Nanoscopic adventures : unraveling macromolecular complexes in infectious diseases via integrative structural biology

Abstract: This thesis focuses on understanding the underlying molecular mechanisms of infectious diseases, which claim nearly 9 million lives annually. The research centers on critical analysis of pathogen mechanisms and drug resistance. I have mainly focused on two clades of pathogens: Enterococcus faecalis and microsporidia. E. faecalis is a key nosocomial opportunistic pathogen, and microsporidia are a group of emerging fungal pathogens that considerably impact the environment and economy, causing, among other things, the decline of honeybee populations. In this thesis, I have combined biochemistry and cryo-electron microscopy to perform an in-depth molecular analysis of crucial protein complexes that drive the infectivity of these organisms. In E. faecalis, the primary drug efflux pump, EfrCD, is examined to gain insight into its role in antibiotic resistance. Microsporidia often have a drastically reduced genome and display altered macromolecular structures due to their parasitic lifestyle. The research aims to provide insights into the regulation of translational processes in microsporidia by comparing the dormant spore stage to the active intracellular stage and looking closely into the infection mechanism. In the publication “Deep mutational scan of a drug efflux pump reveals its structure– function landscape,” I determined the structure of EfrCD and several of its conformations to understand better how this protein complex contributes to E. faecalis' multidrug resistance. In further research, our focus moved to Microsporidia. During our work on the “Structure of the reduced microsporidian proteasome bound by PI31-like peptides in dormant spores” and “Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome,” we solved the structure of the endogenous microsporidian ribosome as well as multiple versions of the proteasome; the dormant form of 20S proteasome and the active form of the 20S and 26S proteasome. This gave a deeper understanding of how microsporidia could highly reduce those conserved macromolecular complexes. By discovering novel inhibitors, we were also able to understand how those energy- demanding molecular machines can efficiently regulate themselves. Furthermore, we investigated the specialized infection organ of microsporidia, known as the polar tube. As part of the paper titled "Ribosome clustering and surface layer reorganization in the microsporidian host-invasion apparatus," I contributed by performing proteomic analysis of the endogenously affinity-purified polar tubes using a native affinity tag I discovered. Additionally, I identified potential protein-protein interactions of the polar tube proteins. This complemented the work performed on the dynamics and ultrastructure remodeling of the polar tube during germination through light microscopy and cryo-electron tomography. We observed a cargo-filled state with organized arrays of ribosomes clustered along the thin tube wall and an empty post-translocation state with a thicker wall. The findings of this thesis work expand our understanding of pathogen biology and open up new possibilities for addressing drug development and drug resistance, a significant global health challenge.