Periodic Reporting for period 1 - MicroMISTRANS (Error-prone protein synthesis in fungal pathogens Microsporidia: its scope and potential therapeutic targeting)
Reporting period: 2020-03-01 to 2022-02-28
In this study, we explored animal parasites Microsporidia to understand one of the most fundamental problems in evolutionary biology – how parasitic lifestyles affect the activity and evolution of the molecular building blocks of a parasite cell. We believe this knowledge will help us create a new and revolutionary approach to eradicate currently incurable and deadly parasitic infections.
Specifically, because most branches of microbial parasites are very different from each other at the molecular level, we frequently need to develop distinct therapies against distinct groups of parasites. However, emerging evidence shows that nearly all parasites share many similarities in the way they evolve, suggesting a potential basis for broad-range therapies against microbial parasites.
In this study, we aimed to explore these common evolutionary tendencies in parasitic cells, studying protein synthesis machinery (also known as the ribosome) from microsporidian pathogen Encephalitozoon cuniculi. Using cryo-electron microscopy analysis, we described the molecular anatomy of microsporidian ribosomes, thereby answering the long-standing question in parasite biology: how do parasites retain the function of their molecular machines in the face of the extreme reduction of their molecular building blocks?
In fact, for nearly two decades, microbial parasites were known for their extreme reduction of their molecular building blocks. Particularly, proteins and RNA molecules in microbial parasites are reduced to up to 50% of their size in free-living microorganisms. The objects of our study—microsporidian ribosomes—were predicted to lack multiple ribosomal proteins and up to a third of rRNA nucleotides compared to free-living species. How these molecules are nonetheless functional in parasites remained largely a riddle.
Our study showed that parasites use many molecular tricks to retain the biological activity of their ribosomes, providing a better understanding of how parasites cope with a drastic reduction of their molecular building blocks. Collectively, our work helped to better understand the fundamental difference between parasites and their hosts (in terms of how each of these two groups of organisms is designed at the molecular level), providing new knowledge for combating microbial pathogens.
Specifically, because most branches of microbial parasites are very different from each other at the molecular level, we frequently need to develop distinct therapies against distinct groups of parasites. However, emerging evidence shows that nearly all parasites share many similarities in the way they evolve, suggesting a potential basis for broad-range therapies against microbial parasites.
In this study, we aimed to explore these common evolutionary tendencies in parasitic cells, studying protein synthesis machinery (also known as the ribosome) from microsporidian pathogen Encephalitozoon cuniculi. Using cryo-electron microscopy analysis, we described the molecular anatomy of microsporidian ribosomes, thereby answering the long-standing question in parasite biology: how do parasites retain the function of their molecular machines in the face of the extreme reduction of their molecular building blocks?
In fact, for nearly two decades, microbial parasites were known for their extreme reduction of their molecular building blocks. Particularly, proteins and RNA molecules in microbial parasites are reduced to up to 50% of their size in free-living microorganisms. The objects of our study—microsporidian ribosomes—were predicted to lack multiple ribosomal proteins and up to a third of rRNA nucleotides compared to free-living species. How these molecules are nonetheless functional in parasites remained largely a riddle.
Our study showed that parasites use many molecular tricks to retain the biological activity of their ribosomes, providing a better understanding of how parasites cope with a drastic reduction of their molecular building blocks. Collectively, our work helped to better understand the fundamental difference between parasites and their hosts (in terms of how each of these two groups of organisms is designed at the molecular level), providing new knowledge for combating microbial pathogens.
WP1:
To deliver this work package, I cooperated with Dr. Panek, who was another MSCA fellow in the Hirt laboratory hosting and supervising my research. Together, we produced microsporidian spores and purified ribosomes from the microsporidian parasite E. cuniculi. Then, I analyzed these ribosomes using cryo-electron microscopy and determined their structure. Then, in cooperation with the Prof. Trost’s laboratory, I attempted to perform the mass spectrometry analysis of E. cuniculi ribosomes and proteomes to assess mistranslation of microsporidian proteins but could not produce good enough data due to limited quantities and purify of the E. cuniculi ribosomes or proteins. Therefore, I decided to focus on cryo-EM studies, and completed this recently published work:
Deliverable 1:
https://www.nature.com/articles/s41467-022-28281-0
To make experimental data publicly available, I deposited the experimental data in the public repository of protein structures (PDB) and the public repository of electron microscopy data (EMDB):
Deliverable 2:
https://www.rcsb.org/structure/7QEP
To disseminate this study (and acknowledge the MSCA support), I spread the news about our publication on my twitter account and presented this work at two consecutive international conferences “Microsporidia fest 2020” and “Microsporidia fest 2021” (both held in Toronto, Canada) being one of only 13 selected people whose work was selected for talks.
Deliverable 3:
https://nature.altmetric.com/details/121910141
https://www.reinkelab.org/news/2020/9/21/microsporidiafest-2020
Because this study revealed several unexpected features of microsporidian protein synthesis machinery, I followed up on this study by purifying and analyzing ribosomes from another species of microsporidian parasites, T. hominis. This analysis of T. hominis ribosomes will be hopefully soon described as another successful collaboration with Dr. Panek and Prof. Hirt.
WP2:
In this work package, I tested whether unusual properties of the protein synthesis machinery of microsporidian parasites can be used to suppress microsporidian infection of animal cells. For this purpose, we exposed infected host cells (RK13 cells infected with E. cuniculi or T. hominis) to moderate heat shock. In cooperation with Dr. Panek, we produced preliminary data supporting the idea that transient exposure (2-4 hours long) to moderate heat (39-41°C) inhibits parasite growth, suggesting that moderate heat exposure may help prevent the spread of some microsporidian infections of mammalian cells, especially if microsporidian parasites originate from a host with lower body temperatures compared to humans. We are currently following up on this study in my recently established laboratory at Newcastle University (UK), where I continue my cooperation with Prof. Hirt.
To deliver this work package, I cooperated with Dr. Panek, who was another MSCA fellow in the Hirt laboratory hosting and supervising my research. Together, we produced microsporidian spores and purified ribosomes from the microsporidian parasite E. cuniculi. Then, I analyzed these ribosomes using cryo-electron microscopy and determined their structure. Then, in cooperation with the Prof. Trost’s laboratory, I attempted to perform the mass spectrometry analysis of E. cuniculi ribosomes and proteomes to assess mistranslation of microsporidian proteins but could not produce good enough data due to limited quantities and purify of the E. cuniculi ribosomes or proteins. Therefore, I decided to focus on cryo-EM studies, and completed this recently published work:
Deliverable 1:
https://www.nature.com/articles/s41467-022-28281-0
To make experimental data publicly available, I deposited the experimental data in the public repository of protein structures (PDB) and the public repository of electron microscopy data (EMDB):
Deliverable 2:
https://www.rcsb.org/structure/7QEP
To disseminate this study (and acknowledge the MSCA support), I spread the news about our publication on my twitter account and presented this work at two consecutive international conferences “Microsporidia fest 2020” and “Microsporidia fest 2021” (both held in Toronto, Canada) being one of only 13 selected people whose work was selected for talks.
Deliverable 3:
https://nature.altmetric.com/details/121910141
https://www.reinkelab.org/news/2020/9/21/microsporidiafest-2020
Because this study revealed several unexpected features of microsporidian protein synthesis machinery, I followed up on this study by purifying and analyzing ribosomes from another species of microsporidian parasites, T. hominis. This analysis of T. hominis ribosomes will be hopefully soon described as another successful collaboration with Dr. Panek and Prof. Hirt.
WP2:
In this work package, I tested whether unusual properties of the protein synthesis machinery of microsporidian parasites can be used to suppress microsporidian infection of animal cells. For this purpose, we exposed infected host cells (RK13 cells infected with E. cuniculi or T. hominis) to moderate heat shock. In cooperation with Dr. Panek, we produced preliminary data supporting the idea that transient exposure (2-4 hours long) to moderate heat (39-41°C) inhibits parasite growth, suggesting that moderate heat exposure may help prevent the spread of some microsporidian infections of mammalian cells, especially if microsporidian parasites originate from a host with lower body temperatures compared to humans. We are currently following up on this study in my recently established laboratory at Newcastle University (UK), where I continue my cooperation with Prof. Hirt.
In the future, we hope that our work will help create a fundamentally different, more efficient, and more general approach to combating microbial infections. Instead of seeking unique drug targets in parasite cells, we will attempt to learn how to target those defects in molecular architecture that are common for a very broad range of parasites. One type of these defects can be mutations that make parasitic proteins and nucleic acids more sensitive to heat shock. We are currently testing an approach in which parasitic infections can be alleviated by transient exposure of the infected animal cells to moderate heat shock. If successful, this can help improve our public health by creating robust and inexpensive strategies to treat microbial infections in humans and industrially valuable animals.