Periodic Reporting for period 3 - FungalTolerance (Unraveling the complexity of fungal drug tolerance at multiple scales of biology)
Berichtszeitraum: 2024-03-01 bis 2025-08-31
Fungal pathogens kill up to 3.4 million people annually, largely due to infections caused by drug-susceptible fungi. Candida albicans, a fungus that grows within the gut and on the skin of most humans, is the most frequent cause of fungal infections in Western hospitals. The C. albicans strain in one person is very different from strains in other people, with respect to their genetics and to the ways that they respond to drug treatments. Antifungal drug tolerance is a common property of C. albicans strains, and it helps non-resistant isolates survive drug treatments. Tolerance is the ability of some single cells, within a single population of genetically identical cells, to grow slowly even while the drug inhibits other cells. Tolerance involves a range of different genes and pathways -- and we do not understand how to harness these differences in infections to treat them effectively. The goal of this FungalTolerance ERC SYN project is to understand the many different ways that different C. albicans isolates can, and do, tolerate antifungal drug treatments.
As fungi become more common in medical settings, and with only a limited arsenal of antifungal drugs available, there is a growing need for new, effective approaches to personalize antifungal therapies. Ultimately, to improve the clinical outcomes of patients who suffer from life-threatening systemic fungal infections, we aim to design appropriate treatments that specifically stop the type of tolerance used by each specific infecting fungal isolate.
To that end, we are working to reach a fundamental understanding of antifungal tolerance across the range of the scales of C. albicans biology. We are:1) capturing the diversity of tolerance across the species, using a huge library of C. albicans isolates, and characterizing it at the genomic and proteomic level; 2) identifying isolate-level metabolic pathways and molecular mechanisms that drive antifungal drug tolerance; and 3) probing processes and compounds that differ between genetically identical cells. By working out the mechanisms that drive tolerance and fungal single-cell diversity, we are working to identify drug targets that could reduce tolerance, providing a paradigm shift in anti-fungal treatment strategies.
As fungi become more common in medical settings, and with only a limited arsenal of antifungal drugs available, there is a growing need for new, effective approaches to personalize antifungal therapies. Ultimately, to improve the clinical outcomes of patients who suffer from life-threatening systemic fungal infections, we aim to design appropriate treatments that specifically stop the type of tolerance used by each specific infecting fungal isolate.
To that end, we are working to reach a fundamental understanding of antifungal tolerance across the range of the scales of C. albicans biology. We are:1) capturing the diversity of tolerance across the species, using a huge library of C. albicans isolates, and characterizing it at the genomic and proteomic level; 2) identifying isolate-level metabolic pathways and molecular mechanisms that drive antifungal drug tolerance; and 3) probing processes and compounds that differ between genetically identical cells. By working out the mechanisms that drive tolerance and fungal single-cell diversity, we are working to identify drug targets that could reduce tolerance, providing a paradigm shift in anti-fungal treatment strategies.
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.
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.
One focus of our work has been on ploidy (the number of chromosome sets per cell) and aneuploidy (the presence of unusual chromosome numbers per cell); both increased ploidy and aneuploidy have been found in C. albicans strains that acquired drug resistance and/or tolerance. In addition to a proof-of-principle study of over 1,000 diverse baker’s yeast isolates published last year in Nature, we found that ~ 20% of C. albicans strains are aneuploid. Thus, it appears that there is a similar level of aneuploidy in baker’s yeast and C. albicans isolates across the two species.
Using tools to analyze gene expression in individual cells, we analyzed differences in drug responses, protein levels, and metabolic interactions between genetically identical cells in a population. This work revealed that, when exposed to drug, C. albicans yields two clear subpopulations of cells with very different expression patterns.
Two-thirds of C. albicans genes have not been characterized at all. With the goal of understanding their functions, we generated proteomic data for 91 representative strains from our collection, across 11 physiologically relevant growth and stress conditions. This study will identify proteins whose expression levels are coordinated (co-vary) under specific conditions, to infer the functions of these C. albicans proteins and the genes that encode them.
We reached insights about general issues of: genome diversity, proteome diversity, prevalence of aneuploidy, segmental chromosome amplifications or losses, the degree to which in vitro assays of filamentous growth are associated with different infection niches, and about specific genes and/or pathways that are associated with growth under different types of stresses, including antifungal drug stresses. We expect that the computational analyses of these very large data sets will drive the discovery of many novel insights into the diversity of antifungal drug tolerance mechanisms within different isolates and how we can know which strains use which mechanisms. With that information in hand, we will be in a good position to improve infection treatment strategies by interfering with the specific tolerance mechanisms found in a specific subset of strains. Essentially, this will enable a ‘personalized medicine’ approach to deal with the specific C. albicans strains that drive a particular infection.
We also studied the role of several commonly prescribed drugs and their effect on C. albicans growth in vitro, and in the Galleria mellonella larval model. Much of this work is submitted or being prepared for publication. In addition, we have studied the effect of drug-drug interactions under the types of conditions found in different human infection niches (e.g. blood, oral, vaginal) and identified specific drivers of different responses in the different niches.
Using tools to analyze gene expression in individual cells, we analyzed differences in drug responses, protein levels, and metabolic interactions between genetically identical cells in a population. This work revealed that, when exposed to drug, C. albicans yields two clear subpopulations of cells with very different expression patterns.
Two-thirds of C. albicans genes have not been characterized at all. With the goal of understanding their functions, we generated proteomic data for 91 representative strains from our collection, across 11 physiologically relevant growth and stress conditions. This study will identify proteins whose expression levels are coordinated (co-vary) under specific conditions, to infer the functions of these C. albicans proteins and the genes that encode them.
We reached insights about general issues of: genome diversity, proteome diversity, prevalence of aneuploidy, segmental chromosome amplifications or losses, the degree to which in vitro assays of filamentous growth are associated with different infection niches, and about specific genes and/or pathways that are associated with growth under different types of stresses, including antifungal drug stresses. We expect that the computational analyses of these very large data sets will drive the discovery of many novel insights into the diversity of antifungal drug tolerance mechanisms within different isolates and how we can know which strains use which mechanisms. With that information in hand, we will be in a good position to improve infection treatment strategies by interfering with the specific tolerance mechanisms found in a specific subset of strains. Essentially, this will enable a ‘personalized medicine’ approach to deal with the specific C. albicans strains that drive a particular infection.
We also studied the role of several commonly prescribed drugs and their effect on C. albicans growth in vitro, and in the Galleria mellonella larval model. Much of this work is submitted or being prepared for publication. In addition, we have studied the effect of drug-drug interactions under the types of conditions found in different human infection niches (e.g. blood, oral, vaginal) and identified specific drivers of different responses in the different niches.