Stefan Baumeister

The DEAH-box helicase DHX36 mediates dendritic localization of the neuronal precursor-microRNA-134

Specific microRNAs (miRNAs), including miR-134, localize to neuronal dendrites, where they control synaptic protein synthesis and plasticity. However, the mechanism of miRNA transport is unknown. We found that the neuronal precursor-miRNA-134 (pre-miR-134) accumulates in dendrites of hippocampal neurons and at synapses in vivo. Dendritic localization of pre-miR-134 is mediated by the DEAH-box helicase DHX36, which directly associates with the pre-miR-134 terminal loop. DHX36 function is required for miR-134-dependent inhibition of target gene expression and the control of dendritic spine size. Dendritically localized pre-miR-134 could provide a local source of miR-134 that can be mobilized in an activity-dependent manner during plasticity.

Growth Inhibition of Toxoplasma gondii and Plasmodium falciparum by Nanomolar Concentrations of 1-Hydroxy-2-Dodecyl-4(1H)Quinolone, a High-Affinity Inhibitor of Alternative (Type II) NADH Dehydrogenases

ABSTRACT Both apicomplexan parasites Toxoplasma gondii and Plasmodium falciparum lack type I NADH dehydrogenases (complex I) but instead carry alternative (type II) NADH dehydrogenases, which are absent in mammalian cells and are thus considered promising antimicrobial drug targets. The quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) was recently described as a high-affinity inhibitor of fungal alternative NADH dehydrogenases in enzymatic assays, probably by interfering with the ubiquinol binding site of the enzyme. We describe here that HDQ effectively inhibits the replication rates of P. falciparum and T. gondii in tissue culture. The 50% inhibitory concentration (IC50) of HDQ for T. gondii was determined to be 2.4 ± 0.3 nM with a growth assay based on vacuole sizes and 3.7 ± 1.4 nM with a growth assay based on beta-galactosidase activity. Quantification of the P. falciparum replication rate using a fluorometric assay revealed an IC50 of 14.0 ± 1.9 nM. An important feature of the HDQ structure is the length of the alkyl side chain at position 2. Derivatives with alkyl side chains of C6, C8, C12 (HDQ), and C14 all displayed excellent anti-T. gondii activity, while a C5 derivative completely failed to inhibit parasite replication. A combined treatment of T. gondii-infected cells with HDQ and the antimalarial agent atovaquone, which blocks the ubiquinol oxidation site of cytochrome b in complex III, resulted in synergism, with a calculated fractional inhibitory concentration of 0.16 nM. Interference of the mitochondrial ubiquinone/ubiquinol cycle at two different locations thus appears to be a highly effective strategy for inhibiting parasite replication. HDQ and its derivatives, particularly in combination with atovaquone, represent promising compounds with a high potential for antimalarial and antitoxoplasmal therapy.

Fosmidomycin Uptake into Plasmodium and Babesia-Infected Erythrocytes Is Facilitated by Parasite-Induced New Permeability Pathways

Background Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii. Methodology/Principal Findings Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite. Conclusions/Significance Our findings provide an explanation for the observed differences in activity of fosmidomycin and FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery.

Phosphocholine-containing, zwitterionic glycosphingolipids of adult Onchocerca volvulus as highly conserved antigenic structures of parasitic nematodes.

Human Onchocerca volvulus infection sera were found to recognize zwitterionic glycolipids of O. volvulus and to cross-react with those of other parasitic nematodes (Ascaris suum, Setaria digitata and Litomosoides sigmodontis). By the use of an epitope-specific monoclonal antibody, zwitterionic glycolipids of all these nematode species were observed to contain the antigenic determinant phosphocholine. A hyperimmune serum specific for arthro-series glycolipid structures reacted with the various neutral glycolipids of all these nematodes, which demonstrated that their oligosaccharide moieties belonged to the arthro-series of protostomial glycolipids. These results indicated that arthro-series glycosphingolipids carrying, in part, phosphocholine substituents, represent highly conserved, antigenic glycolipid markers of parasitic nematodes. Three glycolipid components of the O. volvulus zwitterionic fraction were structurally characterized by matrix-assisted laser-desorption/ionization time-of-flight MS, methylation analysis and exoglycosidase treatment. Their chemical structures were elucidated to be phosphocholine-6GlcNAc(beta1-3)Man(beta1-4)Glc(1-1)ceramide, GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta1-4)Glc(1-1) ceramide and Gal(alpha1-3)GalNAc(beta1-4)[phosphocholine-6]GlcNAc(beta1-3)Man(beta 1-4)Glc(1-1)ceramide for the zwitterionic ceramide tri-, tetra- and penta-hexosides respectively. The ceramide composition was found to be dominated by 2-hydroxylated docosanoic (C(22h:0)), tricosanoic (C(23h:0)) and tetracosanoic (C(24h:0)) acids, and C(17) sphingosine (C(d17:1)) (where (h) is hydroxylated and (d) is dihydroxylated).

Evidence for the involvement of Plasmodium falciparum proteins in the formation of new permeability pathways in the erythrocyte membrane

The intraerythrocytic developmental stages of the malaria parasite Plasmodium falciparum are responsible for the clinical symptoms associated with malaria tropica. The non‐infected human erythrocyte is a terminally differentiated cell that is unable to synthesize proteins and lipids de novo, and it is incapable of importing a number of solutes that are essential for parasite proliferation. Approximately 12–15 h after invasion the parasitized cell undergoes a marked increase in its permeability to a variety of different solutes present in the extracellular milieu. The increase is due to the induction in the erythrocyte membrane of ‘new permeability pathways’ which have been characterized in some detail in terms of their transport and electrophysiological properties, but which are yet to be defined at a molecular level. Here we show that these pathways are resistant to trypsin but are abolished by treatment of intact infected erythrocytes with chymotrypsin. On resuspension of chymotrypsinized cells in chymotrypsin‐free medium the pathways progressively reappear, a process that can be inhibited by cytotoxic agents, and by brefeldin A which inhibits protein secretion. Our results provide evidence for the involvement of parasite encoded proteins in the generation of the pathways, either as components of the pathways themselves or as auxiliary factors.

A high specificity and affinity interaction with serum albumin stimulates an anion conductance in malaria-infected erythrocytes.

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl- and lactate outward currents with an EC50 of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

CHARACTERIZATION AND CLONING OF THE GENE ENCODING THE VACUOLAR MEMBRANE PROTEIN EXP-2 FROM PLASMODIUM FALCIPARUM

As a contribution to the characterization of the parasitophorous vacuolar membrane from Plasmodium falciparum we have begun the identification of vacuolar membrane proteins. Exported protein-2 (EXP-2) is a vacuolar membrane protein exposed into the vacuolar space. To further characterize EXP-2, it was purified, and the 45 N-terminal amino acids were determined by micro-sequencing. Based on this information, partial cDNA and genomic fragments were amplified by PCR and used as probes for the isolation of complete cDNA and genomic DNA clones. The single copy gene is located on chromosome 14, and is transcribed during the ring stage of parasite development. The open reading frame encodes an N-terminal signal sequence which is cleaved from the mature protein. The amino acid composition of EXP-2 is characterized by charged amino acids, with a high abundance of aspartate residues in the C-terminal portion of the protein. In contrast to EXP-1, an integral protein of the vacuolar membrane, EXP-2 lacks a typical hydrophobic transmembrane domain. We suggest that EXP-2 may associate with the vacuolar membrane via an amphipathic helix located in the N-terminal half of the protein.

Digestive vacuole of Plasmodium falciparum released during erythrocyte rupture dually activates complement and coagulation.

Severe Plasmodium falciparum malaria evolves through the interplay among capillary sequestration of parasitized erythrocytes, deregulated inflammatory responses, and hemostasis dysfunction. After rupture, each parasitized erythrocyte releases not only infective merozoites, but also the digestive vacuole (DV), a membrane-bounded organelle containing the malaria pigment hemozoin. In the present study, we report that the intact organelle, but not isolated hemozoin, dually activates the alternative complement and the intrinsic clotting pathway. Procoagulant activity is destroyed by phospholipase C treatment, indicating a critical role of phospholipid head groups exposed at the DV surface. Intravenous injection of DVs caused alternative pathway complement consumption and provoked apathy and reduced nociceptive responses in rats. Ultrasonication destroyed complement-activating and procoagulant properties in vitro and rendered the DVs biologically inactive in vivo. Low-molecular-weight dextran sulfate blocked activation of both complement and coagulation and protected animals from the harmful effects of DV infusion. We surmise that in chronic malaria, complement activation by and opsonization of the DV may serve a useful function in directing hemozoin to phagocytic cells for safe disposal. However, when the waste disposal system of the host is overburdened, DVs may transform into a trigger of pathology and therefore represent a potential therapeutic target in severe malaria.

Digestive vacuoles of Plasmodium falciparum are selectively phagocytosed by and impair killing function of polymorphonuclear leukocytes

Sequestration of parasitized erythrocytes and dysregulation of the coagulation and complement system are hallmarks of severe Plasmodium falciparum malaria. A link between these events emerged through the discovery that the parasite digestive vacuole (DV), which is released together with infective merozoites into the bloodstream, dually activates the intrinsic clotting and alternative complement pathway. Complement attack occurs exclusively on the membrane of the DVs, and the question followed whether DVs might be marked for uptake by polymorphonuclear granulocytes (PMNs). We report that DVs are indeed rapidly phagocytosed by PMNs after schizont rupture in active human serum. Uptake of malaria pigment requires an intact DV membrane and does not occur when the pigment is extracted from the organelle. Merozoites are not opsonized and escape phagocytosis in nonimmune serum. Antimalarial Abs mediate some uptake of the parasites, but to an extent that is not sufficient to markedly reduce reinvasion rates. Phagocytosis of DVs induces a vigorous respiratory burst that drives the cells into a state of functional exhaustion, blunting the production of reactive oxygen species (ROS) and microbicidal activity upon challenge with bacterial pathogens. Systemic overloading of PMNs with DVs may contribute to the enhanced susceptibility of patients with severe malaria toward invasive bacterial infections.

pfmdr2 Confers Heavy Metal Resistance to Plasmodium falciparum

Heavy metals are required by all organisms for normal function, but high levels of heavy metals are toxic. Therefore, homeostasis of these metals is crucial. In the human malaria-causing agent Plasmodium falciparum, the mechanisms of heavy metal transport have yet to be characterized. We have developed a P. falciparum line resistant to heavy metals from a wild-type line sensitive to heavy metals. A molecular and biochemical analysis of the involvement of the P. falciparum multidrug resistance 2 (pfmdr2) gene, an ABC-type transporter, in heavy metal homeostasis was studied. Using a novel uptake assay applied on these two strains, it was demonstrated that, when exposed to heavy metals, the sensitive line accumulates metal, whereas no accumulation was observed in the resistant line. The accumulation occurs within the parasite itself and not in the cytoplasm of the red blood cell. This difference in the accumulation pattern is not a result of amplification of the pfmdr2 gene or of a change in the expression pattern of the gene in the two lines. Sequencing of the gene from both lines revealed a major difference; a stop codon is found in the sensitive line upstream of the normal termination, resulting in a truncated protein that lacks 188 amino acids that contain a portion of the essential cytoplasmatic transporter domain, thereby rendering it inactive. In contrast, the resistant line harbors a full-length, active protein. These findings strongly suggest that the PFMDR2 protein acts as an efflux pump of heavy metals.