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

项目总结/文摘

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.