Secreted Virulence Proteins and biofilm formation in Candida

描述(由申请人提供)： 白色念珠菌是一种重要的医院内机会性致病菌。念珠菌现在是医院获得性血液感染的第四大常见原因，也是退伍军人中发病率和死亡率的主要原因。尽管抗真菌治疗有所改进，但念珠菌感染导致的高归因性死亡率并不比20年前好，而且在诊断方面，传统的血培养对检测侵袭性念珠菌病的灵敏度只有50%。因此，我们预防、诊断和治疗念珠菌感染的能力仍然需要极大的提高。为了寻找新的诊断和药物靶点，人们研究了一些与白念珠菌毒力相关的蛋白，其中许多是分泌型蛋白，如分泌型天冬氨酸蛋白酶(SAPS)和磷脂酶。此外，最近对假丝酵母菌生物膜的研究也做了相当大的努力，这是念珠菌致病的另一个关键组成部分。这些生物膜使念珠菌能够在受保护的地方定居，从那里可以传播感染和保护免受抗真菌治疗。因此，我们的总体目标是：(I)确定哪些基因对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的形成机制和识别新的药物靶点可能会被证明是非常有价值的。在以前的研究中，我们研究了空泡前分泌途径在白念珠菌毒力相关蛋白的分泌和生物膜形成中的作用，受到空泡蛋白分选基因VPS1、Vps4和PEP12的调节。令人感兴趣的是，一个白色念珠菌Pep12突变体在体内毒力方面存在缺陷，并形成了一种生物膜，在极小的干扰下戏剧性地碎片化。在这些研究的基础上，我们将通过检查由胞囊蛋白复合体调节的胞吐作用的关键最后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，本项目将检验如下关键假设：(I)白念珠菌胞吐突变体将蛋白质错误地分选到细胞外空间，导致生物膜形成缺陷；(Ii)在胞吐作用的关键步骤中，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是确定白念珠菌胞吐基因突变在极化分泌的关键步骤中的影响及其对生物被膜形成的贡献。具体目的2是在体外和体内直接检测白念珠菌胞吐基因对毒力的要求。具体目标3是确定是否需要白念珠菌PEP12的下游效应器来维持生物膜的完整性。因此，我们的总体目标是确定这些重要的分泌型毒力蛋白的关键运输基因和途径，并确定它们在生物被膜形成中的作用，以便了解发病机制和寻找新的药物靶点。

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.