Candida species have become among the most common causes of nosocomial infections in the United States and worldwide. An estimated 60-70,000 cases of disseminated candidiasis occur per year in the US alone. Unfortunately, even with modern antifungal therapy, disseminated candidiasis has an unacceptable attributable mortality of 40%-50%. New prophylactic and therapeutic strategies are required. A key to the development of new strategies to prevent and treat Candida infections is the elucidation of the organism's pathogenetic strategies. Mouse models are the standards for defining candidal pathogenesis and the efficacy of antifungal strategies for candidal infections. However, a crucial limitation of murine models of candidiasis is the need to sacrifice animals to determine tissue distribution of the fungus. Because of this limitation, it is impossible to prospectively correlate tissue fungal distribution with success or failure at clearing the infection. In contrast, for models of bacterial infection, novel luciferase technology has recently enabled real-time tracking of pathogenic organisms in rodents by use of whole animal imagers that detect light-emitting strains of bacteria. Luciferase technology has been utilized in Candida strains; however, these candidal strains contain genes encoding firefly or Renilla luciferase, which require exogenous substrates to emit light. Unfortunately, while the substrates penetrate candidal blastospore cell walls, they are unable to penetrate hyphal cell walls. As a result, the candidal strains constructed to date are not reliable for in vivo studies of pathogenesis or therapeutic efficacy. In contrast, the bacterial lux system contains genes encoding its own substrate. Therefore, cloning of the bacterial lux system into Candida will allow constitutive emission of light that would enable the strain to be utilized in in vivo pathogenesis or therapeutic studies. We propose to determine the feasibility of such a system by introducing a yeast-adapted bacterial lux system into a well characterized, pathogenic strain of C. albicans. Internal ribosomal entry sequences (IRES) will allow translation of multiple open reading frames from single messenger RNA (mRNA) strands. Candidal promoters will be used in lieu of S. cerevisiae promoters, and CUG codons will be optimized for expression in Candida. We will then confirm that the bioimager detects the constructed strain in vitro and in vivo during infection in mice. Establishment of a successful candidal luciferase system would be in demand for use by a variety of academic and commercial investigators. Furthermore, the strain would enable the partnering company to establish a fee-for-service, contract-based pathogenesis unit to test antifungal therapies and modified candidal strains in animal models of candidiasis. If feasibility of the system is confirmed in this phase I STTR, subsequent studies in phase II will focus on establishing the accuracy and fidelity of the system for tracking tissue fungal burden and response to therapy in a variety of animal models of candidiasis. Candida is a fungus that has become among the most common causes of life-threatening hospital- acquired infections in the United States and worldwide. A key to the development of new strategies to prevent and treat Candida infections is to develop a greater understanding of how the fungus causes disease. We propose to dramatically enhance the ability of scientists to study how Candida causes disease by adapting a cutting edge technology in which a Candida strain is created that emits light. Use of a light-emitting strain of Candida will enable scientists to use a device that can literally see where the Candida goes in the mouse during invasive infection, thereby dramatically improving our knowledge of how the fungus causes disease.
