Development of protease activity-based detector substrates for diagnosing 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， 附着在物体表面的密集细胞群。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（~50%），（2）生物膜是主要的 这些感染的来源，以及（3）生物膜也对当前的抗真菌药物具有耐药性，可以快速早期检测 生物膜的形成对于改善疾病结果至关重要。加州大学旧金山分校的克雷克实验室（ 最近开发了一种新的基于质谱的筛选技术， 蛋白酶在复杂生物混合物中的底物特异性。这项技术，称为多路复用 质谱底物分析（MSP-MS），允许无偏和同时检测所有 蛋白酶活性在给定的样品;它采用了合理设计的肽底物和监测库 他们的乳沟。我们应用MSP-MS来鉴定生物膜特异性、抗肿瘤特异性、双特异性和泛- 来自七种致病性念珠菌属的念珠菌蛋白酶活性（BioSynesis保留对 UCSF的专利）。基于这些结果，我们开发了几种检测器基板，用于个人 C.白色念珠菌这些检测器是自猝灭的，但是当被切割时， 信号了我们的长期目标是将这些检测器底物开发成快速灵敏的酶检测 念珠菌属生物膜和播散性感染试剂盒。作为概念证明，我们已经证明 我们的第一代探测器基板之一，这是特定的C。白色念珠菌Sap 6分泌的蛋白酶， 检测从植入导管感染C.白色念珠菌生物膜 （未感染的对照为阴性）。在这一成果的基础上，我们建议继续发展更多的 检测底物，并优化那些已经在手，以检测蛋白酶分泌的C。白色念珠菌和 其他七种致病念珠菌将不断开发和测试优化的探测器基板 他们能够准确地检测念珠菌生物膜和体外生长的嗜酸性细胞（Aim 1）， 使用临床前鼠导管生物膜和播散性感染模型（Aim 2）。的结果 临床前样品将用于选择和优化有前景的检测器基质，以在未来的临床试验中进行测试。 样品总的来说，第一阶段提案的结果将为开发优化的 用于快速诊断生物膜和播散性念珠菌感染的检测底物试剂盒。

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 that attach to surfaces. 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 (~50%), (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 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. We applied MSP-MS to identify biofilm-specific, planktonic-specific, dual-specific, and pan- Candida protease activities from seven pathogenic Candida species (BioSynesis retains an exclusive license on this patent from UCSF). Based on these results we developed several detector substrates for individual proteases secreted by C. albicans. These detectors are self-quenched but, when cleaved, release a fluorescent signal. Our long-term goal is to develop these detector substrates into a rapid and sensitive enzymatic detection kit for Candida species biofilm and disseminated infections. As a proof of concept, we have demonstrated that one of our first-generation detector substrates, which is specific for the C. albicans Sap6 secreted protease, can detect this activity in serum isolated from rats that have an implanted catheter infected with a C. albicans biofilm (uninfected controls were negative). Building on this result, we propose to continue the development of additional detector substrates and optimize those already in hand in order to detect proteases secreted by C. albicans and seven other pathogenic Candida species. Optimized detector substrates will be continually developed and tested for their abilities to accurately detect Candida biofilms and planktonic cells grown in vitro (Aim 1), and in vivo using preclinical murine catheter biofilm and disseminated infection models (Aim 2). The results from the preclinical samples will be used to choose and optimize promising detector substrates to test on future clinical samples. Overall, the results from this Phase I proposal will set the stage for the development of an optimized detector substrate kit for the rapid diagnosis of both biofilm and disseminated Candida infections.