Secreted Virulence Proteins and biofilm formation in Candida

描述（由申请人提供）： 真菌白色念珠菌已成为一种重要的医院内机会致病菌。念珠菌属现在是医院获得性血流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。尽管抗真菌治疗有所改进，但念珠菌感染的高归因死亡率并不比二十年前好，并且在诊断上，传统的血培养检测侵袭性念珠菌病的灵敏度仅为50%。因此，我们预防、诊断和治疗念珠菌感染的能力仍然需要极大的提高。在寻找新的诊断和药物靶点的努力中，许多C.已经研究了白色念珠菌毒力相关蛋白，并且这些蛋白中的许多是分泌性蛋白，例如分泌的N-乙酰基蛋白酶（Saps）和磷脂酶。此外，相当多的最近的努力已经取得了研究念珠菌生物膜，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定殖在受保护的部位，从该部位可以发生感染的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Saps和其他毒力蛋白的运输和分泌至关重要，以及（ii）定义分泌在生物膜形成中的作用。如果我们能够确定这些关键基因，这些信息可能对理解生物膜形成机制和确定新的药物靶点非常有用。在以前的研究中，我们检测了前液泡分泌途径在C.白色念珠菌，由液泡蛋白分选基因VPS 1，VPS 4和PEP 12调节。令人感兴趣的是，一个C。白念珠菌pep 12突变体在体内毒力缺陷，并形成生物膜，其在最小干扰下显著碎裂。在这些研究的基础上，我们将通过检查由外囊蛋白复合物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pep 12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，本研究将探讨以下主要假设：（i）C.白色念珠菌胞吐突变体将蛋白质错配到细胞外空间，导致有缺陷的生物膜形成，和（ii）在胞吐的关键步骤中分泌途径基因的突变将导致体内毒力减弱，和（iii）C.白色念珠菌PEP 12是生物膜完整性的关键介质。 具体目标1是确定C.白念珠菌胞吐基因在极化分泌的关键步骤及其对生物膜形成的贡献。具体目标二是直接考察C.白念珠菌胞吐基因的毒力在体外和体内。具体目标3是确定C.白色念珠菌PEP 12是维持生物膜完整性所必需的。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，并确定它们在生物膜形成中的作用，以了解发病机制和确定新的药物靶点。

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.