Endocytosis in Candida filamentation, biofilm formation and virulence

真菌白色念珠菌是医院获得性血液感染的第四大常见原因 (BSI)，是静脉导管相关性感染、尿路感染、皮肤和粘膜的主要原因 在我们的老病人群体中，感染和侵袭性疾病。尽管医疗保健取得了进步，但 侵袭性念珠菌感染导致的高死亡率并不比20年前好。因此，我们有能力 预防、诊断和治疗侵袭性念珠菌感染仍然需要极大的改进。虽然C. 白念珠菌是一种正常的人类殖民者，它具有通过各种特殊属性致病的能力。 这些与毒力相关的因子包括分泌有助于组织入侵的降解酶， 在称为成丝的过程中形成细长的菌丝结构，并建立复合体 被称为生物膜的结构，它保护这种真菌免受抗微生物药物和宿主免疫防御的影响。 在以前的研究中，我们研究了空泡前分泌途径在分泌 受空泡蛋白分泌调节的白念珠菌毒力相关蛋白和生物膜的形成 基因VPS1、Vps4和PEP12。我们证明了这种空泡前期分泌途径有助于 分泌一种关键的降解酶，分泌天冬氨酸蛋白酶，并参与丝状化的各个方面， 生物膜的形成和致病力。例如，我们发现白色念珠菌Pep12零突变体形成了一个 生物膜在极小的干扰下戏剧性地碎裂，并且在体内的毒性方面存在缺陷。 在这些研究的基础上，我们接下来通过检查关键的最后步骤来研究分泌的后期 胞吐作用受胞囊蛋白复合体调节。在这项工作中，我们证明了与外囊相关的 SNARE蛋白Sso2p和Sec9p对于白色念珠菌的生存是必不可少的，并且是 天冬氨酸蛋白酶和脂肪酶的分泌，以及菌丝的形成。相比之下，几个主要组件 胞囊复合体，包括Exo70p和Exo84p，以及调节蛋白Sro77p似乎不是 分流所必需的。 接下来，我们开始研究分泌途径的另一个重要组成部分，即内吞作用。 和内吞途径。这种高度调控的顺序途径参与了细胞外的摄取。 质膜蛋白和分泌途径的其他成分的物质和循环。我们的 主要目的是确定内吞作用在分泌、丝状化、生物膜形成和 致命性。此外，我们将从机械学的角度研究内吞作用对丝状化的具体贡献。 立场。因此，该项目将研究以下关键假设：(I)关键步骤中的特定基因 内吞作用是丝状化和生物膜形成所必需的，(Ii)内吞作用途径基因在 关键步骤将导致在体外和体内的毒力减弱，以及(Iii)白色念珠菌具有新的基因和 参与内吞作用的途径。 我们将首先研究白念珠菌END3、SYP1、PAL1、ENT1和YAP1801的特定功能，它们是 预计将参与内吞作用的关键早期步骤。我们还将研究RHO1，以确定是否存在 白念珠菌中存在的内吞作用的新途径，如酵母菌中所描述的那样。接下来，我们将学习一组 在缺少人类或酵母菌对应的八个新基因中，涉及内吞或分泌， 我们使用计算/比较基因组学方法进行了鉴定。然后我们将寻求识别小说 通过筛选几个白念珠菌突变文库收集参与内吞作用的基因。所有这些研究 将使用遗传、生化、分子和细胞生物学方法来完成。 预期结果：我们希望确定与癌症关键阶段相关的特定基因的功能作用 白念珠菌中的内吞作用，并定义它们与丝状化和毒力的联系，以便了解 发病机制的分子机制和寻找新的药物靶点进行进一步干预。

The fungus Candida albicans is the 4th most common cause of hospital-acquired bloodstream infections (BSI), and is a major cause of intravenous catheter-associated infections, urinary infections, skin and mucosal infections, and invasive disease in our veteran patient population. Despite advancements in medical care, the high mortality rate due to invasive Candida infections is no better than two decades ago. Thus, our ability to prevent, diagnose, and treat invasive Candida infections is still in need of great improvement. Although C. albicans is a normal human colonizer, it has the ability to cause disease through various specialized attributes. These virulence-associated factors include secretion of degradative enzymes that assist in tissue invasion, formation of elongated hyphal structures in a process termed filamentation, and establishment of complex structures called biofilms, which protect this fungus from antimicrobial drugs and host immune defenses. In previous studies, we examined the role of the pre-vacuolar secretory pathway in the secretion of virulence-associated proteins and biofilm formation in C. albicans, regulated by the vacuolar protein secretion genes VPS1, VPS4, and PEP12. We demonstrated that this pre-vacuolar secretory pathway contributes to secretion of a key degradative enzyme, secreted aspartyl protease, and is involved in aspects of filamentation, biofilm formation, and virulence. For example, we discovered that the C. albicans pep12 null mutant formed a biofilm that dramatically fragmented with minimal disturbance, and was defective in virulence in vivo. Expanding upon these studies, we next studied the late stages of secretion by examining key final steps in exocytosis regulated by the exocyst protein complex. In this work, we demonstrated that the exocyst-related SNARE proteins Sso2p and Sec9p were essential for viability in C. albicans, and were required for the secretion of aspartyl proteases and lipases, and hyphal formation. In contrast, several major components of the exocyst complex, including Exo70p and Exo84p, and the regulatory protein Sro77p did not appear to be required for filamentation. Next, we have begun studies of another essential component of the secretory pathway, that is, endocytosis and the endocytic pathway. This highly regulated, sequential pathway is involved in intake of extracellular materials and recycling of plasma membrane proteins and other components of the secretory pathway. Our main objective is to determine the role of endocytosis in secretion, filamentation, biofilm formation, and virulence. Further, we will study the specific contributions of endocytosis to filamentation from a mechanistic standpoint. This project will therefore examine the key hypotheses that: (i) specific genes in key steps of endocytosis are required for filamentation and biofilm formation, (ii) mutations in endocytosis pathway genes at key steps will result in attenuated virulence in vitro and in vivo, and (iii) C. albicans has novel genes and pathways that are involved in endocytosis. We will first study the specific functions of C. albicans END3, SYP1, PAL1, ENT1, and YAP1801, which are predicted to be involved in key early steps in endocytosis. We will also study RHO1 to determine if there is a novel route of endocytosis that exists in C. albicans, as described in Saccharomyces. Next, we will study a set of eight novel genes lacking human or Saccharomyces counterparts, involved in endocytosis or secretion, which we identified using a computational/comparative genomics approach. Then we will seek to identify novel genes involved in endocytosis by screening several C. albicans mutant library collections. All of these studies will be accomplished using genetic, biochemical, molecular and cell biology approaches. Expected Results: We expect to determine the functional role of specific genes related to key stages of endocytosis in in C. albicans, and define their link to filamentation and virulence in order to understand molecular mechanisms of pathogenesis and identify novel drug targets for further intervention.