A zinc transporter and carbonic anhydrase related protein play important roles in the lytic cycle of toxoplasma gondii
Chasen, Nathan Michael
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The Apicomplexa phylum includes a large number of human pathogens for which we lack effective vaccines and treatments. The pathogenesis of T. gondii is linked to its lytic cycle, which starts when the tachyzoite (the fast growing, asexual stage of the parasite) invades a host cell and secretes a battery of distinct proteins from specialized organelles. This system is composed of multiple organelles: rhoptries, micronemes, and dense granules. These organelles release their contents when the parasite invades a host cell. Rhoptries are specialized secretory organelles that contain a vast number of proteins, many of which are secreted during invasion. These proteins have important roles not only during the initial interaction between parasite and host but also in the formation of the PV and later on in the modification of the host cell. The α-carbonic anhydrases (α-CAs) are zinc metalloenzymes found in a variety of organisms that catalyze the reversible hydration of CO2, which is important for a number of biological functions, including respiration, photosynthesis, renal tubular acidification, and bone resorption (10-12). In addition to these enzymatically active CAs, there are CA isoforms known as carbonic anhydrase-related proteins or CARPs, which share sequence and structural similarity to active CAs, but lack enzymatic activity. Their lack of activity is due to the absence of one or more histidine amino acids that are required in the active site for zinc ion (Zn2+) coordination. My dissertation investigates the role of an carbonic anhydrase related protein in the rhoptries by the generation of knockout mutants, phenotype assays, and microscopy methods. My findings reveal that the T. gondii carbonic anhydrase related protein (TgCARP) is glycophosphatidylinsolitol anchored and plays an important role in the biogenesis of the rhoptries. I demonstrate that mutants lacking TgCARP have atypical rhoptry morphology and reduced virulence both in-vitro and in-vivo. Zinc is an essential element in biological systems, that acts as a cofactor for a large number of enzymes and regulatory proteins. Zinc must be tightly regulated, as both deficiency and overabundance of intracellular free Zn2+ are harmful to cells. I investigate the function of the only annotated ZnT family zinc transporter (TgZnT) of T. gondii, and determine that is capable of rescuing zinc-tolerance when exogenously expressed in zinc-sensitive yeast mutants. I also demonstrate that knockdown of TgZnT in T. gondii causes defects in the lytic cycle, including significantly slower intracellular growth and invasion.