Endocytosis in Candida filamentation, biofilm formation and virulence

念珠菌丝状形成、生物膜形成和毒力中的内吞作用

基本信息

批准号：
10266031
负责人：
SAMUEL AUSTIN LEE
金额：
--
依托单位：
WHITE RIVER JUNCTION VA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10266031
关键词：
Antifungal Agents Antifungal Therapy Aspartic Endopeptidases Attenuated Biochemical Biology Caenorhabditis elegans Candida Candida albicans Candidiasis Caring Catheters Cell membrane Cellular biology Collection Complex Critical Pathways Data Defect Diagnosis Disease Disseminated candidiasis Endocytosis Endocytosis Pathway Enzymes Exocytosis Filament Fungemia Future Genes Genetic Genomic approach Growth Health Care Costs Hospitals Human Immune In Vitro Infection Intake Intervention Intravenous Investigation Length of Stay Libraries Link Lipase Mediating Mediator of activation protein Medical Membrane Proteins Methods Microbial Biofilms Modeling Molecular Molecular Biology Morphogenesis Mucous Membrane Mus Mutation Pathogenesis Pathway interactions Phenotype Play Process Protein Secretion Proteins Recycling Resistance Role Route SNAP receptor Saccharomyces Saccharomyces cerevisiae Secretory Component Sepsis Skin Structure Tail Tissues Urinary tract infection Veins Veterans Virulence Work antimicrobial drug biological adaptation to stress clinically significant comparative genomics extracellular forward genetics fungus genetic approach genetic regulatory protein in vitro Model in vivo in vivo Model lucifer yellow macrophage mortality mutant new therapeutic target novel pathogenic fungus patient population prevent protein complex screening secretion process trafficking

项目摘要

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.

真菌白色念珠菌是医院获得性血流感染的第四大常见原因 (BSI)，是静脉导管相关感染、泌尿道感染、皮肤和粘膜感染的主要原因我们的老患者群体中的感染和侵袭性疾病。尽管医疗保健取得了进步，侵袭性念珠菌感染造成的高死亡率并不比二十年前好。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很大的改进。虽然C. 白色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引起疾病的能力。这些毒力相关因素包括有助于组织侵袭的降解酶的分泌，在称为丝化的过程中形成细长的菌丝结构，并建立复杂的称为生物膜的结构，可以保护这种真菌免受抗菌药物和宿主免疫防御的侵害。在之前的研究中，我们研究了液泡前分泌途径在分泌白色念珠菌中毒力相关蛋白和生物膜形成，受液泡蛋白分泌调节基因 VPS1、VPS4 和 PEP12。我们证明了这种液泡前分泌途径有助于分泌一种关键的降解酶，即分泌型天冬氨酰蛋白酶，并参与丝化的各个方面，生物膜的形成和毒力。例如，我们发现白色念珠菌 pep12 无效突变体形成了生物膜在最小的干扰下就急剧破碎，并且体内毒力有缺陷。扩展这些研究，我们接下来通过检查关键的最后步骤来研究分泌的后期阶段胞吐作用由胞吐蛋白复合物调节。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至关重要，并且是天冬氨酰蛋白酶和脂肪酶的分泌以及菌丝形成。相比之下，几个主要组成部分外囊复合体，包括 Exo70p 和 Exo84p，以及调节蛋白 Sro77p 似乎不存在成丝所需的。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涉及细胞外物质的摄入质膜蛋白和分泌途径其他成分的材料和回收。我们的主要目的是确定内吞作用在分泌、丝状形成、生物膜形成和毒力。此外，我们将从机制上研究内吞作用对丝化的具体贡献立场。因此，该项目将检验以下关键假设：(i) 关键步骤中的特定基因丝状化和生物膜形成需要内吞作用，（ii）内吞作用途径基因的突变关键步骤将导致体外和体内毒力减弱，并且 (iii) 白色念珠菌具有新的基因和参与内吞作用的途径。我们首先研究白色念珠菌END3、SYP1、PAL1、ENT1和YAP1801的具体功能，它们是预计参与内吞作用的关键早期步骤。我们还将研究 RHO1 以确定是否存在白色念珠菌中存在的新的内吞作用途径，如酵母菌中所述。接下来我们来研究一组八个缺乏人类或酵母菌对应物的新基因，参与内吞作用或分泌，我们使用计算/比较基因组学方法确定了这一点。然后我们将寻求识别小说通过筛选几个白色念珠菌突变体文库来筛选参与内吞作用的基因。所有这些研究将使用遗传、生物化学、分子和细胞生物学方法来完成。预期结果：我们期望确定与关键阶段相关的特定基因的功能作用白色念珠菌的内吞作用，并定义它们与丝状化和毒力的联系，以便理解发病机制的分子机制并确定进一步干预的新药物靶点。