TOXOPLASMA GONDII NUTRIENT ACQUISITION AND UTILIZATION

Toxoplasma gondii is a widely pervasive parasite that infects 1/3 of the world’s population. It is capable of invading any mammalian cell it encounters and can exist in a dormant state for the entirety of the host’s life, waiting for the immune landscape to become favorable to activate into a destructive stage of its life cycle, capable of lysing precious neurons, cardiac tissue, skeletal muscle, or cells of the eye and more. Although this danger exists, very little is known about how this parasite exploits its host to survive for decades undetected, and therefore our inquiry aims to understand the nutrients Toxoplasma needs for its survival, how it acquires them, and what is the full scope of its utilization of them. We do this first by focusing on nutrients essential for Toxoplasma gondii’s growth, for example cholesterol and phosphate. We follow these essential nutrients from the extra-parasitic environment and into the parasite to gain valuable insight into its utilization in hopes of uncovering essential pathways that could potentially become drug targets for intervention. We show how cholesterol is scavenged through the selective hijacking of the host’s endocytic pathway by recruitment of subpopulations of Rab11 vesicles which are characterized by different effectors. To elucidate the full scope of utilization of scavenged cholesterol, we study two proteins implicated in steroidogenesis that have homology with proteins that can interact with cholesterol or other cholesterol-derived byproducts. We also show how phosphate is acquired via a single phosphate transporter that serves multiple roles, bulk phosphate acquisition and mobilization of internal phosphate stores for osmoregulation. We create methodologies and tools for the study of this stage in physiologically relevant and a hugely understudied setting of infection, the heart. Lastly, we aim to understand chronic infection more fully by creating in-vivo models capable of measuring cardiomyopathies that exist due to chronic infection. Untangling the complex web of nutrient acquisition and metabolism, specifically during encystation, can equip us with an understanding of pathways yet discovered, which could hopefully lead to advancements in drug discovery that have thus far been stagnant for several decades

A glycosylphosphatidylinositol-anchored carbonic anhydrase-related protein of Toxoplasma gondii is important for rhoptry biogenesis and virulence

Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries

A plastid two-pore channel essential for inter-organelle communication and growth of Toxoplasma gondii.

Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca2+-dependent events. Little is known about TPCs in unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca2+ indicator targeted to the apicoplast, we show that Ca2+ signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca2+ uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge

Acidocalcisome-Mitochondrion Membrane Contact Sites in Trypanosoma brucei

Membrane contact sites are regions of close apposition between two organelles, typically less than 30 nanometers apart, that facilitate transfer of biomolecules. The presence of contact sites has been demonstrated in yeast, plants, and mammalian cells. Here, we investigated the presence of such contact sites in Trypanosoma brucei. In mammalian cells, endoplasmic reticulum-mitochondria contact sites facilitate mitochondrial uptake of Ca2+ released by the ER-located inositol 1,4,5-trisphosphate receptor (InsP3R). However, the InsP3R in trypanosomes localizes to acidocalcisomes, which serve as major Ca2+ stores in these parasites. In this work, we have used super-resolution structured illumination microscopy and electron microscopy to identify membrane contact sites that exist between acidocalcisomes and mitochondria. Furthermore, we have confirmed the close association of these organelles using proximity ligation assays. Characterization of these contact sites may be a necessary starting point towards unraveling the role of Ca2+ in regulating trypanosome bioenergetics

A single Na+-Pi cotransporter in Toxoplasma plays key roles in phosphate import and control of parasite osmoregulation.

Inorganic ions such as phosphate, are essential nutrients required for a broad spectrum of cellular functions and regulation. During infection, pathogens must obtain inorganic phosphate (Pi) from the host. Despite the essentiality of phosphate for all forms of life, how the intracellular parasite Toxoplasma gondii acquires Pi from the host cell is still unknown. In this study, we demonstrated that Toxoplasma actively internalizes exogenous Pi by exploiting a gradient of Na+ ions to drive Pi uptake across the plasma membrane. The Na+-dependent phosphate transport mechanism is electrogenic and functionally coupled to a cipargarmin sensitive Na+-H+-ATPase. Toxoplasma expresses one transmembrane Pi transporter harboring PHO4 binding domains that typify the PiT Family. This transporter named TgPiT, localizes to the plasma membrane, the inward buds of the endosomal organelles termed VAC, and many cytoplasmic vesicles. Upon Pi limitation in the medium, TgPiT is more abundant at the plasma membrane. We genetically ablated the PiT gene, and ΔTgPiT parasites are impaired in importing Pi and synthesizing polyphosphates. Interestingly, ΔTgPiT parasites accumulate 4-times more acidocalcisomes, storage organelles for phosphate molecules, as compared to parental parasites. In addition, these mutants have a reduced cell volume, enlarged VAC organelles, defects in calcium storage and a slightly alkaline pH. Overall, these mutants exhibit severe growth defects and have reduced acute virulence in mice. In survival mode, ΔTgPiT parasites upregulate several genes, including those encoding enzymes that cleave or transfer phosphate groups from phosphometabolites, transporters and ions exchangers localized to VAC or acidocalcisomes. Taken together, these findings point to a critical role of TgPiT for Pi supply for Toxoplasma and also for protection against osmotic stresses

The Toxoplasma Vacuolar H+-ATPase Regulates Intracellular pH and Impacts the Maturation of Essential Secretory Proteins

Summary: Vacuolar-proton ATPases (V-ATPases) are conserved complexes that couple the hydrolysis of ATP to the pumping of protons across membranes. V-ATPases are known to play diverse roles in cellular physiology. We studied the Toxoplasma gondii V-ATPase complex and discovered a dual role of the pump in protecting parasites against ionic stress and in the maturation of secretory proteins in endosomal-like compartments. Toxoplasma V-ATPase subunits localize to the plasma membrane and to acidic vesicles, and characterization of conditional mutants of the a1 subunit highlighted the functionality of the complex at both locations. Microneme and rhoptry proteins are required for invasion and modulation of host cells, and they traffic via endosome-like compartments in which proteolytic maturation occurs. We show that the V-ATPase supports the maturation of rhoptry and microneme proteins, and their maturases, during their traffic to their corresponding organelles. This work underscores a role for V-ATPases in regulating virulence pathways. : Stasic et al. characterize the function of the vacuolar proton ATPase in the life cycle of Toxoplasma gondii, a widespread parasite that infects almost one-third of the world’s population. The work presents molecular evidence of the pump’s role in the synthesis of virulence factors of a highly successful pathogen. Keywords: toxoplasma, vacuolar-H+-ATPase, intracellular pH, proton transport, plant-like vacuole, VAC, rhoptries, micronemes, lytic cycle, lysosom