Development of Novel Protease Activity-Based Diagnostics for the Rapid Identification of Candida Infections

项目总结/摘要 念珠菌属真菌的成员构成正常人类微生物群的一部分，但也是机会性的 能够引起严重粘膜和全身感染的病原体。念珠菌细胞生长和分裂在 悬浮（无菌）培养，但它们也形成有弹性和耐药性的生物膜-组织紧密， 附着在表面上的密集的细胞群落。生物膜定植在人体的许多小生境中， 它们也在植入的医疗器械上形成，是患者新感染的主要来源。死亡率 念珠菌感染的发病率在免疫功能低下的个体中特别高， 一旦感染扩散到实质器官， 血流因为（1）播散性感染的死亡率很高（约50%），（2） 生物膜是这些感染的主要来源，和（3）生物膜也对目前的抗真菌药物有抗性， 生物膜形成的快速和早期检测对于改善疾病结果至关重要。Craik实验室在 加州大学旧金山分校（该提案的合作者）最近开发了一种新的基于质谱的筛查方法， 技术，以确定全球底物特异性和动力学效率的蛋白酶在复杂的生物 混合物。该技术被称为质谱多重底物分析（MSP-MS），允许 对于给定样品中所有蛋白酶活性的无偏和同时检测;它采用 合理设计肽底物并监测其切割。在与克雷克实验室协商后， 我们已经应用了这种全局分析策略来鉴定生物膜特异性的、抗肿瘤特异性的和广泛的- 光谱蛋白酶活性，目的是开发蛋白酶可裂解的荧光底物， 使生物膜和广泛传播的感染的快速和灵敏的酶检测成为可能 假丝酵母属通过对C.白念珠菌分泌蛋白质组，我们已经确定了两个 分泌的丝氨酸蛋白酶Sap 5和Sap 6，以及枯草杆菌蛋白酶样丝氨酸蛋白酶Kex 2，它们由 C.白色念珠菌生物膜我们已经开发出第一代荧光底物的具体每一个活动; 遗传实验（使用C.对于给定蛋白酶缺失的白色念珠菌菌株）已经证实，这些 底物对它们的靶蛋白酶显示出高特异性。我们还表明，这些相同的基质可以 检测其他致病性念珠菌的蛋白水解活性。最后，我们证明了 sap 6-可切割的荧光底物可以检测从患有 一个被感染了C.白色念珠菌生物膜根据这些初步结果，在第一阶段， R43建议，我们建议开发一个蛋白酶分析管道，用于发现其他蛋白酶 分泌自C.白色念珠菌和其它致病性念珠菌。优化的蛋白酶可切割荧光 将不断开发、改进和测试基质准确检测念珠菌的能力 使用临床前小鼠导管生物膜模型在体外（Aim 1）和体内生长的生物膜和增殖细胞 和鼠播散性感染模型（Aim 2）。在本奖项的第二阶段，我们将翻译这些 通过评估我们基于蛋白酶活性的方法对念珠菌的临床疗效， 生物膜和传播性感染检测。最终的目标是开发一种优化的基板 用于快速诊断生物膜相关和播散性感染的试剂盒。

PROJECT SUMMARY/ABSTRACT Members of the Candida genus of fungi form part of the normal human microbiota but are also opportunistic pathogens capable of causing serious mucosal and systemic infections. Candida cells grow and divide in suspension (planktonic) cultures, but they also form resilient and drug resistant biofilms – organized, tightly- packed communities of cells attached to a surface. Biofilms colonize many niches of the human body and can also form on implanted medical devices, where they are a major source of new infections in patients. Mortality rates from Candida infections are particularly high in immunocompromised individuals, where life-threatening colonization and invasion of parenchymal organs can occur once the infection has disseminated through the bloodstream. Because (1) the mortality rate of disseminated infections is high (approximately 50 percent), (2) biofilms are a major source of these infections, and (3) biofilms are also resistant to current antifungal drugs, rapid and early detection of biofilm formation is critical for improving disease outcome. The Craik laboratory at UCSF (collaborators on this proposal) recently developed a novel mass spectrometry-based screening technology to identify the global substrate specificity and kinetic efficiency of proteases in complex biological mixtures. This technology, referred to as Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS), allows for unbiased and simultaneous detection of all protease activities in a given sample; it employs a library of rationally designed peptide substrates and monitors their cleavage. In consultation with the Craik laboratory, we have applied this global profiling strategy to identify biofilm-specific, planktonic-specific, and broad- spectrum protease activities with the goal of developing protease-cleavable fluorogenic substrates that will enable the rapid and sensitive enzymatic detection of biofilm and disseminated infections from a broad range of Candida species. Through this profiling of the C. albicans secreted proteome, we have identified two secreted aspartyl proteases, Sap5 and Sap6, and a subtilisin-like serine protease, Kex2, that are produced by C. albicans biofilms. We have developed first-generation fluorogenic substrates specific for each activity; genetic experiments (using C. albicans strains deleted for a given protease) have confirmed that these substrates show high specificity for their target protease. We have also shown that these same substrates can detect proteolytic activity from other pathogenic Candida species. Finally, we have demonstrated that the Sap6-cleavable fluorogenic substrate can detect infection-specific activity in serum isolated from rats that have an implanted catheter infected with a C. albicans biofilm. Based on these preliminary results, in Phase I of this R43 proposal, we propose to develop a protease-profiling pipeline for the discovery of additional proteases secreted from C. albicans and other pathogenic Candida species. Optimized protease-cleavable fluorogenic substrates will be continually developed, refined, and tested for their ability to accurately detect Candida biofilms and planktonic cells grown in vitro (Aim 1) and in vivo using a preclinical murine catheter biofilm model and a murine disseminated infection model (Aim 2). In Phase II of this award, we will translate these discoveries to humans by evaluating the clinical efficacy of our protease activity-based approach for Candida biofilm and disseminated infection detection. The eventual goal is the development of an optimized substrate kit for the rapid diagnosis of biofilm-associated and disseminated infections.