ROLE OF VACUOLE EXPANSION IN THE ORAL PATHOGEN CANDIDA ALBICANS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 背景： 念珠菌是困扰人类的最常见的机会性病原体，其中白色念珠菌最常见。白色念珠菌可引起一系列不同的粘膜疾病，包括口腔和食道鹅口疮以及假牙口炎。超过90%的HIV+患者会引发口腔念珠菌病，这些感染是高度复发的。这需要在这些患者中长期使用抗真菌药物治疗，从而出现抗真菌耐药菌株。念珠菌也可以引起威胁生命的系统性真菌感染，迫切需要新的治疗策略来对抗这些感染。白念珠菌可逆地在酵母菌和菌丝生长形态之间切换的能力对致病性至关重要。高度极化的菌丝形态被认为有助于组织入侵，而较小的圆形酵母细胞可能有助于传播。在从酵母菌向菌丝生长的转变过程中，细胞质物质保持在顶端隔室，而真菌液泡经历了戏剧性的扩张，产生了高度‘液泡化’的亚顶端隔室。这代表了一种新的分化机制。 目标： 1)。目的：探讨白念珠菌菌丝细胞空泡化的分子机制。这将通过对Vps21p和Ypt7p蛋白的功能分析来实现，这两个蛋白是液泡前隔室(PVC)和液泡生物发生的关键调节因子。 2)。以确定破坏真菌液泡功能的治疗潜力。这将通过在体外“毒力分析”、组织侵袭模型和基于动物的感染模型中测试缺乏PVC(Vps21D)和空泡生物发生(Ypt7D)的突变株来建立。 结果：我们获得了绿色荧光蛋白(GFP)标记的Vps21p和Ypt7p版本。GFP-Vps21p和GFP-Ypt7p的定位分析表明，这些蛋白如预测的那样定位于PVC和液泡室。Western印迹分析还表明，Ypt7p在菌丝生长过程中的表达水平高于酵母。此外，虽然Vps21p的表达水平相似，但在菌丝生长过程中检测到了第二种形式的分子量增加。这可能反映了在菌丝生长过程中调节Vps21p活性的翻译后修饰。我们还使用体外模型系统证明了vps11D突变体(在PVC和空泡生物发生方面存在缺陷)不能侵袭口腔组织。此外，在系统性疾病的小鼠模型中，缺乏pvc(Vps21D)或空泡生物发生(Ypt7D)的突变株高度减弱。 讨论：这些数据表明，Vps21p和Ypt7p的活性是液泡生物发生的关键调节因子，它们都在向组织侵袭菌丝形式的转变过程中受到调节。此外，PVC或液泡生物发生中的缺陷导致了该病原菌毒力的丧失。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background: Candida species are the most frequent opportunistic pathogens afflicting humans, with C. albicans the most prevalent. C. albicans can cause a diverse array of mucosal disease including oral and esophageal thrush, and denture stomatitis. More than 90% of HIV+ patients will incur oral candidiasis and these infections are highly recurrent. This has necessitated long term treatment with antifungals in these patients, with the consequent emergence of antifungal resistant strains. Candida can also cause life threatening systemic fungal infections, and new therapeutic strategies are badly needed to combat these infections. The capacity of C. albicans to reversibly switch between yeast and hyphal growth forms is crucial for pathogenicity. The highly polarized hyphal form is thought to facilitate tissue invasion, while the smaller, rounded yeast cells may aid dissemination. During the transition from yeast to hyphal growth, cytoplasmic material is maintained in the apical compartment while the fungal vacuole undergoes a dramatic expansion to generate highly 'vacuolated' sub-apical compartments. This represents a novel mechanism of differentiation. Objectives: 1). To establish the molecular mechanism of hyphal cell vacuolation in C. albicans. This will be achieved through the functional analysis of proteins Vps21p and Ypt7p which are key regulators of pre-vacuole compartment (PVC) and vacuole biogenesis. 2). To determine the therapeutic potential of disrupting the function of the fungal vacuole. This will be established though testing mutant strains deficient in PVC (vps21D) and vacuolar biogenesis (ypt7D) in in vitro 'virulence assays', models of tissue invasion, and animal based models of infection. Results: We have generated Green Fluorescent Protein (GFP) tagged versions of Vps21p and Ypt7p. Localization analysis of GFP-Vps21p and GFP-Ypt7p has demonstrated these proteins localize to the PVC and vacuole compartments as predicted. Western blot analysis has also revealed that Ypt7p is expressed at a higher level during hyphal than yeast growth. Furthermore, while Vps21p expression levels are similar, a second form of increased molecular weight was detected specifically during hyphal growth. This may reflect a post-translational modification which modulates Vps21p activity during hyphal growth. We have also demonstrated that a vps11D mutant (defective in PVC and vacuole biogenesis) is incapable of oral tissue invasion using an in vitro model system. Furthermore, mutants deficient in PVC (vps21D) or vacuole biogenesis (ypt7D) are highly attenuated in a mouse model of systemic disease. Discussion: These data suggest that the activity of Vps21p and Ypt7p, which are key regulators of vacuole biogenesis, are both modulated during the transition to the tissue invasive hyphal form. Moreover, that defects in either PVC or vacuole biogenesis lead to the loss of virulence in this pathogen.