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

荚膜组织胞浆菌是一种热二型真菌，是一种致病的胞内致病菌。 巨噬细胞。HC在土壤中以多细胞菌丝形式生长。一旦吸入，HC对哺乳动物身体产生反应 温度通过转化为单细胞酵母形式并启动毒力基因的表达 巨噬细胞定植。我们有丰富的经验来阐明基因网络 在酵母细胞中被转录诱导。在我们发表的工作中，我们对酵母阶段的转录组进行了注释 细胞，并发现了一个小的(≤200aA)家族预测的分泌蛋白，显示出保守的C- 末端，6-半胱氨酸间隔图案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，这表明 它们在感染过程中扮演着重要的角色。进一步的分析发现，在这个家族中有26个HC ORF，每个 含有预测的半胱氨酸结(或结蛋白)结构域。相比之下，大多数真菌物种含有预测的0-2个 它们基因组中的结蛋白。打结蛋白结构域由3个相互交织的二硫键组成，形成一个 已知的最小的稳定的球状结构域，使这些蛋白质对化学、热和 蛋白水解性压力。我们的初步数据显示，突变株缺乏个体的显著结果 在HC感染的小鼠模型中，结蛋白显示出毒力降低。所有这些突变体都有部分缺陷。 在刺激宿主巨噬细胞的裂解时，一些但不是所有的巨噬细胞内的生长都减弱了， 这表明结蛋白在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分子遗传学研究单个和多个打结蛋白在发病中的作用 HC发病机制。我们提出了以下目标：第一，利用我们已经产生的突变株， 利用我们已经采用的CRISPR技术来高效地产生更多的突变菌株，我们 将进一步研究单个和多个结蛋白在巨噬细胞HC发病机制中的作用 以及感染的小鼠模型。第二，我们发表的工作证实了HC激活了受感染的细胞的凋亡 巨噬细胞通过在这些细胞中触发综合应激反应(ISR)。我们会将转录后的 巨噬细胞对野生型与突变型结蛋白感染的应答特征 ISR和宿主分子对HC反应的其他方面的单个结。此外，由于子集 结蛋白突变体显示巨噬细胞内生长减少，我们将确定结蛋白是否影响 HC阻止吞噬小体成熟的能力是细胞内生存的关键步骤。最后，为了阐明 打结蛋白作用的分子机制，我们将使用我们实验室的标准管道来确定 巨噬细胞感染HC过程中部分结蛋白的亚细胞定位和蛋白相互作用。这些 该方法将首次探索结蛋白在哺乳动物真菌发病中的作用，并将 对结蛋白在丙型肝炎发病机制中的作用给予关键的洞察。

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.