A systems-level investigation of the vesicle formation machinery in Giardia intestinalis
Creators: Pipaliya, Shweta
Publisher: name: University of Alberta Library
Year: 2022

<p>The endomembrane system is one of the hallmark features of all eukaryotes that distinguishes them from prokaryotes. Compared to bacteria and archaea, eukaryotic cells consist of biochemically and functionally distinct membranous compartments such as the endoplasmic reticulum, the Golgi apparatus, endosomes, lysosomes, and the plasma membrane. Cargo is transported to and from these organelles in distinct vesicle carriers whose formation and fusion at donor and recipient organellar membranes is facilitated by vesicle formation and fusion trafficking proteins. Together, these compartments with this molecular machinery form a complex interconnected network termed the membrane trafficking system that mediates extracellular export, intracellular import, and cargo-sorting between the cell and its environment. Proper functioning of this system is fundamental to the survival of all eukaryotes, and breakdown results in disease states or the inability to inhabit niche environments. Eukaryotic pathogens heavily rely on their membrane trafficking system for host-pathogen interactions, to transition between different lifecycle stages, and secrete virulence factors for immune avoidance and establishing disease. Giardia intestinalis is one such enteric microbial parasite of humans and animals that relies on its cargo secretory and endocytic processes to cause diarrheal infection within the animal gut and for environmental survival and propagation. Microscopically, Giardia’s endomembrane organization is strikingly different from that present in model eukaryotes such as yeast, plants, animals, or even other eukaryotic parasites. It lacks conventional stacked Golgi and endo-lysosomal compartments, and instead, possesses numerous Giardia-specific organelles. These are the peripheral vacuoles, which are static-state vesicular compartments, a labyrinth tubulovesicular endoplasmic reticulum, and stage-specific encystation-specific vesicles. Giardia belongs to the sub-phylum Fornicata within the eukaryotic supergroup Metamonada. Fornicata is a lineage that consists of free-living heterotrophic flagellates such as Carpediemonas membranifera and Carpediemonas-like organisms, endobionts of ruminant animals such as the retortamonads, and the largely parasitic Diplomonadida, which comprises bi-nucleated parasites such as Giardia. The endomembrane organization in fornicates is variable in its complexity and one that is gradually reduced. Within the Giardia genus, different Giardia intestinalis assemblages have been proposed to be distinct species that cause disease in humans and animals. This thesis examined the molecular evolution of the membrane trafficking system, specifically the vesicle formation machinery, to understand how the minimal trafficking system in Giardia arose and what evolutionary processes were at interplay. It also investigated the differences in the vesicle formation machinery between the different Giardia intestinalis assemblages, especially those implicated in causing human infections, through a population-level survey. Finally, upon identifying critical components from several important vesicle formation protein complexes, their molecular functions were assessed to study their roles in this parasite’s divergent endomembrane system.   A systems approach using evolutionary bioinformatics (comparative genomics and phylogenetics), immunofluorescent microscopy, proteomics, and large-scale genome assembly determined how the vesicle formation machinery evolved in fornicates from an ancestral state, leading into parasitism and within the Giardia lineage, and what organelles they associate in the Giardia trophozoite cells. The culmination of work produced in this thesis determined that Giardia’s reduced trafficking system is a by-product of ancient losses, parasitism-associated streamlining, and Giardia-specific adaptations. A population-level survey of the vesicle formation proteins across isolates of the human-infecting Giardia intestinalis also revealed inter-assemblage differences in the molecular complement of these proteins. Finally, in vitro microscopy and proteomics investigations with key membrane trafficking proteins in the lab strain of Giardia intestinalis AWB (C6) revealed associations of these proteins primarily with the peripheral vacuoles, marking them as a singular yet a multi-dynamic destination for endo-lysosomal trafficking in this parasite. Unexpectedly, promiscuous roles of some machinery were also elucidated at the parasite mitosomes and the ER and have shed light on the plasticity of trafficking system proteins in eukaryotes in general. Overall, findings from this thesis unveiled new modes and tempo by which cargo transport processes evolved and take place in this parasite that is of substantial public health, clinical, and biomedical importance. </p>