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

开发用于诊断念珠菌感染的基于蛋白酶活性的检测器底物

• 批准号: 10676162
• 负责人: Matthew B. Lohse
• 金额: $ 29.61万
• 依托单位: BIOSYNESIS, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-03 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10676162
• 关键词: Animal Model; Animals; Antifungal Agents; Biological; Biological Assay; Blood Circulation; Candida; Candida albicans; Catheters; Cells; Clinical; Communities; Complex; Detection; Development; Diagnosis; Diagnostic; Diagnostic Reagent Kits; Disease; Disease Outcome; Drug resistance; Early Diagnosis; Fluorogenic Substrate; Foundations; Future; Generations; Goals; Hand; Hospitals; Human; Human body; Immune system; Immunocompromised Host; In Vitro; Individual; Indwelling Catheter; Infection; Invaded; Laboratories; Legal patent; Libraries; Licensing; Life; Mass Spectrum Analysis; Medical; Medical Device; Microbial Biofilms; Modeling; Molecular Profiling; Monitor; Mucous Membrane; Mus; Mycoses; Organ; Pathogenicity; Patients; Peptide Hydrolases; Peptide Library; Peptides; Performance; Phase; Procedures; Property; Rattus; Resistance; Sampling; Scanning; Serum; Signal Transduction; Skin Tissue; Small Business Technology Transfer Research; Soft Tissue Infections; Source; Specificity; Substrate Specificity; Surface; Suspensions; Systemic infection; Technology; Testing; Virulent; candida biofilm; cell community; clinical practice; commercialization; cost; design; detector; drug testing; fluorophore; fungus; human microbiota; improved; in vitro Assay; in vivo; medical implant; member; mortality; mouse model; novel; opportunistic pathogen; pathogenic fungus; peripheral blood; pre-clinical; prospective; rapid detection; rapid diagnosis; rational design; resilience; screening

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.

项目概要/摘要 真菌念珠菌属的成员是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在其中生长和分裂。 悬浮（浮游）培养物，但它们也形成有弹性和耐药性的生物膜——有组织的、紧密的 附着在表面的细胞群落聚集在人体的许多部位，并且可以生长。 它们也形成于植入式医疗设备上，是患者新感染的主要来源。 在免疫功能低下的个体中，念珠菌感染的发生率特别高，这些人会危及生命 一旦感染通过肠道传播，就会发生实质器官的定植和侵袭。 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，并且（3）生物膜也对当前的抗真菌药物具有抗药性，可以快速、早期检测 生物膜形成对于改善疾病结果至关重要。 该提案）最近开发了一种新颖的基于质谱的筛选技术来识别全球 复杂生物混合物中蛋白酶的底物特异性，这种技术称为 Multiplex。 通过质谱 (MSP-MS) 进行底物分析，可以无偏且同时检测所有物质 它采用合理设计的肽底物库和监测器来检测给定样品中的蛋白酶活性； 我们应用 MSP-MS 来识别生物膜特异性、浮游特异性、双特异性和泛性。 来自七种致病性念珠菌物种的念珠菌蛋白酶活性（BioSynesis 保留了 这项来自 UCSF 的专利）基于这些结果，我们开发了几种适合个人的探测器基板。 白色念珠菌分泌的蛋白酶是自猝灭的，但在裂解时会释放荧光。 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，我们已经证明了这一点。 我们的第一代检测器底物之一，专门针对白色念珠菌 Sap6 分泌的蛋白酶，可以 检测从植入感染白色念珠菌生物膜的导管的大鼠中分离出的血清中的这种活性 （未感染的对照呈阴性）在此结果的基础上，我们建议继续开发更多的产品。 检测器底物并优化现有的底物，以检测白色念珠菌分泌的蛋白酶， 将不断开发和测试其他七种致病性念珠菌物种。 因其能够准确检测体外（目标 1）和体内生长的念珠菌生物膜和浮游细胞 使用临床前小鼠导管生物膜和播散性感染模型（目标 2）的结果。 临床前样品将用于选择和优化有前途的检测器基板，以测试未来的临床 总体而言，第一阶段提案的结果将为优化的开发奠定基础。 检测器底物套件，用于快速诊断生物膜和播散性念珠菌感染。