Development of protease activity-based detector substrates for diagnosing 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 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.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.