Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY Fungal infections significantly impact human health, both in terms of mortality and treatment cost. While anti- fungal drugs have been the leading therapy for fungal infections, there is an increasing incidence of resistant fungal infections that are difficult to treat. An alternative approach to disrupting fungal cell wall synthesis with drugs is the active degradation of fungal cell wall polysaccharides. However, there is a substantial knowledge gap in regards to the requirements for effective fungal cell wall degradation. This shortfall prevents the development of new anti-fungal therapies that could be used alone or in combination with current drug treatments. The long-term goal of this project is to develop mechanistic understanding of polysaccharide deconstruction to produce medically relevant enzymes. The objective of this particular proposal is focused on identifying and characterizing the mechanisms for the degradation of fungal cell wall polysaccharides by the bacterium Cellvibrio japonicus. Our central hypothesis is that a coordinated suite of enzymes is required to effectively degrade the glucan and chitin components of fungal cell walls. We will test this hypothesis with three Specific Aims: (1) Multiomic analyses during degradation of fungal cell wall polysaccharides, (2) Functional analysis of genes that encode enzymes essential for fungal cell wall deconstruction, and (3) Quantitative enzymology of fungal cell wall degrading enzymes. For the first Aim, we will use established transcriptomic and proteomic methods to decipher the complex gene and protein expression patterns of C. japonicus when actively degrading the fungal cell walls of Aspergillus nidulans and Saccharomyces cerevisiae. Novel targets will be placed in a functional context by subsequent genetic analysis. The second Aim will determine the contribution of individual gene products for the deconstruction of fungal cell walls. We have established both transposon and high-throughput targeted mutational approaches to identify and analyze genes that are essential for polysaccharide degradation in C. japonicus. We will test the fitness of mutant strains lacking these genes with growth assays using insoluble fungal cell wall polysaccharides and intact fungal biomass. For the third Aim, we will purify and characterize enzymes capable of degrading fungal cell wall polysaccharides to determine their substrate specificity, kinetic parameters, and to assess enzyme synergy. The use of fungal biomass as a substrate will allow us to determine what enzyme combinations are maximally effective at deconstructing intact fungal cell walls. These approaches are innovative because we use a bacterium that has a robust polysaccharide degrading capability coupled with a novel screen that uses intact fungal biomass, which includes filamentous fungi and yeasts. This project is significant because it will characterize enzymes with medically-relevant properties, give mechanistic insight into the requirements for the effective disruption of fungal cell walls, and generate a powerful system for the discovery of enzymes that have anti-fungal potential.
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