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Background: Microsporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence similarity. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes.

Results: In this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin named ANNOTEX (Annotation Extension for ChimeraX), allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted protein-coding DNA sequences, of which 89% were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes.

Conclusion: We provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.

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. 

Mechanosensitive ion channels play an essential role in reacting to environmental signals and sustaining cell integrity by facilitating ion flux across membranes. For obligate intracellular pathogens like microsporidia, adapting to changes in the host environment is crucial for survival and propagation. Despite representing a eukaryote of extreme genome reduction, microsporidia have expanded the gene family of mechanosensitive ion channels of small conductance (mscS) through repeated gene duplication and horizontal gene transfer. All microsporidian genomes characterized to date contain mscS genes of both eukaryotic and bacterial origin. Here, we investigated the cryo-electron microscopy structure of the bacterially derived mechanosensitive ion channel of small conductance 2 (MscS2) from Nematocida displodere, an intracellular pathogen of Caenorhabditis elegans. MscS2 is the most compact MscS-like channel known and assembles into a unique superstructure in vitro with six heptameric MscS2 channels. Individual MscS2 channels are oriented in a heterogeneous manner to one another, resembling an asymmetric, flexible six-way cross joint. Finally, we show that microsporidian MscS2 still forms a heptameric membrane channel, however the extreme compaction suggests a potential new function of this MscS-like protein.

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.

Malaria, caused by Plasmodium parasites, claims over 600,000 deaths annually. Parasite invasion of red blood cells (RBCs) involves protein secretion from specialised organelles— micronemes, rhoptries, and dense granules—to facilitate host cell entry and establish a protective parasitophorous vacuole (PV). Despite the critical role of rhoptry proteins in infection, many remain poorly characterised due to the absence of recognisable trafficking motifs and dispensability in vitro. Here, we leverage spatial proteomics from Plasmodium falciparum to identify two novel Plasmodium berghei ortholog proteins associated with the PV (MAP1, PBANKA_1425900 and RhoSH, PBANKA_1001500) both containing hydrolase domains. Ultra-expansion microscopy reveals their localisation to the rhoptries in late schizogony, while co-immunoprecipitation shows their interaction. In vivo studies demonstrate that these proteins help the parasite evade spleen-mediated clearance and contribute to disease progression. One protein, MAP1, mediates sequestration to adipose tissue, and conditional knockdown of its P. falciparum ortholog results in reduced CD36-mediated cytoadhesion, suggesting a mechanism for immune evasion and sustained infection. Our findings identify MAP1 and RhoSH as key mediators of Plasmodium virulence. Takingadvantage of an in vivo approach, this work provides valuable insights toward global malaria eradication efforts as it lays the groundwork for novel therapeutic strategies, positioning mainly MAP1 but also RhoSH as promising targets, including their use as antigens in recombinant vaccines, attenuated live vaccine candidates, or enzyme-inhibiting drugs.