Host reticulocytes provide metabolic reservoirs that can be exploited by malaria parasites

Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5′-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites

Conditional U1 gene silencing in Toxoplasma gondii

The functional characterisation of essential genes in apicomplexan parasites, such as Toxoplasma gondii or Plasmodium falciparum, relies on conditional mutagenesis systems. Here we present a novel strategy based on U1 snRNP-mediated gene silencing. U1 snRNP is critical in pre-mRNA splicing by defining the exon-intron boundaries. When a U1 recognition site is placed into the 3’-terminal exon or adjacent to the termination codon, pre-mRNA is cleaved at the 3’-end and degraded, leading to an efficient knockdown of the gene of interest (GOI). Here we describe a simple method that combines endogenous tagging with DiCre-mediated positioning of U1 recognition sites adjacent to the termination codon of the GOI which leads to a conditional knockdown of the GOI upon rapamycin-induction. Specific knockdown mutants of the reporter gene GFP and several endogenous genes of T. gondii including the clathrin heavy chain gene 1 (chc1), the vacuolar protein sorting gene 26 (vps26), and the dynamin-related protein C gene (drpC) were silenced using this approach and demonstrate the potential of this technology. We also discuss advantages and disadvantages of this method in comparison to other technologies in more detail

In the human malaria parasite Plasmodium falciparum,polyamines are synthesized by a bifunctional ornithine decarboxylase, S-adenosylmethionine decarboxylase

The polyamines putrescine, spermidine, and spermine are crucial for cell differentiation and proliferation. Interference with polyamine biosynthesis by inhibition of the rate-limiting enzymes ornithine decarboxylase (ODC) andS-adenosylmethionine decarboxylase (AdoMetDC) has been discussed as a potential chemotherapy of cancer and parasitic infections. Usually both enzymes are individually transcribed and highly regulated as monofunctional proteins. We have isolated a cDNA from the malaria parasite Plasmodium falciparumthat encodes both proteins on a single open reading frame, with the AdoMetDC domain in the N-terminal region connected to a C-terminal ODC domain by a hinge region. The predicted molecular mass of the entire transcript is 166 kDa. The ODC/AdoMetDC coding region was subcloned into the expression vector pASK IBA3 and transformed into the AdoMetDC- and ODC-deficient Escherichia coli cell line EWH331. The resulting recombinant protein exhibited both AdoMetDC and ODC activity and co-eluted after gel filtration on Superdex S-200 at ~333 kDa, which is in good agreement with the molecular mass of ~326 kDa determined for the native protein from isolated P. falciparum. SDS-polyacrylamide gel electrophoresis analysis of the recombinant ODC/AdoMetDC revealed a heterotetrameric structure of the active enzyme indicating processing of the AdoMetDC domain. The data presented describe the occurrence of a unique bifunctional ODC/AdoMetDC in P. falciparum, an organization which is possibly exploitable for the design of new antimalarial drugs

Isolation of a novel flavanonol and an alkylresorcinol with highly potent anti-trypanosomal activity from Libyan propolis

Twelve propolis samples from different parts of Libya were investigated for their phytochemical constituents. Ethanol extracts of the samples and some purified compounds were tested against Trypanosoma brucei, Plasmodium falciparum and against two helminth species, Trichinella spiralis and Caenorhabditis elegans, showing various degrees of activity. Fourteen compounds were isolated from the propolis samples, including a novel compound Taxifolin-3-acetyl-4’-methyl ether (4), a flavanonol derivative. The crude extracts showed moderate activity against T. spiralis and C. elegans, while the purified compounds had low activity against P. falciparum. Anti-trypanosomal activity (EC50 = 0.7 µg/mL) was exhibited by a fraction containing a cardol identified as bilobol (10) and this fraction had no effect on Human Foreskin Fibroblasts (HFF), even at 2.0 mg/mL, thus demonstrating excellent selectivity. A metabolomics study was used to explore the mechanism of action of the fraction and it revealed significant disturbances in trypanosomal phospholipid metabolism, especially the formation of choline phospholipids. We conclude that a potent and highly selective new trypanocide may be present in the fraction

Zum Einfluß der Dexamethason-Dosis auf Chemotherapie-induzierte akute Nausea und Emesis

20 mg Dexamethason oder eine dessen Wirkung äquivalente Dosis eines Corticosteroids- in Verbindung mit einem 5-HT3-Rezeptorantagonisten- erscheint derzeit als Goldstandardt der Prophylaxe zytostatisch induzierter Emesis. Neben der negativen Auswirkung auf die Tumorbiologie, sei es ein beschleunigtes Zellwachstum oder eine verminderte Tumorzellvernichtung durch die körpereigene Immunabwehr, besteht begründeter Verdacht, daß eine hohe Cortisol-Dosierung am Tag der Chemotherapie vermehrt Nausea und Emesis in der verzögerten Phase begünstigt. Deshalb erscheint es sinnvoll, den Stellenwert einer mittleren Steroiddosierung in der Prophylaxe der akuten Emesis zu überprüfen. In einer prospektiven, einfachblinden randomisierten Studie wurden zwei Dexamethason-Dosierungen -20 mg Dexamethason gegen 8 mg Dexamethason- jeweils in Verbindung mit dem 5-HT3-Rezeptorantagonisten Tropisetron in Cisplatin-haltigen Therapien anhand objektiver und subjektiver Parameter verglichen. Nach einer Zwischenanalyse von 121 Fällen (69 Patientinnen), in der keine signifikanten Unterschiede zwischen den beiden Behandlungsstrategien festgestellt werden konnten, wurde die Untersuchung beendet. Hohe Dexamethason-Dosierungen (20 mg) als antiemetische Prophylaxe ergeben weder in der Anzahl der Emesis-Episoden, noch bei den subjektiven Parameter der Rotterdam Symptoms Checklist Vorteile gegenüber einer mittleren (8 mg) Dosis. Dagegen wird gezeigt, daß das endogene Cortisol, vor der Chemotherapie und nach der Dexamethason- Applikation untersucht, die Ausprägung von Nausea und Emesis mitbeeinflußt. Die Patientinnen, deren Cortisol-Werte vor Therapiebeginn unterhalb des errechneten Medians von 169 [ng / ml] liegen, leiden im Vergleich mit den Patientinnen, deren Wert oberhalb dieser Schwelle liegen, verstärkt unter Übelkeit (p = 0,038). Weiterhin tritt bei den Patientinnen, deren Serum-Cortisol an Tag 2 unterhalb des Medians von 21 [ng/ml] liegt, subjektiv erheblichere Übelkeit (p = 0,020) und Emesis (p = 0,046) auf. Aufgrund dieser Ergebnisse ist die antiemetische Prophylaxe mit der Dosis 8 mg Dexamethason der Dosis 20 mg Dexamethason vorzuziehen, um in Verbindung mit einem 5-HT3 Rezeptorantagonisten in hoch emetogenen Chemotherapien von nicht-hämatologischen Tumoren Übelkeit und Erbrechen zu verhindern

Molecular mechanisms of drug resistance in natural Leishmania populations vary with genetic background

The evolution of drug-resistance in pathogens is a major global health threat. Elucidating the molecular basis of pathogen drug-resistance has been the focus of many studies but rarely is it known whether a drug-resistance mechanism identified is universal for the studied pathogen; it has seldom been clarified whether drug-resistance mechanisms vary with the pathogen's genotype. Nevertheless this is of critical importance in gaining an understanding of the complexity of this global threat and in underpinning epidemiological surveillance of pathogen drug resistance in the field. This study aimed to assess the molecular and phenotypic heterogeneity that emerges in natural parasite populations under drug treatment pressure. We studied lines of the protozoan parasite Leishmania (L.) donovani with differential susceptibility to antimonial drugs; the lines being derived from clinical isolates belonging to two distinct genetic populations that circulate in the leishmaniasis endemic region of Nepal. Parasite pathways known to be affected by antimonial drugs were characterised on five experimental levels in the lines of the two populations. Characterisation of DNA sequence, gene expression, protein expression and thiol levels revealed a number of molecular features that mark antimonial-resistant parasites in only one of the two populations studied. A final series of in vitro stress phenotyping experiments confirmed this heterogeneity amongst drug-resistant parasites from the two populations. These data provide evidence that the molecular changes associated with antimonial-resistance in natural Leishmania populations depend on the genetic background of the Leishmania population, which has resulted in a divergent set of resistance markers in the Leishmania populations. This heterogeneity of parasite adaptations provides severe challenges for the control of drug resistance in the field and the design of molecular surveillance tools for widespread applicability

Chemical and antimicrobial profiling of propolis from different regions within Libya.

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 μg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 μg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts

Attacking Blood-Borne Parasites with Mathematics

Central carbon metabolism is important to cells as it supplies free energy in the form of ATP and the building blocks for new cells. Parasites harvest many of the components they require from their hosts, but they still have to generate ATP themselves, making the metabolic pathways that generate ATP essential to the parasites' survival and thereby potential target pathways for antiparasitic drugs. Metabolic networks often consist of many components that interact with each other via nonlinear kinetics.The behavior of the network arises from the interaction of the components within and outside the network. To understand network behavior, experimental measurements on the components should be integrated through computational approaches. In this chapter, we present an overview of how experiment-driven mathematical models have provided insights on important aspects of parasite metabolism and have aided in elucidating potent antiparasitic drug targets within metabolism