DIMORPHISM AND VIRULENCE IN HISTOPLASMA CAPSULATUM

荚膜组织胞浆菌是一种二形性真菌，可引起人类疾病， 临床表现多种多样。 本次活动的长远目标 应用程序是为了开发一种方法来改善结果 基于我们的生化和分子技术的组织胞浆菌病 早期证明剧毒感染需要相变 宿主体内温度为 37°C。 我们想要阐明基因是如何编码的 应激蛋白和过渡特异性 (ts) 蛋白有助于 形态转变和不同程度的致病性 从患者和土壤中分离出来。 这些研究将具有价值 开发干扰感染建立的药物。 具体目标有以下三个： 1. 研究参与相变早期阶段的基因： a.热适应 - 热休克基因 (hsg)：克隆并表征 热休克 83 基因并确定其表达和调控的方式 在不同温度下的相变过程中的成熟 分离株与耐热性和致病性有关。 确定 hs蛋白的诱导在不同的生理作用中发挥作用 耐热程度、形态发生、线粒体膜完整性 和毒力。 b.相变 - 克隆并表征 ts 基因的表达 菌丝体向酵母分化过程的前 6 小时 确定它们在菌株中的表达模式，包括 过渡缺陷 PCMS 菌株，表现出不同水平的 耐热性和致病性。 在这个组中我们期望找到 在其调控区域中含有热休克元件的基因。 2. 确定参与该阶段的基因破坏的影响 转变和毒力通过： a.建立转换协议 - 设计一个程序 产生一个有效的转化系统，以分析其作用 体外突变基因在形态发生和致病性中发挥作用。 b.体外诱变以确定其对相变和 适应 - 分析突变对生化事件的影响 温度变化后发生的情况。 3. 通过以下方式确定 hs 和 ts 基因在毒力中的作用： a.评估小鼠体内的体外突变菌株。

Histoplasma capsulatum, a dimorphic fungus, causes disease in humans that has a wide range of clinical manifestations. The long-term goal of this application is to develop an approach towards improving the outcome of histoplasmosis using biochemical and molecular techniques based on our earlier demonstration that virulent infection requires a phase transition at 37 degrees C in the host. We want to elucidate how genes coding for stress proteins and transition-specific (ts) proteins contribute to the morphologic transition and to the different degrees of pathogenicity in isolates from patients and soil. These studies will be of value in development of drugs that interfere with establishment of infection. There are three specific aims: 1. STudy genes involved in the early stages of the phase transition: a. Thermal adaptation - heat shock genes (hsg): clone and characterize the heat shock 83 gene and determine how regulation of its expression and maturation during phase transition at various temperatures in different isolates is related to thermotolerance and pathogenicity. Determine the physiological role the induction of hs proteins play in the different degrees of thermotolerance, morphogenesis, mitochondrial membrane integrity and virulence. b. Phase transition - clone and characterize ts gene that are expressed in the first 6 hrs of the mycelium to yeast differentiation process and determine the pattern of their expression in strains, including the transitional-defective PCMS strain, that show different levels of thermotolerance and pathogenicity. Within this group we expect to find genes that contain heat shock elements in their regulatory regions. 2. Determine the effect of disruption of genes involved in the phase transition and in virulence by: a. Establishing a transformation protocol - design a procedure that will yield an efficient transformation system in order to analyze the role in vitro-mutated genes play in morphogenesis and pathogenicity. b. In vitro mutagenesis to determine its effect on phase transition and adaption - analyze the effect mutagenesis has on the biochemical events that take place after the temperature shift. 3. Determine the role that hs and ts genes play in virulence by: a. Evaluating the in vitro-mutated strains in mice.