Fluctuations of the cytosolic free calcium (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cytosolic Ca2+ is highly regulated by a large number of molecules and mechanisms and alterations in these homeostatic mechanisms are the cause of many important diseases in humans, including heart failure or neurodegenerative diseases. In Toxoplasma Ca2+ signaling is involved in the stimulation of critical and essential steps of the lytic cycle (invasion, replication and egress) of the parasite. However, there is only fragmented information about the mechanisms and molecules involved. We study Ca2+ influx in T. gondii and its role in stimulating virulence traits like attachment, and invasion of the host cell. There is a strong link between the lytic cycle and the pathogenesis of T. gondii and Ca2+ signaling is wired into key lytic cycle features.
Our laboratory created a number of genetic tools (See Fig. 1 and 2) to study calcium signaling in Toxoplasma and we have in hand parasite clones expressing calcium indicators in the mitochondria, the apicoplast and the plasma membrane. We recently discovered a number of inhibitors of calcium signaling and parasite growth, and we are presently using forward genetics approaches to discover the molecules involved. This is a new approach in our laboratory and we are very excited about the possibility of discovering new signaling players that could fill the information gap about the signaling toolkit in Toxoplasma, and also provide information about the evolution of complex signaling pathways in other cells.
THE PLANT-LIKE VACUOLE AND ITS ROLE IN THE MATURATION OF SECRETORY PROTEINS
Our laboratory reported the identification of an organelle within the endo-exocytic system of T. gondii that we named the plant-like vacuole (PLV). This vacuole is a large multi-vesicular structure that possesses similarities to the central vacuole found in plant cells. This organelle labels with antibodies against proteins with great similarity to vacuolar plant pumps and channels, such as a K+-sensitive V-H+-PPase (TgVP1), and an aquaporin or water channel (TgAQP1). Physiological evidence revealed further
similarities to plant vacuoles such as the presence of a V-H+-ATPase, Na+/H+ and Ca2+/H+ exchangers, and the storage of calcium. As in plant vacuoles, the PLV also contains proteases such a cathepsin L (TgCPL). The PLV is specially prominent in extracellular tachyzoites and with multiple potential functions, such as a role in resistance to environmental stresses. Recent work on the characterization of the Vacuolar-H+-ATPase, the enzyme responsible for the acidification of the PLV, revealed new roles for the PLV. Secretion of both rhoptries and micronemes are affected when the V-H+-ATPase is downregulated. Secretion of micronemes, rhoptries and dense granule are essential for the host cell invasion and underscores the importance of protein trafficking in the lytic cycle. Our work showed an essential role of the PLV for the virulence of the parasite. We are presently searching for potential specific inhibitors of the V-H+-ATPase that do not inhibit the mammalian enzyme.
STUDIES ON THE ISOPRENOID PATHWAY
Our laboratory is interested in studying metabolic pathways in Toxoplasma that could be exploited as targets for chemotherapy. We think that our findings will also be applicable for other apicomplexan parasites of medical importance like Plasmodium and Cryptosporidium. Management of most parasitic diseases rests heavily on chemotherapy and for most of them available treatments face multiple challenges. Many drugs in use are only active during one phase of the disease, many show harmful side effects after continuous use, and multiple drug resistant strains have emerged naturally. A constant source of new drugs and potential drug targets is needed to stay ahead of the threat posed by these pathogens.
Our interest in studying the Toxoplasma gondii isoprenoid pathway was triggered when we first discovered the inhibitory effect of bisphosphonates (pyrophosphate analogues) on the growth to these parasites. At the time we thought that the target was a different pyrophosphate-utilizing enzyme but later work quite conclusively showed that these compounds were inhibiting the isoprenoid pathway. We cloned, expressed and characterized the central enzyme of this pathway; the farnesyl diphosphate synthase (TgFPPS) and most recently we were able to isolate knockout clones for this enzyme. Our work with these mutants showed that intracellular parasites are able to import isoprenoid metabolites in addition to synthesizing their own. This was interesting and was the basis for testing inhibitors of both parasite and host isoprenoid pathways and establishing their synergistic interaction. These findings about the interaction of these compounds is the product of several years of work on the characterization of this pathway and we certainly believe that it will introduce a novel chemotherapeutic strategy (Fig. 4). Currently our laboratory is testing combinations of bisphosphonate derivatives with statins against T. gondii-infected mice in the acute and chronic model of toxoplasmosis.