Periodic Reporting for period 2 - FungalTolerance (Unraveling the complexity of fungal drug tolerance at multiple scales of biology)
Période du rapport: 2022-09-01 au 2024-02-29
Fungal pathogens kill ~1.6 million people annually despite testing as susceptible/sensitive to anti-fungal drugs that are used in the clinic. Candida albicans is a fungus that grows within the gut and on the skin of most humans; C. albicans is also the most prevalent fungal pathogen in western hospitals. C. albicans strains isolated from different people can be very different from one another, in their genetics and in the ways that they respond to drugs. One of the ways Candida albicans manages to survive drug treatments is by utilizing antifungal tolerance–the ability of some cells within a genetically identical population to grow slowly in the presence of drug while other cells do not grow. This ability can involve a range of different regulatory and metabolic routes and we do not understand how these routes affect our ability to treat fungal infections. The goal of the FungalTolerance ERC SYN project is to understand the many different routes that different isolates of C. albicans can, and do, use to tolerate antifungal drug treatments.
As fungi become more prevalent in medical settings, and with only a limited arsenal of anti-fungal drugs available, it is important to develop effective approaches to treating fungal infections and especially infections with the most prevalent fungal pathogens. Ultimately, we want to exploit our understanding of antifungal drug tolerance to design appropriate treatments that specifically stop the type of tolerance used by each specific infecting fungal isolate. We hope to greatly improve the clinical outcomes of patients who suffer from life-threatening systemic infections.
Our overall goal is to reach a fundamental understanding of antifungal tolerance across the range of its biological scales by: 1) capturing the diversity of tolerance, by creating a huge library of C. albicans isolates, and characterizing it at the genomic and proteomic level; 2) identifying metabolic pathways and molecular mechanisms that drive antifungal drug tolerance within isolates; and 3) probing processes and compounds that affect phenotypic heterogeneity between cells and suppress antifungal drug tolerance. By elucidating the mechanisms that drive tolerance and fungal single-cell diversity, we propose to render tolerance targetable, providing a paradigm shift in anti-fungal treatment strategies.
As fungi become more prevalent in medical settings, and with only a limited arsenal of anti-fungal drugs available, it is important to develop effective approaches to treating fungal infections and especially infections with the most prevalent fungal pathogens. Ultimately, we want to exploit our understanding of antifungal drug tolerance to design appropriate treatments that specifically stop the type of tolerance used by each specific infecting fungal isolate. We hope to greatly improve the clinical outcomes of patients who suffer from life-threatening systemic infections.
Our overall goal is to reach a fundamental understanding of antifungal tolerance across the range of its biological scales by: 1) capturing the diversity of tolerance, by creating a huge library of C. albicans isolates, and characterizing it at the genomic and proteomic level; 2) identifying metabolic pathways and molecular mechanisms that drive antifungal drug tolerance within isolates; and 3) probing processes and compounds that affect phenotypic heterogeneity between cells and suppress antifungal drug tolerance. By elucidating the mechanisms that drive tolerance and fungal single-cell diversity, we propose to render tolerance targetable, providing a paradigm shift in anti-fungal treatment strategies.
First, we collected a diverse set of C. albicans isolates, including clinical, human commensal, environmental, and animal sources. We originally proposed to assemble a collection of 1,000 isolates, but we have exceeded this initial plan with a total of over 2,000 isolates collected from all major infection niches. 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 efficient high throughput DNA sequencing protocols and obtained whole genome sequences for all 2,000 strains, which are being assembled using multiple de novo assembly approaches. We established high throughput protocols for assays measuring drug susceptibility and growth properties, in liquid and on agar medium, and measured growth in a range of antifungal drugs and other stresses for all 2,000 isolates.
Second, we studied the role of aneuploidy, which refers to an unusual number of chromosomes, in drug resistance and/or tolerance in C. albicans. We focused on aneuploidy because it is often associated with these traits. As a proof of principle project, we explored the role of aneuploidy in over 1,000 diverse budding yeast isolates; we are now extending the methods to C. albicans isolates. Additionally, we developed protocols and methods for generating peptide preparations and proteomic methods optimized for the analysis of fungal proteomes. We also studied the role of colony heterogeneity in the appearance of tolerance to azole l drugs, both in C. albicans and in budding yeast.
Third, we analyzed how genetically identical cells from a single isolate can differ in their drug responses, protein levels, and metabolic interactions. We developed tools for studying gene expression in individual cells, which revealed that when exposed to drug, C. albicans yields two clear subpopulations of cells with very different expression patterns. We also optimized microscopy protocols for single-cell analysis using 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 S. cerevisiae cells as a model.
Fourth,we established a pipeline for the acquisition and analyses of the rapid dynamics of genetically encoded nanoparticles (GEMs) in C. albicans, which enables visualization of multiple fluorescent proteins simultaneously. We foundn that, coincident with the acquisition of fluconazole tolerance and the appearance of aberrant cell morphologies, cytoplasmic crowding/viscosity dramatically decreases. At high fluconazole concentrations (10X MIC), the decrease in cytoplasmic crowding/viscosity is observed in virtually all cells. Similarly, altering the function of Erg11, the target of fluconazole, results in significantly decreased cytoplasmic crowding/viscosity, indicating that antifungal drug tolerance is associated with cytoplasmic phase separation.
We developed efficient high throughput DNA sequencing protocols and obtained whole genome sequences for all 2,000 strains, which are being assembled using multiple de novo assembly approaches. We established high throughput protocols for assays measuring drug susceptibility and growth properties, in liquid and on agar medium, and measured growth in a range of antifungal drugs and other stresses for all 2,000 isolates.
Second, we studied the role of aneuploidy, which refers to an unusual number of chromosomes, in drug resistance and/or tolerance in C. albicans. We focused on aneuploidy because it is often associated with these traits. As a proof of principle project, we explored the role of aneuploidy in over 1,000 diverse budding yeast isolates; we are now extending the methods to C. albicans isolates. Additionally, we developed protocols and methods for generating peptide preparations and proteomic methods optimized for the analysis of fungal proteomes. We also studied the role of colony heterogeneity in the appearance of tolerance to azole l drugs, both in C. albicans and in budding yeast.
Third, we analyzed how genetically identical cells from a single isolate can differ in their drug responses, protein levels, and metabolic interactions. We developed tools for studying gene expression in individual cells, which revealed that when exposed to drug, C. albicans yields two clear subpopulations of cells with very different expression patterns. We also optimized microscopy protocols for single-cell analysis using 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 S. cerevisiae cells as a model.
Fourth,we established a pipeline for the acquisition and analyses of the rapid dynamics of genetically encoded nanoparticles (GEMs) in C. albicans, which enables visualization of multiple fluorescent proteins simultaneously. We foundn that, coincident with the acquisition of fluconazole tolerance and the appearance of aberrant cell morphologies, cytoplasmic crowding/viscosity dramatically decreases. At high fluconazole concentrations (10X MIC), the decrease in cytoplasmic crowding/viscosity is observed in virtually all cells. Similarly, altering the function of Erg11, the target of fluconazole, results in significantly decreased cytoplasmic crowding/viscosity, indicating that antifungal drug tolerance is associated with cytoplasmic phase separation.
The project will certainly push the boundaries of genomics and proteomics of fungal pathogens, both because we will obtain a far more comprehensive picture of the genetic and phenotypic diversity of the most prevalent human fungal pathogen, C. albicans, but also because we will conduct, for the first time, a proteomic analysis of thousands of clinical isolates of any fungal pathogen. We expect that the computational analyses of the very large data sets generated will drive the discovery of many novel insights into the range of antifungal drug tolerance mechanisms and how we could interfere with them in order to improve infection treatment strategies.
Examples of these insights include:
1) Discovery of genes and proteins connected to different stress responses and to different isolate types.
2) Insights into the role of cell-cell heterogeneity in stress survival
3) Identification of lead compounds or repurposed drugs and drug combinations to combat antifungal drug tolerance.
Examples of these insights include:
1) Discovery of genes and proteins connected to different stress responses and to different isolate types.
2) Insights into the role of cell-cell heterogeneity in stress survival
3) Identification of lead compounds or repurposed drugs and drug combinations to combat antifungal drug tolerance.