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

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 infectionsCandida cells grow and divide in suspensionplanktonicculturesbut they also form resilient and drug resistant biofilmsorganizedtightlypacked communities of cells attached to a surfaceBiofilms colonize many niches of the human body and can also form on implanted medical deviceswhere they are a major source of new infections in patientsMortality rates from Candida infections are particularly high in immunocompromised individualswhere life threatening colonization and invasion of parenchymal organs can occur once the infection has disseminated through the bloodstreamBecausethe mortality rate of disseminated infections is highapproximatelypercentbiofilms are a major source of these infectionsandbiofilms are also resistant to current antifungal drugsrapid and early detection of biofilm formation is critical for improving disease outcomeThe Craik laboratory at UCSFcollaborators on this proposalrecently developed a novel mass spectrometry based screening technology to identify the global substrate specificity and kinetic efficiency of proteases in complex biological mixturesThis technologyreferred to as Multiplex Substrate Profiling by Mass SpectrometryMSP MSallows for unbiased and simultaneous detection of all protease activities in a given sampleit employs a library of rationally designed peptide substrates and monitors their cleavageIn consultation with the Craik laboratorywe have applied this global profiling strategy to identify biofilm specificplanktonic specificand broadspectrum 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 speciesThrough this profiling of the Calbicans secreted proteomewe have identified two secreted aspartyl proteasesSapand Sapand a subtilisin like serine proteaseKexthat are produced by Calbicans biofilmsWe have developed first generation fluorogenic substrates specific for each activitygenetic experimentsusing Calbicans strains deleted for a given proteasehave confirmed that these substrates show high specificity for their target proteaseWe have also shown that these same substrates can detect proteolytic activity from other pathogenic Candida speciesFinallywe have demonstrated that the Sapcleavable fluorogenic substrate can detect infection specific activity in serum isolated from rats that have an implanted catheter infected with a Calbicans biofilmBased on these preliminary resultsin Phase I of this Rproposalwe propose to develop a protease profiling pipeline for the discovery of additional proteases secreted from Calbicans and other pathogenic Candida speciesOptimized protease cleavable fluorogenic substrates will be continually developedrefinedand tested for their ability to accurately detect Candida biofilms and planktonic cells grown in vitroAimand in vivo using a preclinical murine catheter biofilm model and a murine disseminated infection modelAimIn Phase II of this awardwe will translate these discoveries to humans by evaluating the clinical efficacy of our protease activity based approach for Candida biofilm and disseminated infection detectionThe eventual goal is the development of an optimized substrate kit for the rapid diagnosis of biofilm associated and disseminated infections