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

开发用于快速鉴定念珠菌感染的新型蛋白酶活性诊断方法

• 批准号: 9335705
• 负责人: Matthew B. Lohse
• 金额: $ 30万
• 依托单位: BIOSYNESIS, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-15 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9335705
• 关键词: Antifungal Agents; Aspartic Endopeptidases; Award; Biological; Biological Assay; Blood Circulation; Candida; Candida albicans; Catheters; Cells; Characteristics; Clinical; Communities; Complex; Consultations; Detection; Development; Diagnostic; Disease; Disease Outcome; Distant; Drug resistance; Early Diagnosis; Fluorogenic Substrate; Gastrointestinal tract structure; Generations; Genetic; Goals; Harvest; Human; Human body; Immune system; Immunocompromised Host; Implant; In Vitro; Individual; Infection; Kinetics; Laboratories; Libraries; Life; Mass Spectrum Analysis; Medical; Medical Device; Methods; Microbial Biofilms; Modeling; Molecular Profiling; Monitor; Mus; Mycoses; Organ; Pathogenicity; Patients; Peptide Hydrolases; Peptide Library; Peptides; Performance; Phase; Pre-Clinical Model; Procedures; Property; Proteolysis; Proteome; Rattus; Reproducibility; Resistance; Sampling; Scanning; Serine Protease; Serum; Site; Skin Tissue; Small Business Innovation Research Grant; Soft Tissue Infections; Source; Specificity; Substrate Specificity; Subtilopeptidase A; Surface; Suspensions; Systemic infection; Technology; Testing; Time; Translating; Translations; Validation; Virulent; base; c new; candida biofilm; cell type; clinical efficacy; clinical practice; clinically relevant; commercialization; design; drug testing; experimental study; extracellular; fungus; improved; in vitro Assay; in vitro activity; in vivo; medical implant; member; microbiota; mortality; mouse model; novel; pathogen; phase 1 study; pre-clinical; rapid detection; rapid diagnosis; 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 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.

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（约 50%），(2) 生物膜是这些感染的主要来源，并且（3）生物膜也对当前的抗真菌药物具有耐药性， 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（该提案的合作者）最近开发了一种新型的基于质谱的筛查 识别复杂生物中蛋白酶的全局底物特异性和动力学效率的技术 该技术称为多重底物质谱分析 (MSP-MS)，可实现多种混合物的分析。 为了公正且同时检测给定样品中的所有蛋白酶活性，它使用了一个库； 与 Craik 实验室协商，合理设计肽底物并监测其裂解。 我们应用了这种全球分析策略来识别生物膜特异性、浮游生物特异性和广泛的 光谱蛋白酶活性，目标是开发蛋白酶可裂解的荧光底物， 能够快速、灵敏地对生物膜和广泛范围的播散性感染进行酶检测 通过对白色念珠菌分泌蛋白质组的分析，我们鉴定了两种念珠菌。 分泌型天冬氨酰蛋白酶 Sap5 和 Sap6，以及枯草杆菌蛋白酶样丝氨酸蛋白酶 Kex2，它们由 我们开发了针对每种活性的第一代荧光底物； 基因实验（使用针对给定蛋白酶删除的白色念珠菌菌株）已证实这些 底物对其目标蛋白酶表现出高特异性。我们还表明，这些相同的底物可以。 检测其他致病性念珠菌物种的蛋白水解活性。 Sap6 可裂解的荧光底物可以检测从患有以下疾病的大鼠中分离出的血清中的感染特异性活性 根据这些初步结果，在第一阶段中，植入了感染白色念珠菌生物膜的导管。 R43提案，我们建议开发一个蛋白酶分析管道来发现其他蛋白酶 由白色念珠菌和其他致病性念珠菌物种分泌，经过优化的蛋白酶可裂解荧光。 将不断开发、改进和测试底物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（目标 1）和体内生长生物膜和浮游细胞 以及小鼠传播感染模型（目标 2）。在该奖项的第二阶段，我们将翻译这些模型。 通过评估我们基于蛋白酶活性的念珠菌方法的临床功效，为人类带来了发现 最终目标是开发优化的基质。 用于快速诊断生物膜相关和播散性感染的试剂盒。