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.

荚膜组织胞浆菌 (Hc) 是一种热二态性真菌，也是一种细胞内病原体。 Hc 以多细胞菌丝形式生长在土壤中，一旦被吸入，Hc 就会对哺乳动物的身体产生反应。 通过转化为单细胞酵母形式并启动毒力重要基因的表达来调节温度 我们在阐明巨噬细胞定植的基因网络方面拥有丰富的经验。 在我们发表的工作中，我们注释了酵母阶段的转录组。 细胞并发现了一个小型（≤ 200 个 AA）预测分泌蛋白家族，这些蛋白表现出保守的 C- 末端，6-半胱氨酸间隔模式让人想起一些昆虫毒素编码这些蛋白质的转录本。 与转录组的其余部分相比，在酵母细胞中显示出高度差异的表达，表明 进一步分析表明，该家族中有 26 个 Hc ORF，每个都在感染过程中发挥重要作用。 包含预测的胱氨酸结（或结蛋白）结构域，相比之下，大多数真菌物种包含 0-2 个预测的结构域。 Knottin 蛋白在其基因组中由 3 个相互交织的二硫键组成。 已知最小的稳定球状结构域，使这些蛋白质对化学、热和 我们的初步数据揭示了突变菌株缺乏个体的显着结果。 结蛋白在 Hc 感染的小鼠模型中显示出毒力降低。所有这些突变体都部分缺陷。 刺激宿主巨噬细胞的裂解，并且一些但不是全部显示巨噬细胞内生长减弱， 表明结蛋白在 Hc-宿主相互作用中发挥关键作用，我们将利用我们在 Hc- 方面的专业知识。 巨噬细胞相互作用和 Hc 分子遗传学探讨单个和多个结蛋白在 Hc发病机制我们提出以下目标：首先，使用我们已经产生的突变株， 并利用我们已经适应的 CRISPR 技术来有效地产生更多突变菌株，我们 将进一步研究单个和多个结蛋白对巨噬细胞中Hc发病机制的贡献 其次，我们发表的研究表明，Hc 可以激活感染细胞的细胞凋亡。 巨噬细胞通过在这些细胞中触发整合应激反应（ISR），我们将比较转录。 巨噬细胞对野生型与突变型结蛋白菌株感染的特征，以阐明 单个结蛋白对 ISR 和宿主分子对 Hc 反应的其他方面的影响。 的节蛋白突变体显示巨噬细胞内生长减少，我们将确定节蛋白是否影响 Hc 阻断吞噬体成熟的能力，这是细胞内存活的关键步骤。 结蛋白功能的分子机制，我们将使用我们实验室的标准管道来确定 Hc 巨噬细胞感染期间选定结蛋白的亚细胞定位和蛋白质相互作用组。 这些方法将首次探索结蛋白在哺乳动物真菌发病机制中的作用，并将 深入了解结蛋白对 Hc 发病机制的贡献。