隆基因在H.荚膜酵母 人工染色体（YAC）文库。