Adaptive Evolution of Candida Biofilms

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PROJECT SUMMARY There is a critical need to identify important parameters involved in adaptation to environmental stresses in fungal biofilms, in order to develop effective therapeutic strategies against fungal infections. Treatment of fungal infections by Candida spp, including the emerging pathogen Candida glabrata (C. glabrata), remains a clinical challenge especially in immunocompromised individuals. C. glabrata is now the second most frequently isolated Candida spp in North America. In vivo, microbes mostly exist in biofilms, which serve as protective layers. Cells in biofilms exhibit increased resistance to environmental stresses. Since biofilms are an integral part of pathogenesis, the adaptation of fungal pathogens inside a biofilm is an important aspect that requires a deeper understanding. However, existing knowledge of fungal adaptation in biofilms is limited, partly due to the lack of established experimental methods for the long-term propagation of fungal biofilms. With a long-term goal of contributing to the development of therapeutic strategies for candidiasis, the overall objective of the project is to develop a fungal biofilm propagation method for use in experimental adaptive evolution, and to apply the system to identify essential genes involved in C. glabrata biofilms. In Aim 1, a biofilm culture system suitable for long- term in vitro evolution will be developed and characterized. A key feature of the method being developed is the ability to grow multiple biofilms from the same seed biofilm, allowing multiple procedures for analysis and characterization of biofilms at each passage during in vitro evolution. Aim 2 will Implement the fungal biofilm propagation system for in vitro evolution of C. glabrata to environmental stressors. Key properties, such as amount of biofilm formation and changes in biofilm structure will be monitored. Molecular mechanisms associated with adaptation to environmental stressors in fungal biofilms will be elucidated based on genome- sequencing and phenotypic analyses. Aim 3 will combine the biofilm propagation method with transposon sequencing to identify essential genes involved in biofilm formation in C. glabrata. The method being developed can be broadly applied to other microbial pathogens to better identify how pathogens adapt and evolve in a more host-relevant environment, and enables the identification of potential therapeutic strategies against difficult-to- treat biofilms. This R16 will fund the research of approximately 20 undergraduates (UGs) over four years, including many minority trainees. UGs will perform the majority of the proposed work, with training and mentorship from a technician and the PI. This funding would allow the PI to continue to develop a strong track record in research, give meaningful research experiences to underrepresented minority students, and enhance the research capacity at San Jose State University.