Dissecting the regulation of ATP-dependent lipid transporters from the pathogenic fungus Cryptococcus neoformans at the single molecule level

• Applicant: Dr. Sarina Veit
• Subject Area: Biophysics ; Biochemistry
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 555663153

The fungal pathogen Cryptococcus neoformans causes more than 100,000 deaths per year in immunocompromised HIV patients and recently an increase in its fungicide resistance was observed. Due to the unique capsule, which is the primary virulence factor, secretory pathways play an essential role for the survival of this fungi in the host and thus its pathogenicity. Lipid transporters play a crucial role in these secretion mechanisms, vesicular trafficking and lipid homeostasis by driving lipid transport across membranes and would therefore be perfect candidates for new antifungal targets. Cryo-electron microscopy has recently resolved the structures of several ATP-dependent lipid transporters from other organisms, but functional characterisation remains a major challenge. Current concepts suggest that the activity of lipid transporters is regulated by lipid bilayer composition, membrane curvature, co-factors and membrane potential. However, direct experimental evidence for these concepts is lacking. On the one hand, this is due to the difficulties in handling integral membrane proteins, their delicacy in production, purification and characterisation of their assembly with membrane lipids. On the other hand, sophisticated techniques are required to study membrane protein activity at the molecular level. Our approach will tackle this problem from both sides by establishing defined reconstituted vesicle systems based on large unilamellar liposomes and state-of-the-art single-molecule techniques to study lipid transporters of the P4 subfamily of P-type ATPases at different levels of lipid compositional complexity (from simple to ‘cell-like’). Single vesicle fluorescence microscopy can extract useful information hidden in traditional ensemble-averaged biophysical or biochemical studies that are hampered by the sample heterogeneity. Vesicles are immobilized and imaged in a lipid transport assay setup in a high-throughput fashion, generating fluorescence intensity data for each vesicle individually. The extracted intensities can be used to compute vesicle size, lamellarity, tightness, protein distribution, protein orientation and due to the wide range of spatial and temporal resolution capabilities of the setup, single protein kinetics can be recorded. We expect that these lipid transport studies at the single vesicle level will reveal (i) the effect of the lipid composition and on lipid transporter activity, (ii) the underlying regulatory mechanism controlling lipid transport. Here, we are focussing especially on the regulation by kinases and by a proton gradient. In conclusion, the powerful combination of membrane protein biochemistry and imaging techniques proposed here should provide important new insights into the molecular characteristics and working mechanisms of lipid transporters, allowing the development of new treatment strategies for

• DFG Programme: Research Grants