| 1. |
- Ehrenbolger, Kai, et al.
(författare)
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Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome
- 2020
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Ingår i: PLoS biology. - : Public Library of Science. - 1544-9173 .- 1545-7885. ; 18:10
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Tidskriftsartikel (refereegranskat)abstract
- Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.
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| 2. |
- Ehrenbolger, Kai Patric, 1991-
(författare)
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Nanoscopic adventures : unraveling macromolecular complexes in infectious diseases via integrative structural biology
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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.
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| 3. |
- Jespersen, Nathan, et al.
(författare)
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Structure of the reduced microsporidian proteasome bound by PI31-like peptides in dormant spores
- 2022
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Proteasomes play an essential role in the life cycle of intracellular pathogens with extracellular stages by ensuring proteostasis in environments with limited resources. In microsporidia, divergent parasites with extraordinarily streamlined genomes, the proteasome complexity and structure are unknown, which limits our understanding of how these unique pathogens adapt and compact essential eukaryotic complexes. We present cryo-electron microscopy structures of the microsporidian 20S and 26S proteasome isolated from dormant or germinated Vairimorpha necatrix spores. The discovery of PI31-like peptides, known to inhibit proteasome activity, bound simultaneously to all six active sites within the central cavity of the dormant spore proteasome, suggests reduced activity in the environmental stage. In contrast, the absence of the PI31-like peptides and the existence of 26S particles post-germination in the presence of ATP indicates that proteasomes are reactivated in nutrient-rich conditions. Structural and phylogenetic analyses reveal that microsporidian proteasomes have undergone extensive reductive evolution, lost at least two regulatory proteins, and compacted nearly every subunit. The highly derived structure of the microsporidian proteasome, and the minimized version of PI31 presented here, reinforce the feasibility of the development of specific inhibitors and provide insight into the unique evolution and biology of these medically and economically important pathogens.
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| 4. |
- Meier, Gianmarco, et al.
(författare)
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Deep mutational scan of a drug efflux pump reveals its structure–function landscape
- 2023
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Ingår i: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 19, s. 440-450
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Tidskriftsartikel (refereegranskat)abstract
- Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational scanning (DMS) on the bacterial ABC transporter EfrCD to determine the drug efflux activity profile of more than 1,430 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity toward positively charged ethidium, whereas additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryogenic electron microscopy structure of EfrCD uncovered a high-affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert EfrCD into a drug-specific ABC importer. [Figure not available: see fulltext.]
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| 5. |
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| 6. |
- Sharma, Himanshu, et al.
(författare)
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Ultrastructural insights into the microsporidian infection apparatus reveal the kinetics and morphological transitions of polar tube and cargo during host cell invasion
- 2024
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 22:2
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Tidskriftsartikel (refereegranskat)abstract
- During host cell invasion, microsporidian spores translocate: their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the Vairimorpha necatrix polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and uncovers new aspects of ribosome regulation and transport.
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