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Pathogens have developed sophisticated strategies to thrive in hostile host environments. They use species-specific innovations and virulence factors to successfully invade and replicate in host organisms, including humans. Many pathogen infections cause disease and sometimes death, which affects not only the medical but also the agricultural and environmental sectors. 

A strategy to combat infectious disease is to better understand the pathogen’s biology at a molecular level. In particular, the identification, structural, and biochemical characterization of virulence factors that are essential for infection can provide a solid basis for antimicrobial drug development. 

This thesis focuses on two types of pathogens that have evolved ingenious mechanisms to adapt to their hosts: Microsporidia, which are fungal-like, obligate intracellular pathogens that streamlined their genomes for optimal parasitism, and Vibrio cholerae, a Gram-negative bacterium with numerous virulence genes, and the etiological agent of cholera disease. 

In the first project of my thesis, we developed a tool for the functional genome annotation of divergent organisms like microsporidia. To overcome the problematic annotation of divergent genomes, due to low sequence similarity, our tool complements traditional sequence-based annotation with structural similarity matching. We used this method to annotate the newly sequenced genome of Vairimorpha necatrix, a microsporidian parasite of Lepidoptera (e.g., butterflies and moths). The addition of structural similarity matching improved the quality and accuracy of the genome annotation. Further, the resulting annotation can serve as a reference to curate other microsporidian genomes. This will increase our understanding of the parasites’ proteomic repertoire and help us to identify potential virulence factors that can be studied experimentally. 

In the second study, we analyzed the cryogenic electron microscopy (cryo-EM) structure of the mechanosensitive ion channel of small conductance 2 (MscS2) from Nematocida displodere, a microsporidian pathogen of Caenorhabditis elegans. Microsporidia acquired mscS2 from bacteria via horizontal gene transfer and drastically shortened its sequence length, leading to the loss of the mechanosensation domain. In our in vitro setting, MscS2 oligomerizes into a unique superstructure where six heptameric MscS2 channels form an asymmetric, flexible six-way cross joint. We show that, despite its drastic reduction, MscS2 still forms a homo-heptameric membrane-associated channel. However, the loss of the sensory domain indicates that microsporidia evolved MscS2 to fulfill a new function. 

In the third project, we solved the cryo-EM structure of the V. cholerae toxin motility-associated killing factor A (MakA). MakA is part of the ɑ-pore-forming toxin (ɑ-PFT) MakBAE, which inserts into host cell membranes and is cytotoxic. However, in the absence of MakB and MakE, at low pH, and in the presence of membrane vesicles, MakA by itself changes its conformation and oligomerizes into a helical structure. This helix comprises MakA dimer pairs that spiral around a central, circular cavity. Near the cavity and the MakA transmembrane helices, we observed an annular lipid bilayer. This suggests that MakA depletes membranous vesicles of their lipids. While we believe this helical assembly is a non- physiological artifact, our analyses demonstrate that MakA changes its conformation and inserts itself into cell membranes, where it oligomerizes, ultimately leading to cell death in vitro. Further, we observed four different conformations of MakA within the dimer pairs, which displays the protein’s dynamic behavior. We assume that MakA adopts one of these conformations when forming an ɑ-PFT with MakB and MakE. 

The findings of my thesis contribute to the identification and understanding of protein families and virulence factors that microsporidia and Vibrio cholera employ to conquer and exploit their hosts. In conclusion, the thesis provides key pieces of knowledge that can help the future development of inhibitors against these pathogens.