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Slideshow

The Toxoplasma gondii Vacuolar H+-ATPase regulates intracellular pH and calcium and impacts the maturation of essential secretory proteins

A. J. Stasic
A. J. Stasic
Moreno Laboratory
UGA Microbiology
Room S175, Paul D. Coverdell Center
Type of Event:
Student Seminars

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.

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