Target acquired: transcriptional regulators as drug targets for protozoan parasites.

Protozoan parasites are single-celled eukaryotic organisms that cause significant human disease and pose a substantial health and socioeconomic burden worldwide. They are responsible for at least 1 million deaths annually. The treatment of such diseases is hindered by the ability of parasites to form latent cysts, develop drug resistance, or be transmitted by insect vectors. Additionally, these pathogens have developed complex mechanisms to alter host gene expression. The prevalence of these diseases is predicted to increase as climate change leads to the augmentation of ambient temperatures, insect ranges, and warm water reservoirs. Therefore, the discovery of novel treatments is necessary. Transcription factors lie at the junction of multiple signalling pathways in eukaryotes and aberrant transcription factor function contributes to the progression of numerous human diseases including cancer, diabetes, inflammatory disorders and cardiovascular disease. Transcription factors were previously thought to be undruggable. However, due to recent advances, transcription factors now represent appealing drug targets. It is conceivable that transcription factors, and the pathways they regulate, may also serve as targets for anti-parasitic drug design. Here, we review transcription factors and transcriptional modulators of protozoan parasites, and discuss how they may be useful in drug discovery. We also provide information on transcription factors that play a role in stage conversion of parasites, TATA box-binding proteins, and transcription factors and cofactors that participate with RNA polymerases I, II and III. We also highlight a significant gap in knowledge in that the transcription factors of some of parasites have been under-investigated. Understanding parasite transcriptional pathways and how parasites alter host gene expression will be essential in discovering innovative drug targets.

Overexpression of a mutant form of EhRabA, a unique Rab GTPase of Entamoeba histolytica, alters endoplasmic reticulum morphology and localization of the Gal/GalNAc adherence lectin.

Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery and liver abscess. Vesicle trafficking events, such as phagocytosis and delivery of plasma membrane proteins, have been implicated in pathogenicity. Rab GTPases are proteins whose primary function is to regulate vesicle trafficking; therefore, understanding the function of Rabs in this organism may provide insight into virulence. E. histolytica possesses a number of unique Rabs that exhibit limited homology to host Rabs. In this study we examined the function of one such Rab, EhRabA, by characterizing a mutant overexpressing a constitutively GTP-bound version of the protein. Overexpression of mutant EhRabA resulted in decreased adhesion to and phagocytosis of human red blood cells and in the appearance of large tubular organelles that could be stained with endoplasmic reticulum (ER)-specific but not Golgi complex-specific antibodies. Consistent with the adhesion defect, two subunits of a cell surface adhesin, the galactose/N-acetylgalactosamine lectin, were mislocalized to the novel organelle. A cysteine protease, EhCP2, was also localized to the ER-like compartment in the mutant; however, the localization of two additional cell surface proteins, Igl and SREHP, remained unchanged in the mutant. The phenotype of the mutant could be recapitulated by treatment with brefeldin A, a cellular toxin that disrupts ER-to-Golgi apparatus vesicle traffic. This suggests that EhRabA influences vesicle trafficking pathways that are also sensitive to brefeldin A. Together, the data indicate that EhRabA directly or indirectly influences the morphology of secretory organelles and regulates trafficking of a subset of secretory proteins in E. histolytica.

Entamoeba histolytica: lipid rafts are involved in adhesion of trophozoites to host extracellular matrix components.

Adhesion is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Lipid rafts, cholesterol-rich domains, function in compartmentalization of cellular processes. In E. histolytica, rafts participate in parasite-host cell interactions; however, their role in parasite-host extracellular matrix (ECM) interactions has not been explored. Disruption of rafts with a cholesterol extracting agent, methyl-beta-cyclodextrin (MbetaCD), resulted in inhibition of adhesion to collagen, and to a lesser extent, to fibronectin. Replenishment of cholesterol in MbetaCD-treated cells, using a lipoprotein-cholesterol concentrate, restored adhesion to collagen. Confocal microscopy revealed enrichment of rafts at parasite-ECM interfaces. A raft-resident adhesin, the galactose/N-acetylgalactosamine-inhibitable lectin, mediates interaction to host cells by binding to galactose or N-acetylgalactosamine moieties on host glycoproteins. In this study, galactose inhibited adhesion to collagen, but not to fibronectin. Together these data suggest that rafts participate in E. histolytica-ECM interactions and that raft-associated Gal/GalNAc lectin may serve as a collagen receptor.

Entamoeba histolytica: comparison of the role of receptors and filamentous actin among various endocytic processes.

Entamoeba histolytica is the causative agent of amoebic dysentery. Uptake of iron is critical for E. histolytica growth and iron-bound human transferrin (holo-transferrin) has been shown to serve as an iron source in vitro. Although a transferrin-binding protein has been identified in E. histolytica, the mechanism by which this iron source is taken up by this pathogen is not well understood. To gain insight into this process, the uptake of fluorescent-dextran, -holo-transferrin, and human red blood cells (hRBCs) was compared. Both dextran and transferrin were taken up in an apparent receptor-independent fashion as compared to hRBCs, which were taken up in a receptor-mediated fashion. Interestingly, the uptake of FITC-dextran and FITC-holo-transferrin differentially relied on an intact actin cytoskeleton suggesting that their internalization routes may be regulated independently.

Entamoeba histolytica: FYVE-finger domains, phosphatidylinositol 3-phosphate biosensors, associate with phagosomes but not fluid filled endosomes.

Endocytosis is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Although a number of E. histolytica proteins that regulate this process have been identified, less is known about the role of lipids. In other systems, phosphatidylinositol 3-phosphate (PI3P), a product of phosphatidylinositol 3-kinase (PI 3-kinase), has been shown to be required for endocytosis. FYVE-finger domains are protein motifs that bind specifically to PI3P. Using a PI3P biosensor consisting of glutathione-S-transferase (GST) fused to two tandem FYVE-finger domains, we have localized PI3P to phagosomes but not fluid-phase pinosomes in E. histolytica, suggesting a role for PI3P in phagocytosis. Treatment of cells with PI 3-kinase inhibitors impaired GST-2 x FYVE-phagosome association supporting the authenticity of the biosensor staining. However, treatment with PI 3-kinase inhibitors did not inhibit E. histolytica-particle interaction, indicating that PI3P is not required for the initial step, but is required for subsequent steps of phagocytosis.

The WASp-like protein scar regulates macropinocytosis, phagocytosis and endosomal membrane flow in Dictyostelium.

Scar, a member of the WASp protein family, was discovered in Dictyostelium discoideum during a genetic screen for second-site mutations that suppressed a developmental defect. Disruption of the scar gene reduced the levels of cellular F-actin by 50%. To investigate the role of Scar in endocytosis, phagocytosis and endocytic membrane trafficking, processes that depend on actin polymerization, we have analyzed a Dictyostelium cell line that is genetically null for Scar. Rates of fluid phase macropinocytosis and phagocytosis are significantly reduced in the scar- cell-line. In addition, exocytosis of fluid phase is delayed in these cells and movement of fluid phase from lysosomes to post-lysosomes is also delayed. Inhibition of actin polymerization with cytochalasin A resulted in similar phenotypes, suggesting that Scar-mediated polymerization of the actin cytoskeleton was important in the regulation of these processes. Supporting this conclusion, fluorescence microscopy revealed that some endo-lysosomes were ringed with F-actin in control cells but no F-actin was detected associated with endo-lysosomes in Scar null cells. Disruption of the two genes encoding the actin monomer sequestering protein profilin in wild-type cells causes defects in the rate of pinocytosis and fluid phase efflux. Consistent with a predicted physical interaction between Scar and profilin, disrupting the scar gene in the profilin null background results in greater decreases in the rate of fluid phase internalization and fluid phase release compared to either mutant alone. Taken together, these data support a model in which Scar and profilin functionally interact to regulate internalization of fluid and particles and later steps in the endosomal pathway, probably through regulation of actin cytoskeleton polymerization.

Early and late endosomal compartments of Entamoeba histolytica are enriched in cysteine proteases, acid phosphatase and several Ras-related Rab GTPases.

Pure populations of early and late endosomes of Entamoeba histolytica were isolated by magnetic fractionation and characterized. It was shown that these vesicles were enriched in acid phosphatase and cysteine protease activities. An important virulence factor, a 27-kDa cysteine protease, was also enriched in early and late endosomes of E. histolytica. These data suggest that E. histolytica hydrolases reside in compartments that are part of or communicate with the endosomal pathway. To begin to identify the role of Rab GTPases in E. histolytica, an oligonucleotide approach was employed to screen an E. histolytica cDNA library for genes encoding Rab-like proteins. cDNAs encoding a Rab11-like protein (EhRab11) and a novel Rab protein (EhRabA) were isolated and characterized. The EhRab11 cDNA predicts a polypeptide of at least 206 amino acids with a molecular mass of at least 23.2 kDa. Phylogenetic analysis and alignment of EhRab11 with other Rab proteins demonstrated that EhRab11 shared significant homology at the amino acid level with Rab11-like proteins from a number of other eukaryotes, suggesting that EhRab11 is a Rab11 homolog for E. histolytica. The EhRabA clone predicts a polypeptide of 219 amino acids with a molecular mass of at least 24.5 kDa. EhRabA shared only limited homology at the amino acid level with other Rab proteins, suggesting that it is a novel member of this family of GTP-binding proteins. Finally, Western blot analysis demonstrated that EhRab11 and a previously described Rab7-like GTPase from E. histolytica was enriched in magnetically purified endosomal compartments of this organism.

Trehalase of Dictyostelium discoideum: inhibition by amino-containing analogs of trehalose and affinity purification.

The inhibitory effects of three nitrogen containing analogs of trehalose, validamycin A, MDL 25,637 and castanospermine, on Dictyostelium discoideum trehalase were examined. Prior to this study, the effects of glycohydrolase inhibitors on D discoideum trehalase have not been reported. Validamycin A, MDL 25,637 and castanospermine were found to be potent, reversible, competitive inhibitors of D discoideum vegetative trehalase in vitro with IC50 values of 1 x 10(-9) M, 2 x 10(-8) M and 1.25 x 10(-4) M, respectively. Validamycin A and MDL 25,637 also exhibited time-dependent inhibition of D discoideum trehalase, whereby the potencies of these two inhibitors were observed to increase when pre-incubated with the enzyme for up to 60 min. The competitive natures of validamycin A and MDL 25,637 were also altered during pre-incubation with enzyme such that the compounds behaved as mixed inhibitors under these conditions. Taken together, these results suggest that the inhibitory action of validamycin A and MDL 25,637 on trehalase is of a slow-binding nature. A trehalase-specific affinity resin was synthesized by covalently coupling validamycin A to Sepharose 6B. This resin was used to purify D discoideum trehalase to near homogeneity in a two-step procedure. SDS-PAGE of affinity-purified trehalase, and silver staining or in situ staining for trehalase activity, revealed a major protein species of 42 kDa, exhibiting trehalase activity, and two minor protein species of approximately 45 and 49 kDa. Since validamycin A demonstrates strict binding specificity for trehalase, validamycin A-Sepharose has potential and novel applications in rapid, large scale, purification of trehalases from a variety of species origins.

Cloning and characterization of a Dictyostelium discoideum cDNA encoding a protein related to the medium chain subunit of clathrin-associated adaptor complexes.

We describe the cloning and characterization of a cDNA, DdApm1, encoding a putative medium chain subunit of a clathrin-associated protein (adaptor or assembly protein [AP]) complex in Dictyostelium discoideum. The DdApm1 clone is predicted to encode a polypeptide of 439 amino acids (aa) with a molecular mass of 49.9 kDa. The predicted translation product (DdApm1p) shares at least 51.7% identity and 76.3% similarity with the medium chain subunits of plasma membrane (mb)-associated clathrin AP complexes from rat and Caenorhabditis elegans. The deduced aa sequence also demonstrates significant but lesser homology to a number of medium chain subunits of Golgi-associated clathrin AP complexes. Since DdApm1p demonstrates significantly greater homology to plasma mb-associated clathrin AP complex medium chains than to their Golgi-associated counterparts, we suggest that DdApm1p may be a medium chain subunit of an AP complex involved in clathrin function at the plasma mb of D. discoideum. Southern blot analysis indicated that DdApm1 gene defines a single copy gene in the D. discoideum genome. Northern blot analysis of RNA purified at different times during growth and development demonstrated that the DdApm1 gene is expressed at relatively constant levels throughout the life cycle of the organism. DdApm1 is the first reported full-length cDNA encoding a subunit of an AP complex in D. discoideum, and thus provides the first evidence for the existence of AP complexes in this organism.

Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum.

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454-3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants.

The role of myosin Is in endosomal trafficking and the lysosomal system was investigated in a Dictyostelium discoideum myosin I double mutant myoB-/C-, that has been previously shown to exhibit defects in fluid-phase endocytosis during growth in suspension culture (Novak et al., 1995). Various properties of the endosomal pathway in the myoB-/C- double mutant as well as in the myoB- and myoC- single mutants, including intravesicular pH, and intracellular retention time and exocytosis of a fluid phase marker, were found to be indistinguishable from wild-type parental cells. The intimate connection between the contractile vacuole complex and the endocytic pathway in Dictyostelium, and the localization of a myosin I to the contractile vacuole in Acanthamoeba, led us to also examine the structure and function of this organelle in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in the myoB-, myoC- or myoB-/C- cell lines. The transport, processing, and localization of a lysosomal enzyme, alpha-mannosidase, were also unaltered in all three mutants. However, the myoB- and myoB-/C- cell lines, but not the myoC- cell line, were found to oversecrete the lysosomal enzymes alpha-mannosidase and acid phosphatase, during growth and starvation. None of the mutants oversecreted proteins following the constitutive secretory pathway. Two additional myosin I mutants, myoA- and myoA-/B-, were also found to oversecrete the lysosomally localized enzymes alpha-mannosidase and acid phosphatase. Taken together, these results suggest that these myosins do not play a role in the intracellular movement of vesicles, but that they may participate in controlling events that occur at the actin-rich cortical region of the cell. While no direct evidence has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fidelity of the actin cortex, and changes in cortical organization could influence lysosomal-related membrane events such as internalization or transit of vesicles to the cell surface.