Development of protease activity-based detector substrates for diagnosing Candida infections

项目摘要/摘要 假丝酵母菌属的成员构成了正常的人类微生物区系的一部分，但也是机会主义的 能够引起严重的粘膜和全身感染的病原体。念珠菌细胞在体内生长和分裂 悬浮(浮游)培养，但它们也形成有弹性和抗药性的生物膜-有组织的，紧密的- 附着在表面上的密集的细胞群落。生物膜定居在人体的许多壁龛中，并可以 在植入的医疗设备上也会形成，在那里它们是患者新感染的主要来源。死亡率 念珠菌感染的比率在免疫功能低下的人中尤其高，在这些人中，危及生命的 一旦感染通过体内扩散，就可能发生实质器官的定植和入侵 血液流动。因为(1)播散性感染的死亡率很高(~50%)，(2)生物被膜是主要的 这些感染的来源，以及(3)生物被膜对目前的抗真菌药物也具有耐药性，快速和早期检测 生物被膜的形成对改善疾病预后至关重要。加州大学旧金山分校的Craik实验室(合作者 该建议)最近开发了一种新的基于质谱学的筛选技术，以识别全球 复杂生物混合物中蛋白酶的底物专一性。这种技术称为多路复用器 底物质量分析(MSP-MS)，允许无偏和同时检测所有 特定样品中的蛋白酶活性；它使用合理设计的多肽底物库并监控 他们的乳沟。我们应用MSP-MS鉴定生物膜专一性、浮游生物专一性、双重专一性和泛-专一性。 七种致病念珠菌的蛋白酶活性(BioSynesis保留了 这项专利来自加州大学旧金山分校)。基于这些结果，我们开发了几种适用于个人的探测器基板 白念珠菌分泌的蛋白酶。这些探测器是自熄灭的，但当被切割时，会释放出荧光 信号。我们的长期目标是将这些检测底物开发成快速、灵敏的酶检测方法 念珠菌生物被膜和播散性感染试剂盒。作为概念的证明，我们已经证明了 我们的第一代检测底物之一，它是针对白念珠菌Sap6分泌的蛋白酶的，可以 检测植入导管感染白念珠菌生物膜的大鼠血清中的这种活性 (未感染对照组均为阴性)。在此基础上，我们建议继续发展额外的 检测底物并优化现有底物，以便检测白念珠菌和白念珠菌分泌的蛋白酶 其他七种致病念珠菌。将继续开发和测试优化的探测器基板 用于准确检测体外和体内生长的念珠菌生物膜和浮游细胞的能力(目标1) 使用临床前小鼠导管生物被膜和播散性感染模型(目标2)。结果来自于 临床前样本将用于选择和优化有前景的检测器底物，以便在未来的临床上进行测试 样本。总体而言，此第一阶段提案的结果将为开发优化的 快速诊断生物被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.