ABSTRACT

Toxoplasma gondii is an Apicomplexan obligate intracellular parasite that infects as much as one-third of the world’s population.  The pathogenesis of Toxoplasma is linked to its lytic cycle which involves egress from a host cell, motility, attachment/invasion, and replication.  While the parasite is preparing for egress, a large lysosomal vacuole forms, which is termed the plant-like vacuole (PLV) and that helps the parasite to resist the changing ionic conditions.    In the PLV there is a vacuolar-H⁺-ATPase (V-ATPase), an evolutionarily conserved multi-subunit complex that couples the hydrolysis of ATP to the pumping of protons across membranes. V-ATPases play diverse roles in eukaryotic cellular physiology including the acidification of intracellular compartments, vesicular trafficking, and creation of a proton gradient that can be used for the exchange of other ions.  To study the T. gondii V-ATPase, I created a conditional mutant of the a1 subunit (iDvha1-HA).  Depletion of the V-ATPase resulted significant defects to all major steps of the lytic cycle, indicating its importance to the parasite. In T. gondii, secretory organelles termed micronemes and rhoptries are required for invasion and modulation of the infected host cell. The maturation of proteins destined to these organelles were decreased and often mislocalized in V-ATPase mutant parasites.  Additionally, proteases responsible for the maturation of microneme and rhoptry proteins were defective in their own maturation.  Further characterization of the iDvha1-HA mutants revealed that the defects in the lytic cycle were in part due to defects in calcium signaling and homeostasis.  It was further demonstrated that the proton gradient derived from the V-ATPase is responsible for calcium entry into the parasite and storage in the PLV and acidocalcisomes, organelles important for acidic calcium and polyphosphate storage.   I demonstrate that loss of the V-ATPase results in defects in calcium uptake to the PLV and acidocalcisome.  Loss of the proton gradient generated by the V-ATPase resulted in defects in synthesis and storage of polyphosphate in the acidocalcisome.  This work underscores a novel role for V-ATPases in regulating the maturation of important proteins involved in virulence pathways, vesicular trafficking, and calcium and polyphosphate storage.

Development of a Dual In vitro/In vivo RNA Synthesis System Using RNA Polymerase Holoenzyme and Alternative Sigma Factors

The roseobacter group comprise up to 25 % of the total bacterial community in the surface waters of the ocean, and its members possess a great deal of physiological and genetic diversity. Taxonomic assignments within the roseobacter group have been primarily based on 16S rRNA gene sequences. However, recent studies have demonstrated that this gene lacks the resolution for accurately assigning organisms within the roseobacter group. To address this problem, whole-genome sequence data was used to construct a taxonomy that accurately depicts evolutionary relationships. The result of these analyses was the taxonomic reassignment of 34 species and the proposal of six novel genera.

Dimethylsulfoniopropionate (DMSP) is abundant in marine surface waters and can reach micromolar concentrations, and Ruegeria pomeroyi, a member of the roseobacter group, is capable of metabolizing it. Previous studies have shown that the methyl carbon and sulfur of DMSP are incorporated into methionine, and this led to the hypothesis that the direct capture of methanethiol was the major pathway for methionine biosynthesis from DMSP. To test this hypothesis, a highly efficient method for synthesizing di(methyl-13C)sulfonio-34S-propionate ([13C][34S]DMSP) was developed. The [13C][34S]DMSP was subsequently fed to R. pomeroyi in chemostat and the resulting isotopic labeling of methionine was examined. These experiments indicated that only one-third of methionine was synthesized via the direct capture of methanethiol while the remainder was synthesized by the random reassembly of the sulfur and methyl atoms. The findings also indicated that DMSP was the major source of sulfur even when present at concentrations <1 μM.