Targeting evolutionarily acquired insertion sequences in Candida species, for development of antifungal drugs

摘要 人类所有侵袭性真菌感染中有一半是由白色念珠菌引起的。它作为病原体的成功是由于它的 能够适应寄主中的各种微环境并茁壮成长。使用前向遗传学筛查，我们发现 一种未鉴定的基因NDU1，其缺失导致白念珠菌失去对替代碳源的生存能力 并使这种微生物在体内完全无毒。我们发现假丝酵母-Ndu1蛋白有三个 进化获得的氨基酸插入片段(~氨基酸)，在所有其他真核生物中缺失 包括人类。我们首次报道了~17%的念珠菌蛋白质组具有“额外的”集合 所有非CTG分支真核生物中都不存在的进化获得的氨基酸插入片段，包括 人类。插入区域在白念珠菌蛋白质组中的作用仍有待解开。我们已经证明了 NDU1插入序列对NDU1‘S的活动至关重要，也是必需的。在目标1中， Ndu1插入片段将通过蛋白质生物化学研究进行研究。插入区段在其他念珠菌中的作用 还将对蛋白质进行评估，以了解插入片段是否对功能具有普遍重要性。 考虑到Ndu1对体内生长和毒力是重要的，我们针对Ndu1及其插入区域 抗真菌分子的研究进展。我们确定了FDA批准的一种改用药物氯硝柳胺(NCL)，以及 开发了该分子的Eudradit EPO纳米颗粒配方，以增强其溶解性和生物利用度。 NCL纳米粒(NCL-EPO-NP)可在替代碳源中抑制念珠菌的生长，穿透 在体外分解成熟的生物膜，并在黏膜念珠菌病的生物膜模型中在体内根除它。这个 NCL-NP的活性是仿制药的6-10倍。在目标2中，我们进一步描述了 评价NCL-EPO-NP在小鼠体内的药代动力学、口服生物分布、毒性及体内疗效 一种口服疗法，用于治疗口腔念珠菌病，如单独使用或与其他抗真菌药物联合使用。

Abstract Candida albicans causes half of all invasive fungal infections in humans. Its success as a pathogen is due to its ability to adapt and thrive in various microenvironments in the host. Using a forward genetics screen, we identified an uncharacterized gene NDU1 whose loss caused C. albicans to lose viability on alternative carbon sources and made the organism completely avirulent in vivo. We found that Candida-Ndu1 protein possesses three evolutionarily acquired stretches of amino acid inserts (~64 amino acids), absent from all other eukaryotes including humans. We reported for the first time that ~17% of the Candida proteome possess “additional” sets of evolutionarily acquired amino-acid inserts, that are absent from all non-CTG clade eukaryotes, including humans. The role of insert regions in C. albicans proteome remains to be unraveled. We have shown that the Ndu1 insertion sequences are vital and required for Ndu1’s activity. In aim 1, the mechanistic contribution of Ndu1 inserts will be investigated by protein biochemistry studies. The role of insert regions in other Candida proteins will also be evaluated , to understand if inserts are universally important for function. Considering Ndu1 is important for growth and virulence in vivo, we targeted Ndu1 and its insert regions for development of antifungal molecules. We identified a repurposed FDA approved drug niclosamide (NCL), and developed Eudragit EPO nanoparticle formulations of this molecule to enhance its solubility and bioavailability. NCL nanoparticles (NCL-EPO-NP) could prevent growth of Candida in alternative carbon sources, penetrate and dismantle mature biofilms in vitro, and eradicate it in vivo in biofilm models of mucosal candidiasis. The activity of NCL-NP was 6-10 fold higher than the generic drug alone. In Aim 2, we further characterize and evaluate NCL-EPO-NP for their pharmacokinetics and oral biodistribution, toxicity profiles and in vivo efficacy in an oral regimen for treatment of oral candidiasis, as standalone or adjunct therapy with other antifungal drugs.