Research Interests

Calcium signaling

One of the interests of our laboratory is calcium signaling and its impact in the lytic cycle of T. gondii. Fluctuations of the cytosolic free calcium (Ca²⁺) concentration regulate a variety of cellular functions in all eukaryotes. Cytosolic Ca²⁺ is highly regulated by many molecules and mechanisms and alterations in these homeostatic mechanisms are the cause of many important diseases in humans, including heart failure or neurodegenerative diseases. Ca²⁺ regulation and homeostasis during the infection cycle of T. gondii has been observed by several laboratories. Ca²⁺ signaling is involved in the stimulation of critical and essential steps of the lytic cycle (invasion, replication and egress) (See Figure 1) of the parasite. However, there is only fragmented information about the mechanisms and molecules involved. We showed that influx of Ca²⁺ in T. gondii enhances virulence traits like attachment, and invasion of the host cell. Considering the strong link between the lytic cycle and the pathogenesis of T. gondii it is highly significant to discover the mechanism by which Ca²⁺ signaling is wired into key lytic cycle features. 

Our laboratory created a number of genetic tools (See Fig. 2 and 3) to study Ca²⁺ signaling in T. gondii and have in hand parasite clones expressing Ca²⁺ indicators in the mitochondria (Fig. 3), the apicoplast and the plasma membrane. We recently characterized two channels with divergent peculiarities that are important for Ca²⁺ signaling in T. gondii. We also discovered inhibitors of Ca²⁺ signaling and parasite growth, and we are presently characterizing new molecular players at the plasma membrane and the endoplasmic reticulum likely involved in the initial Ca²⁺ signals that lead to the stimulation of lytic cycle traits. We are presently collaborating with Ivana Kuo from Loyola Medical School on the electrophysiological characterization of plasma membrane Calcium Channels.

Targeting the mitochondrion of Toxoplasma gondii

The mitochondrion of T. gondii and of other apicomplexan parasites is critical for their survival and several major antiparasitic drugs, such as atovaquone and endochin-like quinolones, act through inhibition of the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase. The coenzyme Q or ubiquinone (UQ) molecule consists of a water soluble quinone head (p-hydroxybenzoic acid or PHBA) and a lipophilic isoprenoid tail that confines the UQ to membranes (Fig. 4). The isoprenoid unit derive from a common precursor, isopentenyl pyrophosphate (IPP), and its isomer, dimethylallyl pyrophosphate (DMAPP), which are synthesized in mammalian cells via the mevalonate pathway, but in T. gondii instead is synthesized via an apicoplast localized 1-deoxy-D-xylulose-5-phosphate (DOXP) pathway. IPP and DMAPP are condensed by the action of a unique farnesyl diphosphate synthase (TgFPPS) into farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) (Fig. 5).

We became interested in the synthesis of isoprenoid metabolites of Toxoplasma gondii when we first discovered the inhibitory effect of bisphosphonates (pyrophosphate analogues that inhibit the isoprenoid pathway) on the growth to these parasites. We characterized the central enzyme for the synthesis of longer units, the farnesyl diphosphate synthase (TgFPPS) and discovered that intracellular tachyzoites in addition to their own synthesis can import isoprenoid metabolites from the host. This interesting finding implied that intracellular parasites would be sensitive to inhibition of the mammalian isoprenoid biosynthesis pathway and tolerate bisphosphonate inhibitors of the T. gondii FPPS, such as zoledronate. More recently we became interested in the enzymes that synthesize longer-chain species which attach the UQ ring to the mitochondrial membrane. We found a bisphosphonate compound (BPH1218, Fig. 6) that specifically targets the first enzyme of the UQ synthesis pathway, and it is highly effective against T. gondii growth in vitro and in vivo (Fig. 6). We are currently investigating other enzymatic steps in the synthesis of UQ as alternate therapeutic strategies. We have developed a strategy for the discovery of potential specific UQ synthesis inhibitors. Some of these drugs are effective for the T. gondii chronic infection and we are presently testing novel mitochondrial inhibitors in an established chronic model for toxoplasmosis.

We are actively screening new compound libraries and collaborate with several chemistry laboratories that provide us with experimental compounds to test in our established in vitro and in vivo models.