Unlocking the cidal activity of echinocandins against Aspergillus fumigatus

侵袭性曲霉病(IA)是由烟曲霉菌(A.fumigatus)引起的最常见的侵袭性霉菌感染。 免疫功能受损的个体，与35-90%的死亡率有关。只有三个级别的反- 曲霉菌药物是存在的。全球对三唑类和高得令人无法接受的患者的耐药性上升 多烯类的毒性限制了其中两种的使用。剩下的一类，棘球菌素，通常是 被认为是有效的治疗药物的目标是真菌专一性的(细胞壁生物合成)，他们展示了 低毒。然而，棘球菌素对曲霉菌并没有杀菌作用，而且它的一个矛盾的效果是 治疗的特点是随着药物浓度的增加，药物有效性降低， 描述了体内和体外的情况。可能由于这些问题，高发突破性感染在 棘球菌素治疗已有报道。因此，棘球菌素在IA中的使用也是有限的。发现了 棘球绦虫应激适应和生存的关键机制有望改善治疗 通过确定未来联合治疗的真菌靶点，这些重要化合物的疗效。至 描述了编排棘球绦虫应激反应的新机制，我们最近完成了 野生型遗传背景下蛋白激酶干扰突变文库的构建。这个图书馆曾经是 使用CRISPR/Cas9基因编辑构建，以扰乱由编码的142个可能的蛋白激酶 烟曲霉菌基因组。总共获得了118个非必要的、独特的基因中断，随后 用于体外棘球绦虫药敏试验。我们的初步分析共鉴定了12种蛋白质 显示最低有效浓度(MEC)降低4倍至32倍的激酶干扰突变体 与亲本菌株相比。我们已经发现了其中两个突变，它们位于之前的 未知的SEPL和SidB激酶，赋予多种棘球菌素抗真菌的抗曲霉菌活性。 这些蛋白激酶被预测为间隔起始网络(SIN)的核心组件，SIN是一种三种不同类型的蛋白。 在真菌中进行分离所必需的激酶级联反应。我们令人兴奋的初步数据显示，阻止分隔 任何单一SIN激酶的丢失都会导致广泛的菌丝损伤和活力丧失 棘球菌素治疗。此外，利用SEPL中断突变体，我们发现棘球绦虫 治疗提高了IA小鼠模型的存活率并消除了残留的组织负担。因为隔膜很重要 对于菌丝的机械伤害是有限的，而烟曲霉菌完全没有特征， 对这一网络的探索有望揭示棘球绦虫应激存活的新效应。我们的目标是 确定解锁棘球菌素杀灭活性所需的核心SIN组分(目标1)，定义时间 间隔阻断引起的增强棘球绦虫活性的要求(目标2)，以及 描述依赖SIN通路的隔膜结构机械(目标3)。我们的工作将辨别罪孽 当靶向与棘球绦虫联合治疗时，具有最高潜在益处的成分。

Invasive aspergillosis (IA), caused mainly by A. fumigatus, is the most prevalent invasive mold infection of immunocompromised individuals and is associated with mortality rates of 35-90%. Only three classes of anti- Aspergillus drugs exist. The global rise of resistance to the triazole class and the unacceptably high patient toxicity of the polyene class limits the use of two of these. The remaining class, the echinocandins, are generally considered effective therapeutic agents as their target is fungus-specific (cell wall biosynthesis) and they exhibit low toxicity. However, the echinocandins are not fungicidal for Aspergillus species and a paradoxical effect of treatment, characterized by decreased drug effectiveness with increasing drug concentrations, has been described both in vivo and in vitro. Likely due to these issues, high incidence breakthrough infections during echinocandin therapy have been reported. Therefore, echinocandin use for IA is also limited. The discovery of mechanisms essential for echinocandin stress adaptation and survival is expected to improve therapeutic efficacy with these important compounds by identifying fungal targets for future combination therapies. To delineate novel mechanisms orchestrating echinocandin stress responses, we recently completed the generation of a protein kinase disruption mutant library in a wild type genetic background. This library was constructed using CRISPR/Cas9 gene-editing to disrupt each of the 142 putative protein kinases encoded by the A. fumigatus genome. A total of 118 non-essential, unique gene disruptions were achieved and subsequently employed for in vitro echinocandin susceptibility assays. Our preliminary assays identified a total of 12 protein kinase disruption mutants displaying 4- to >32-fold decreased minimum effective concentrations (MEC) compared to the parental strain. We have discovered that two of these mutations, residing in the previously uncharacterized SepL and SidB kinases, impart fungicidal anti-Aspergillus activity to multiple echinocandins. These protein kinases are predicted to be core components of the Septation Initiation Network (SIN), a three- kinase cascade that is necessary for septation in fungi. Our exciting preliminary data show that blocking septation via loss of any single SIN kinase causes widespread hyphal damage and loss of viability in response to echinocandin treatment. In addition, employing the sepL disruption mutant, we have found that echinocandin therapy improves survival and eliminates residual tissue burden in a mouse model of IA. As septa are important for the limitation of mechanical injury to hyphae, and the A. fumigatus SIN is completely uncharacterized, exploration of this network is expected to reveal novel effectors of echinocandin stress survival. Our aims are to identify core SIN components required for unlocking echinocandin cidal activity (Aim 1), define temporal requirements for enhancement of echinocandin activity resulting from septation blockade (Aim 2), and to delineate the SIN pathway-dependent septum construction machinery (Aim 3). Our work will identify the SIN components with the highest potential for benefit when targeted in combination with echinocandin therapy.