Role of secreted cystine-knot proteins in Histoplasma-host interactions

分泌型胱氨酸结蛋白在组织胞浆菌-​​宿主相互作用中的作用

• 批准号: 10681823
• 负责人: Anita Sil
• 金额: $ 58.47万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681823
• 关键词: Affect; Affinity Chromatography; Apoptosis; Apoptotic; Binding; Biological; Biology; Body Temperature; C-terminal; CRISPR/Cas technology; Cell Death; Cells; Chemicals; Cysteine; Cystine; Cytolysis; Data; Defect; Disease; Elements; Elongation Factor; Exhibits; Family; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Transcription; Genome; Goals; Growth; Histoplasma; Histoplasma capsulatum; Immune; Immune response; Individual; Infection; Inhalation; Insecta; Knock-out; Knockout Mice; Label; Laboratories; Macrophage; Mammals; Mass Spectrum Analysis; Microbe; Molds; Molecular; Molecular Genetics; Morbidity - disease rate; Mus; Mutation; Open Reading Frames; Organism; Pathogenesis; Pathway interactions; Pattern; Phagosomes; Phase; Phosphorylation; Play; Predisposition; Proliferating; Protein Family; Protein Secretion; Proteins; Publishing; Research; Resistance; Role; Scheme; Sequence Homology; Signal Transduction; Soil; Source; Stress; Time; Toxin; Transcend; Transcript; Translations; Virulence; Virulence Factors; Work; Yeasts; biological adaptation to stress; cytotoxicity; density; differential expression; disulfide bond; experience; experimental study; falls; fascinate; fungus; gene network; genetic approach; human pathogen; improved; insight; mortality; mouse model; mutant; pathogen; response; reverse genetics; ribosome profiling; tool; transcription factor CHOP; transcriptome; transcriptome sequencing; virulence gene

Histoplasma capsulatum (Hc) is a thermally dimorphic fungus and an intracellular pathogen of macrophages. Hc grows in the soil in a multicellular hyphal form. Once inhaled, Hc responds to mammalian body temperature by converting to a unicellular yeast form and initiating the expression of virulence genes important for macrophage colonization. We have extensive experience elucidating the gene networks that are transcriptionally induced in yeast cells. In our published work, we annotated the transcriptome of yeast-phase cells and discovered a family of small (≤ 200 AAs) predicted secreted proteins that exhibit a conserved C- terminal, 6-cysteine spacing pattern reminiscent of some insect toxins. The transcripts encoding these proteins showed highly differential expression in yeast cells compared to the remainder of the transcriptome, suggesting that they play an important role during infection. Further analysis revealed 26 Hc ORFs in this family, each containing a predicted cystine knot (or knottin) domain. In contrast, most fungal species contain 0-2 predicted knottin proteins in their genomes. Knottin domains are comprised of 3 interwoven disulfide bonds that form one of the smallest known stable globular domains, making these proteins extremely resistant to chemical, heat, and proteolytic stresses. Our preliminary data reveal the remarkable result that mutant strains lacking individual knottins show reduced virulence in the mouse model of Hc infection. All of these mutants are partially deficient in stimulating lysis of host macrophages, and some but not all display diminished growth within macrophages, indicating that knottins play key roles in Hc-host interactions. We will take advantage of our expertise in Hc- macrophage interactions and Hc molecular genetics to interrogate the role of individual and multiple knottins in Hc pathogenesis. We propose the following aims: First, using the mutant strains we have already generated, and taking advantage of CRISPR technology we have adapted to efficiently generate more mutant strains, we will further investigate the contribution of individual and multiple knottins to pathogenesis of Hc in macrophage and mouse models of infection. Second, our published work established that Hc activates apoptosis of infected macrophages by triggering an integrated stress response (ISR) in these cells. We will compare the transcriptional signature of macrophages to infection with wild-type vs mutant knottin strains to elucidate the contribution of individual knottins to the ISR and other aspects of the host molecular response to Hc. Additionally, since a subset of knottin mutants display reduced growth within macrophages, we will determine whether knottins affect the ability of Hc to block phagosome maturation, which is a key step in intracellular survival. Finally, to elucidate the molecular mechanism of knottin function, we will use standard pipelines in our laboratory to determine the subcellular localization and protein interactome of selected knottins during macrophage infection with Hc. These approaches will provide the first exploration of the role of knottins in fungal pathogenesis of mammals, and will give critical insight into the contribution of knottins to Hc pathogenesis.

组织囊肿囊肿（HC）是一种热二态真菌，是一种细胞内病原体 巨噬细胞。 HC以多细胞菌丝形式在土壤中生长。一旦继承，HC对哺乳动物的身体做出反应 温度通过转化为单细胞酵母形式并启动病毒基因的表达 用于巨噬细胞定植。我们有丰富的经验，阐明了基因网络 在酵母细胞中引起的转录诱导。在我们发表的工作中，我们注释了酵母相的转录组 细胞并发现了一个小（≤200aas）的家族预测的分泌蛋白，该蛋白暴露了构成的c- 末端，6-半胱氨酸间距模式提醒一些绝缘毒素。编码这些蛋白质的成绩单 与转录组的其余部分相比，在酵母细胞中显示高度差异表达，这表明 他们在感染过程中起着重要作用。进一步的分析揭示了这个家庭中的26个HC ORF 包含一个预测的胱氨酸结（或基因）结构域。相反，大多数真菌物种都包含0-2个预测 其基因组中的蛋白质。 Knottin域包括3个编织二硫键，形成一个 这些蛋白质对化学，热和 蛋白水解应力。我们的初步数据揭示了一个显着的结果，即突变菌株缺乏个体 Knottins在HC感染的小鼠模型中表现出降低的病毒。所有这些突变体都部分不足 在刺激宿主巨噬细胞的裂解中，有些但并非都显示出巨噬细胞的生长减少， 表明诺丁在HC-host互动中扮演关键角色。我们将利用我们在HC-的专业知识 巨噬细胞相互作用和HC分子遗传学询问个体和多个基因斯在 HC发病机理。我们提出以下目的：首先，使用我们已经产生的突变株， 利用CRISPR技术，我们已经适应有效产生更多的突变株，我们 将进一步研究个体和多个基因斯对巨噬细胞中HC发病机理的贡献 和小鼠感染模型。其次，我们发表的工作确定HC激活了感染的凋亡 巨噬细胞通过触发这些细胞中的综合应力反应（ISR）。我们将比较转录 用野生型与突变体菌株感染巨噬细胞的签名，以阐明 单个对ISR和宿主分子对HC的反应的障碍。此外，由于子集 Knottin突变体显示巨噬细胞内的生长降低，我们将确定Knottins是否影响 HC阻断吞噬体成熟的能力，这是细胞内存活的关键步骤。最后，阐明 Knottin功能的分子机制，我们将在实验室中使用标准管道来确定 巨噬细胞感染期间，巨噬细胞感染期间，亚细胞定位和蛋白质相互作用。这些 方法将对knottins在哺乳动物的真菌发病机理中的作用进行第一次探索，并将 对Knottins对HC发病机理的贡献进行批判性洞察力。