Secreted Virulence Proteins and biofilm formation in Candida

念珠菌分泌的毒力蛋白和生物膜的形成

• 批准号: 8141795
• 负责人: SAMUEL AUSTIN LEE
• 金额: --
• 依托单位: ALBUQUERQUE VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8141795
• 关键词: Antifungal Agents; Antifungal Therapy; Area; Aspartic Endopeptidases; Attenuated; Biochemical; Blood; Candida; Candida albicans; Candidiasis; Caring; Catheters; Defect; Diagnosis; Disseminated candidiasis; Drug Delivery Systems; Exocytosis; Extracellular Space; Future; Genes; Genetic; Goals; Golgi Apparatus; Healthcare; Histopathology; Hospitals; In Vitro; Infection; Intervention; Length of Stay; Lipase; Maintenance; Mediator of activation protein; Microbial Biofilms; Modeling; Molecular; Morbidity - disease rate; Mus; Mutation; Pathogenesis; Pathway interactions; Peptide Hydrolases; Phenotype; Phospholipase; Population; Proteins; Role; Saccharomyces cerevisiae; Sepsis; Site; Staging; Stress; Survival Analysis; Tetanus Helper Peptide; Tetracyclines; Vacuolar Protein Sorting; Vesicle; Veterans; Virulence; Yeast Model System; attributable mortality; candida biofilm; candidemia; cost; density; fungus; in vivo; interest; killings; macrophage; mortality; mouse model; mutant; novel; novel diagnostics; pathogen; prevent; protein complex; protein transport; secretory protein; trafficking

DESCRIPTION (provided by applicant): The fungus Candida albicans has emerged as an important opportunistic nosocomial pathogen. Candida species are now the fourth most common cause of hospital-acquired bloodstream infections and are a major cause of morbidity and mortality in the veteran population. Despite improvements in antifungal therapy, the high attributable mortality rate due to Candida infections is no better than two decades ago, and diagnostically, the traditional blood culture has only a 50% sensitivity to detect invasive candidiasis. Thus, our ability to prevent, diagnose, and treat Candida infections is still in need of great improvement. In the effort to identify novel diagnostic and drug targets, a number of C. albicans virulence-associated proteins have been investigated, and many of these are secretory proteins such as the secreted aspartyl proteases (Saps) and phospholipases. In addition, considerable recent effort has been made to study Candida biofilms, another key component of Candida pathogenesis. These biofilms enable Candida to colonize a protected site from which dissemination of infection and protection from anti-fungal therapy can occur. Thus, our overall objectives are to: (i) determine which genes are critical for the trafficking and secretion of Saps and other virulence proteins, and (ii) define the role of secretion in biofilm formation. If we can identify these key genes, this information may prove invaluable for understanding the mechanisms of biofilm formation and for identifying novel drug targets. 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 sorting genes VPS1, VPS4, and PEP12. Of great interest, a C. albicans pep12 mutant was defective in virulence in vivo, and formed a biofilm that dramatically fragmented with minimal disturbance. Expanding on these studies, we will examine the late stages of secretion by examining key final steps in exocytosis regulated by the Exocyst protein complex, and determine its contribution to biofilm formation and virulence. Next, we will examine the molecular mechanisms of biofilm formation by investigating the critical determinants of biofilm integrity in the pep12 mutant. Therefore, this project will examine the key hypotheses that: (i) C. albicans exocytosis mutants missort proteins to the extracellular space resulting in defective biofilm formation, and (ii) mutations in secretory pathway genes at key steps in exocytosis will result in attenuated virulence in vivo, and (iii) C. albicans PEP12 is a key mediator of biofilm integrity. Specific Aim 1 is to determine the effects of mutations in C. albicans exocytosis genes at key steps of polarized secretion and their contribution to biofilm formation. Specific Aim 2 is to directly examine the requirement of C. albicans exocytosis genes for virulence in vitro and in vivo. Specific Aim 3 is to determine whether downstream effectors of C. albicans PEP12 are required for maintenance of biofilm integrity. Thus, our overall objectives are to define the key trafficking genes and pathways of these important secreted virulence proteins, and define their role in biofilm formation in order to understand mechanisms of pathogenesis and identify novel drug targets.

描述（由申请人提供）： 真菌白色念珠菌已成为一种重要的机会性医院病原体。念珠菌现在是医院获得性血液感染的第四大常见原因，也是退伍军人发病和死亡的主要原因。尽管抗真菌治疗取得了进步，但念珠菌感染引起的高归因死亡率并不比二十年前好，而且在诊断上，传统的血培养检测侵袭性念珠菌病的敏感性仅为 50%。因此，我们预防、诊断和治疗念珠菌感染的能力仍然需要很大的提高。为了确定新的诊断和药物靶点，人们对许多白色念珠菌毒力相关蛋白进行了研究，其中许多是分泌蛋白，例如分泌型天冬氨酰蛋白酶 (Saps) 和磷脂酶。此外，最近人们在研究念珠菌生物膜（念珠菌发病机制的另一个关键组成部分）方面做出了相当大的努力。这些生物膜使念珠菌能够在受保护的部位定殖，从该部位可以发生感染传播和抗真菌治疗的保护。因此，我们的总体目标是：(i) 确定哪些基因对于 SAP 和其他毒力蛋白的运输和分泌至关重要，以及 (ii) 确定分泌在生物膜形成中的作用。如果我们能够识别这些关键基因，这些信息对于理解生物膜形成机制和识别新药物靶点可能非常有价值。在之前的研究中，我们研究了白色念珠菌中液泡前分泌途径在毒力相关蛋白的分泌和生物膜形成中的作用，该途径受液泡蛋白分选基因 VPS1、VPS4 和 PEP12 的调节。令人非常感兴趣的是，白色念珠菌 pep12 突变体在体内的毒力存在缺陷，并且形成了一种生物膜，该生物膜在最小的干扰下就会急剧破碎。扩展这些研究，我们将通过检查 Exocyst 蛋白复合物调节的胞吐作用的关键最终步骤来检查分泌的后期阶段，并确定其对生物膜形成和毒力的贡献。接下来，我们将通过研究 pep12 突变体中生物膜完整性的关键决定因素来研究生物膜形成的分子机制。因此，该项目将研究以下关键假设：(i) 白色念珠菌胞吐作用突变体将蛋白质错配到细胞外空间，导致生物膜形成缺陷；(ii) 胞吐作用关键步骤中分泌途径基因的突变将导致体内毒力减弱；(iii) 白色念珠菌 PEP12 是生物膜完整性的关键介质。 具体目标 1 是确定白色念珠菌胞吐基因突变对极化分泌关键步骤的影响及其对生物膜形成的贡献。具体目标2是直接检查白色念珠菌胞吐基因对体外和体内毒力的要求。具体目标 3 是确定维持生物膜完整性是否需要白色念珠菌 PEP12 的下游效应子。因此，我们的总体目标是确定这些重要的分泌毒力蛋白的关键运输基因和途径，并确定它们在生物膜形成中的作用，以便了解发病机制并确定新的药物靶点。