HISTOPLASMA CAPSULATUM AND THE MACROPHAGE

荚膜组织胞浆菌和巨噬细胞

• 批准号: 3139062
• 负责人: WILLIAM E GOLDMAN
• 金额: $ 12.21万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3139062
• 关键词: Histoplasma; acidity /alkalinity; alveolar macrophages; antifungal agents; calcium binding protein; calcium metabolism; cell population study; electron microscopy; fluorescence spectrometry; fluorescent dye /probe; genetic strain; histoplasmosis; host organism interaction; intracellular parasitism; laboratory mouse; laboratory rabbit; laboratory rat; lysosomes; macrophage; microorganism culture; microorganism growth; peptides; phagocytosis; radiotracer; superoxides; tissue /cell culture; vesicle /vacuole; virulence

Histoplasma capsulatum is a dimorphic fungus which can cause a respiratory or disseminated disease in humans. The work proposed here is directed at identifying characteristics which enable this organism to be a " successful" pathogen. Requirements for intracellular survival will be evaluated by a systematic examination of a variety of strains, including matched virulent/avirulent strain pairs. In contrast to other laboratories which are investigating how macrophages respond when confronted with H. capsulatum, this proposal focuses on how H. capsulatum has adapted to survive in the potentially hostile environment within macrophages. The first issue addressed is whether virulent H. capsulatum yeasts are better able to survive within macrophages than avirulent yeasts. Alternatively, avirulent yeasts may survive but fail to multiply or perhaps remain metabolically inactive. These possibilities will be examined through a variety of assays using radiolabel incorporation, vital staining, or colony formation as indicators. Later studies explore the mechanisms by which virulent yeasts survive and proliferate in macrophages. In P388D1 cells, phagosomes containing this organism appear to fuse with lysosomes. This will be confirmed by electron microscopy and evaluated in resident peritoneal and alveolar macrophages as well. Since phagosome-lysosome fusion occurs, H. capsulatum must either resist or inactivate the fungicidal mechanisms found in lysosomes. The sensitivity of this organism to oxidative killing mechanisms will be assessed by exposure to exogeneously generated products of the oxidative burst. Likewise, vulnerability to non-oxidative killing mechanisms will be studied in rat macrophages, which are exceptionally adept in these activities. Effects of the fungicidal lysosomal peptides MCP-1 and MCP-2 on this yeast will also be examined. One means of inactivating intracellular fungicidal mechanisms would be to alter the phagolysosomal environment. While phagosomes normally acidify, preliminary experiments using ph- sensitive fluorescein isothiocyanate indicate that those containing virulent yeasts do not. These studies will be confirmed using other ph-sensitive strains as well as probes which become fluorescent only after cleavage by ph-sensitive lysosomal enzymes. Recent reports have suggested that phagolysosomes contain very little calcium; therefore, fura-2 will be used to determine the concentration of calcium in H. capsulatum phagolysosomes. Additional experiments will address the possibility that this yeast has adapted to environments low in calcium by releasing calcium-binding proteins.

荚膜组织胞浆菌是一种二型真菌， 人类的呼吸道或传播性疾病。 工作提出的 这里针对的是识别能够实现这一点的特征， 一个“成功的”病原体。 要求 细胞内存活将通过系统的 检查各种菌株，包括匹配的 强毒株/无毒毒株对。 与其他实验室相比 他们正在研究巨噬细胞在 面对H。capsulatum，这一建议的重点是如何H。 capsulatum已经适应了在潜在的敌对环境中生存 巨噬细胞内的环境。 第一个问题是是否有毒的H。荚膜酵母 在巨噬细胞内存活的能力比无毒的 酵母 或者，无毒酵母菌可以存活，但不能 繁殖或保持代谢不活跃。 这些 可能性将通过各种试验进行检查， 放射性标记掺入、活体染色或菌落形成， 指标 后来的研究探索了致病酵母 在巨噬细胞中存活和增殖。 在P388 D1细胞中， 含有这种生物的吞噬体似乎与 溶酶体 这将通过电子显微镜证实， 在常驻腹膜和肺泡巨噬细胞中也进行了评价。 由于吞噬体-溶酶体发生融合，H. capsulatum必须 抵抗或灭活中发现的杀真菌机制 溶酶体 这种生物对氧化杀伤的敏感性 将通过暴露于外源性 产生的氧化产物。 同样，脆弱性 非氧化性杀伤机制将在大鼠中进行研究 巨噬细胞，这是特别擅长这些活动。 杀真菌溶酶体肽MCP-1和MCP-2对人表皮细胞的影响 还将检查该酵母。 使细胞内杀真菌机制失活的一种方法 就是改变吞噬溶酶体的环境 而 吞噬体通常酸化，初步实验使用pH- 敏感的异硫氰酸荧光素表明， 但致命的酵母菌则不然。 这些研究将使用 其它pH敏感菌株以及探针 仅在被pH敏感性溶酶体裂解后才荧光 内切酶 最近的报道表明，吞噬溶酶体 含有很少的钙;因此，Fura-2将用于 测定H中钙的浓度。浆菌 吞噬溶酶体 其他实验将解决 这种酵母菌适应环境的可能性低， 通过释放钙结合蛋白来释放钙。