MNT OF HYPHAL POLARITY BY DOPA PROTEIN AND ITS ROLE IN ASPERGILLUS PATHOGENESIS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。烟曲霉(Af)是侵袭性曲霉病(IA)的主要原因，侵袭性曲霉病是一种在日益增长的免疫功能低下人群中经常致命的疾病。伊曲康唑和两性霉素B是目前唯一可用的治疗方法，用于治疗侵袭性菌丝生长逃脱宿主天然免疫系统的情况。然而，它们在体内的有效性较低，对IA患者的预后也很差。寻找(Af)毒力因子一直存在问题，因为明显候选基因的突变不会降低毒力。有趣的是，大多数影响致病的基因减缓了菌丝伸长的速度，也许给了宿主更多的时间来进行防御。AF作为菌丝体生长，通过极化的菌丝生长延伸到整个培养基中。这种生长模式需要不断地将新材料移动到菌丝生长的顶端。我们的实验室一直致力于阐明多巴的功能，它是从真菌到哺乳动物的蛋白质家族的创始成员，在进化上是保守的。我们实验室的早期工作表明，多巴是一种参与蛋白质运输的必要基因。特定目标1将检验这样一种假设，即建立和维持高度极化的菌丝生长的能力是免疫低下患者避免天然免疫的关键机制，也是丝状真菌病原体毒力的主要贡献因素。影响真菌生长的突变将在动物细胞系中进行研究。多巴突变体的毒力将在小鼠侵袭性肺曲霉菌病模型中进行评估。特定目标2将测试这样的假设，即Af多巴(多巴)和多巴蛋白代表细胞蛋白质运输的一种新的和必要的成分，这是极化细胞生长对环境信号的反应所必需的。多巴蛋白将通过GFP融合在活细胞中定位。多巴在定位其他已知的极性决定因素中的作用将被研究。多巴相互作用蛋白将通过多种方法鉴定，以阐明极地生长的机制。这种方法将有助于理解信号与菌丝生长相关的分子机制。具体目标3将检验这样的假设，即Af有一个隐蔽的性周期，可以被操纵来发展减数分裂遗传学作为Af的工具。性遗传学的加入对研究烟曲霉的研究人员来说将是无价的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Aspergillus fumigatus (Af) is the major cause of invasive aspergillosis (IA), an often-fatal disease in the growing population of immunocompromised individuals. Itraconazole and amphotericin B are the only currently available treatments for those cases where invasive hyphal growth escapes the host's innate immune system. However, their effectiveness in vivo is low and the prognosis for individuals with IA is poor. The search for (Af) virulence factors has been problematic because mutations in the obvious candidates do not reduce virulence. Interestingly, the majority of genes that affect pathogenesis slow the rate of hyphal extension, perhaps giving the host more time to mount a defense. Af grows as a mycelium that extends through medium via polarized hyphal growth. This mode of growth requires that new materials be constantly moved to the growing tip of the hypha. Our laboratory has been involved in elucidating the function of DopA, the founding member of a protein family that is evolutionarily conserved from fungi to mammals. Earlier work from our lab indicates that DopA is an essential gene that is involved in protein trafficking. Specific Aim 1 will test the hypothesis that the ability to establish and maintain highly polarized hyphal growth is a key mechanism for avoidance of innate immunity in immunocompromised patients, and is a major contributing factor in virulence of filamentous fungal pathogens. Mutations that affect fungal growth will be studied in animal cell lines. The virulence of dopA mutants will be assessed in the mouse invasive pulmonary aspergillosis model. Specific Aim 2 will test the hypothesis that Af DopA (DopeyA) and An DopA proteins represent a novel and essential component of cellular protein trafficking required for polarized cell growth in response to environmental signaling. DopA protein will be localized in living cells by using GFP fusions. The role of DopA in the localization of other known polarity determinants will be investigated. DopA interacting proteins will be identified by a number of methods to elucidate the mechanisms of polar growth. This approach will contribute to an understanding of the molecular mechanisms that link signaling to hyphal growth. Specific Aim 3 will test the hypothesis that Af has a cryptic sexual cycle that can be manipulated to develop meiotic genetics as a tool for Af. The addition of sexual genetics will be invaluable to investigators studying Aspergillus fumigatus.