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.
Public Health Relevance Statement: Project Narrative Infections by fungal pathogens from the Candida clade can be life-threatening in humans, but no diagnostics are available for the rapid detection of Candida in biofilm or planktonic forms. The goal of this proposal is to validate the enzymatic molecular signatures produced by Candida species in the biofilm and planktonic forms to use in the development of a rapid and sensitive diagnostic kit for detection of diverse Candida species. If successful, this STTR Phase I project will provide the foundation for a novel Candida-specific diagnostic that will change current clinical practices for the detection and treatment of fungal infections.
Project Terms: Animals; Antifungal Agents; Antifungal Drug; Therapeutic Fungicides; anti-fungal; anti-fungal agents; anti-fungal drug; antifungals; Biological Assay; Assay; Bioassay; Biologic Assays; Blood Circulation; Bloodstream; Circulation; Candida; Monilia; Candida albicans; C albicans; C. albicans; C.albicans; Cells; Cell Body; Communities; Diagnosis; Disease; Disorder; Drug resistance; drug resistant; resistance to Drug; resistant to Drug; Fluorogenic Substrate; Foundations; fungus; Future; Goals; Hand; Hospitals; Human; Modern Man; Immune system; allergic/immunologic body system; allergic/immunologic organ system; In Vitro; Infection; Laboratories; Libraries; Medical Device; mortality; Mucous Membrane; Mucosa; Mucosal Tissue; Mus; Mice; Mice Mammals; Murine; Mycoses; Fungus Diseases; fungal infection; fungus infection; Patents; Legal patent; Patients; Esteroproteases; Peptidases; Protease Gene; Proteases; Proteinases; Proteolytic Enzymes; Peptide Hydrolases; Peptides; Common Rat Strains; Rat; Rats Mammals; Rattus; diagnostic kit; test kit; Diagnostic Reagent Kits; Cell Communication and Signaling; Cell Signaling; Intracellular Communication and Signaling; Signal Transduction Systems; Signaling; biological signal transduction; Signal Transduction; Specificity; Mass Photometry/Spectrum Analysis; Mass Spectrometry; Mass Spectroscopy; Mass Spectrum; Mass Spectrum Analyses; Mass Spectrum Analysis; Substrate Specificity; Suspension substance; Suspensions; Technology; Testing; Generations; Microbial Biofilms; biofilm; Immunocompromised Host; Immunocompromised; Immunocompromised Patient; Immunosuppressed Host; immunosuppressed patient; Catheters; Soft Tissue Infections; base; Organ; detector; improved; Procedures; Surface; Clinical; Phase; Biological; biologic; Medical; peripheral blood; Blood Serum; Serum; Individual; Licensing; Human Figure; Human body; Systemic infection; Diagnostic; Peptide Library; Life; Complex; Scanning; Source; early detection; Early Diagnosis; Performance; rapid diagnosis; fluorophore; Animal Models and Related Studies; model of animal; model organism; Animal Model; Disease Outcome; novel; member; cutaneous tissue; Skin Tissue; Modeling; Sampling; Property; Pathogenicity; Detection; Molecular Fingerprinting; molecular profile; molecular signature; Molecular Profiling; in vitro Assay; in vivo; Small Business Technology Transfer Research; STTR; Virulent; Monitor; Development; developmental; pre-clinical; preclinical; candida biofilm; cost; medical implant; rapid detection; design; designing; prospective; Resistance; resistant; Implant; drug testing; drug detection; mouse model; murine model; commercialization; clinical practice; screening; human microbiota; human flora; human microbial communities; human microflora; human-associated microbial communities; human-associated microbiota; pathogenic fungus; fungal pathogen; fungi pathogen; cell community; cellular community; opportunistic pathogen; rational design
