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ONGOING PROJECTS IN THE DOCAMPO-MORENO LABORATORY

Study of the composition, function and biological distribution of acidocalcisomes
Polyphosphate metabolism
Calcium pools
Proteomic analysis of purified acidocalcisomes


Transmission Electron Microscopy (TEM) of Trypanosoma cruzi epimastigotes showing empty vacuoles in the left panel (conventional TEM) and electron dense black granules in the right panel (dircet TEM) that we demonstrated correspond to acidocalcisomes

toxotem
This is a picture of a Toxoplasma gondii tachyzoite obtained by transmission electron microscopy using an energy filter. The electron dense black granules are acidocalcisomes. The photo was taken by Kildare Miranda.

Acidocalcisomes are acidic organelles of high-density -both by weight and by electron microscopy- with a high concentration of phosphorus present as pyrophosphate (PPi) and polyphosphate (poly P) complexed with calcium and other elements. Acidocalcisomes are related to organelles previously named “volutin granules” or “metachromatic granules” or “polyphosphate bodies”, which were thought to function as storage granules. Our discovery that a membrane containing a number of pumps, exchangers and channels surrounds the acidocalcisome suggested a metabolic function. After their identification in trypanosomatids, acidocalcisomes were found in several microorganisms such as Apicomplexan parasites, as well as in the green alga Chlamydomonas reinhardtii and the slime mold Dictyostelium discoideum. Our recent identification of acidocalcisome-like organelles in bacteria (Agrobacterium tumefaciens, Rhodospirillum rubrum) and human platelets (dense granules) indicates that this class of organelles has been conserved during evolution from bacteria to man.
Trypanosomatids and Toxoplasma gondii are excellent models to study the acidocalcisome and our goal is to understand its structure and function. In recent years we have demonstrated that acidocalcisome enzymes have important roles in calcium and pH homeostasis and that the acidocalcisome is important for osmoregulation in trypanosomes. The discovery of novel enzymes in this organelle that are absent from mammalian cells led to the finding of compounds (bisphosphonates) that produced radical cures in animal models of diseases caused by several parasites. Further exploration of the structure and function of acidocalcisomes in these parasites could lead to the identification of novel targets for drug action.
We are currently investigating the composition of Trypanosoma brucei, T. cruzi and T. gondii acidocalcisomes by proteomic analysis, characterizing the proteins identified, and investigating their physiological roles in the parasites. Knock out or overexpression of the genes encoding these proteins could provide important insights on the functions of acidocalcisomes. By manipulating the expression of these genes and the cellular levels of their products, we can create phenotypes that will provide clues to the role of acidocalcisome components in the physiology and development of trypanosomatids and Apicomplexan parasites.
Acidocalcisomes of T. cruzi are involved, through their association with a contractile vacuole, in osmoregulation. T. cruzi possess a robust regulatory volume decrease mechanism that completely reverse cell swelling when submitted to hyposmotic stress. The efflux of amino acids and K+ release could account for only part of this volume reversal. Swelling of acidocalcisomes together with a microtubule and cyclic AMP-mediated translocation of an aquaporin to the contractile vacuole and the resulting water movement are responsible for the volume reversal not accounted for by efflux of osmolytes. Osmoregulation is essential for T. cruzi since the parasite is in contact with a variety of osmotic stresses during its life cycle. We are investigating the signaling pathways involved in sensing osmotic changes in T. cruzi, and studying the molecular mechanisms involved in fusion of acidocalcisomes to the contractile vacuole complex and transfer of aquaporin.
Since acidocalcisomes have been conserved during evolution we are also investigating whether they correspond to other acidic organelles containing calcium described in other organisms.

 

 

 

Study of the role of the inositol phosphate/diacylglycerol signaling pathway in parasitic protozoa


Trypanosoma cruzi epimastigotes overexpressing a T. cruzi phosphatidylinositol phospholipase C with a Green Fluorescent Protein tag (TcPI-PLC-GFP). The Fluorescence allows to observe that the protein localizes to the plasma membrane.

Trypanosoma cruzi trypomastigotes and amastigote forms. These are immunofluorescence pictures showing that the amastigote forms (round forms) express higher levels of TcPI-PLC.

 

 

The graph to the right shows the different types of Phospholipase Cs that have been described in the literature. The X and Y domains are present in all the types. TcPI-PLC does not have a PH domain as the zeta type and a has a myristoylation consensus sequence that it is only found in the T. cruzi, T. brucei, and Leishmania spp. enzymes.

Phosphoinositide-specific phospholipase C (PI-PLC) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG is the physiological activator of protein kinase C (PKC), and IP3 induces the release of Ca2+ from internal stores. Together, these second messengers cause an increase in phosphorylation of proteins, which results in cellular responses. This inositol phosphate/diacylglycerol pathway is known to regulate a large array of cellular processes in eukaryotic cells, including metabolism, secretion, contraction, neural activity, and proliferation. A novel PI-PLC has been described in T. cruzi. This enzyme possesses an N-myristoylation and palmitoylation consensus sequence that had not been described previously in any other PI-PLC from eukaryotic cells (see figure below). It has been confirmed experimentally that the enzyme is myristoylated and palmitoylated . Evidence for the involvement of this enzyme in differentiation of these parasites was based on the increase in its expression during the trypomastigote to amastigote differentiation, and increase in the IP3 levels during trypomastigote to amastigote transformation. Recently, we demonstrated that there is a correlation between the expression levels of the TcPI-PLC and the differentiation of trypomastigotes into amastigotes. The overexpression of TcPI-PLC in the plasma membrane stimulated differentiation and reduction in the TcPI-PLC expression inhibited the process.
In addition, preliminary evidence showed that the enzyme could be involved in shedding of Ssp-4, a GPI-anchored protein containing inositolphosphoceramide in its lipid anchor. This was suggested by the simultaneous localization of TcPI-PLC and Ssp-4 in the external surface of the cells, the ability of TcPI-PLC to hydrolyze inositolphosphoceramide in vitro, the shedding of Ssp-4 without its lipid anchor (as demonstrated by its cross-reactive determinant (CRD) reactivity), and the increase in cellular ceramide when maximal surface expression of TcPI-PLC takes place. Ceramide is also an important second messenger involved in cellular differentiation.
Based on all these findings our hypothesis is that TcPI-PLCs could be responsible for multiple functions as it travels to the outer surface of the cells: (1) hydrolysis of PIP2 and generation of IP3 in the parasites, this effect being important for their differentiation; (2) hydrolysis of the glycoinositolphospholipids of GPI-anchors of parasite glycoproteins, which results in shedding of proteins to the medium; and (3) hydrolysis of PIP2 from the host cells leading to changes in its cytoskeleton and generation of IP3 that could be involved in cell signaling in the host.
We are investigating the role of fatty acid modifications in TcPI-PLC localization and orientation, whether TcPI-PLC is involved in the stress response of the parasite, in the hydrolysis of parasite or mammalian phospholipids, or in cell differentiation and host-parasite interactions and the transport mechanism of TcPI-PLC to the plasma membrane.



The isoprenoid pathway as a target for anti-parasitic drugs

Isoprenoids are an extensive group of natural products with diverse structures consisting of various numbers of five carbon isopentenyl diphosphate (IPP) units. The major building reaction in the pathway is the sequential condensation of IPP with growing allylic polyisoprenoid diphosphates. The isoprenoid pathway has been studied in mammalian cells where the enzyme farnesyl diphosphate synthase (FPPS) plays a central role by producing farnesyl diphosphate (FPP), an important precursor of sterols, dolichols, ubiquinones, and prenylated proteins. FPP, a 15-C isoprenoid unit could further form GGPP, a 20-C isoprenoid unit by the enzyme geranylgeranyl diphosphate synthase (GGPPS). FPPS forms FPP by the sequential condensation of dimethylallyl diphosphate (DMAPP) with two molecules of IPP.

Little is known about the isoprenoid pathway in T. gondii. Taking into account the central role of FPPS, its understanding will reveal important information on this pathway in T. gondii. In addition, our preliminary results indicate that this enzyme is a valid target for drugs since bisphosphonates, which are specific FPPS inhibitors, inhibited parasite growth in vitro and in vivo. The gene for the T. gondii farnesyl diphosphate synthase (TgFFPS) was cloned, sequenced and the protein characterized. This gene was present as a single copy in the tachyzoite haploid genome, although by RT-PCR two transcripts were found and named TgFPPS and TgFPPSi. Both isoforms of TgFPPS were expressed in a baculovirus expression system and the corresponding proteins purified for their biochemical characterization. Only TgFPPS had enzymatic activity and, interestingly, this enzyme was able to form both products, FPP and GGPP. This unique characteristic was previously found only in enzymes of the Archaea. Another interesting finding was the mitochondrial localization of this enzyme, which agrees with the presence of a mitochondrial-targeting signal in the protein sequence. This is also a unique feature of this protein since other FPPS are normally found in the cytosol. It has been demonstrated that the FPPS is the target for bisphosphonates, FDA-approved pyrophosphate analogs currently used in the treatment of bone resorption disorders. These compounds inhibited the proliferation of T. gondii in vitro and in vivo. Our preliminary results show that there was a significant difference between the sensitivity of the TgFPPS enzyme to bisphosphonates when compared with the human counterpart. Our hypothesis is that the isoprenoid pathway constitutes a major novel target for the treatment of toxoplasmosis. Therefore we are investigating the TgFPPS by site-directed mutagenesis to understand its bifunctionality, performing structural studies to facilitate drug design, and investigating the importance of TgFPPS products in the parasite biology. In addition we are investigating the effect of pyrophosphate analogs in vitro against T. gondii, and performing structure-activity relationship studies to facilitate drug design.

The isoprenoid pathway has also been particularly useful for the identification of new targets against T. cruzi. Enzymes studied so far involved in the synthesis of sterols and farnesyl diphosphate, and in protein prenylation, have been reported to be excellent drug targets against these parasites. The farnesyl-diphosphate synthase (TcFPPS), for example, has been demonstrated to be the target of nitrogen-containing bisphosphonates that have activity in vitro and in vivo against T. cruzi. The gene encoding this enzyme has been cloned and sequenced and the protein expressed and biochemically chacterized. In addition, we have recently reported the crystal structure determination of TcFPPS at 2 A resolution. Recent results indicate the presence of another important prenyltransferase in T. cruzi, a solanesyl diphosphate synthase (TcSPPS), which is involved in the synthesis of ubiquinone, and is another potential target for chemotherapy. Therefore we are performing structural studies to investigate the importance of TcSPPS products in the parasite biology, and we are investigating the effect of pyrophosphate analogs against T. cruzi FPPS and SPPS, and in vitro against T. cruzi, and performing structure-activity relationship studies to facilitate drug design.

The isoprenoid pathway showing that in some organisms the isopentenyl diphosphate is produced through the mevalonate pathway and in other organisms is made through the DOXP pathway. Further down in the pathway IPP is used by the enzyme farnesyl diphosphate Synthase to form farnesyl diphosphate which can be converted into GGPP by the GGPPS. These metabolites are precursor for important molecules like ubiquinones, heme a, sterols, etc.

bps

Bisphosphonates are pyrophosphate analogues that target enzymes of the isoprenoid pathway. Derivatives are tested in the laboratory against Trypanosomes and also Toxoplasma gondii. Some compounds are highly active in killing parasites without affecting the host cells.

Ribbon representation of the Trypanosoma cruzi farnesyl diphosphate synthase (TcFPPS) dimer. A mesh representation of the envelope of the active site is shown in both monomers (yellow and blue). From Gabelli et al., Proteins 62, 80-88 (2006)