This report covers the first 3 years of the project. We collected over 1700 diverse C. albicans isolates, including clinical, human commensal, environmental, and animal sources. We established a database that stores all relevant information and data for each isolate, including the source (geographical, clinical niche, etc.), DNA sequence, and phenotypic and proteomic data in the presence and absence of drug. We developed and implemented efficient high-throughput DNA sequencing protocols and obtained whole genome sequences for all strains; we analyzed relationships between the strains and generated a family tree with 24 clades (branches). We have collected data for properties for each of the strains, including growth properties under different nutritional and stress conditions and cell structural details based on microscopic analysis of thousands of cells per strain. Additionally, we developed protocols and methods to generate peptide preparations and to collect proteomic data for the analysis of fungal proteomes from baker's yeast (the conventional model yeast) and Candida albicans. We also studied the role of cell-to-cell and colony-to-colony heterogeneity in the appearance of tolerance to azole drugs, both in C. albicans and in Baker’s yeast. Thus, the FungalTolerance project has collected genomic, proteomic, and phenotypic data from >1700 isolates. This vast data set is providing a comprehensive picture of the genetic and phenotypic diversity of the most prevalent human fungal pathogen, C. albicans. This is at least 10X more than has been published previously. We are currently writing a major paper describing the genomes and basic phenotypes.
We also optimized microscopy protocols for single-cell analysis using genetically encoded nanoparticles (GEMs), a method to detect cellular crowding and molecular diffusion, first in the lab strain and then in clinical and other isolates. In addition, we developed a method to measure metabolite exchange within isogenic cell populations. Using a pipeline that we established, we acquired and analyzed the rapid dynamics of GEMS to visualize the movement of multiple fluorescent proteins in the C. albicans cytoplasm. Importantly, the acquisition of fluconazole tolerance coincides with the emergence of aberrant cell morphologies and dramatically decreased cytoplasmic crowding/viscosity in virtually all cells. Similarly, mutations in the drug target significantly decreased cytoplasmic crowding/viscosity. Thus, this implies that antifungal drug tolerance is associated with cytoplasmic phase separation.
As part of our dissemination activities, we issued a series of high-profile press releases highlighting key research findings and developments supported by the project. These included articles on the potential use of blood markers in infectious disease, rapid protein fingerprinting technologies. Further releases addressed how microorganisms defend against free radicals, how cooperation among cells can enhance longevity, and the systematic mapping of previously unknown gene functions. Additional highlights covered microbial cooperation in fungal infections leading to drug tolerance and the mechanisms by which cells manage extra chromosomes. In addition, an article on Prof. Judith Berman’s work highlighted the importance of understanding fungal infections. A dedicated feature also celebrated Prof. Markus Ralser's election as an EMBO member. These press releases were widely distributed through the Host Institutions official channels, and the project received further media attention through radio and TV interviews and a the production of a generic promotional video on studying infectious disease. This broad outreach effort has significantly raised the public profile of the research and its societal relevance.
