Comparison of the in vitro invasive capabilities of Plasmodium falciparum schizonts isolated by Percoll gradient or using magnetic based separation

<p>Abstract</p> <p>Background</p> <p>Percoll gradient centrifugation is often used for synchronization, enrichment, or isolation of a particular stage of <it>Plasmodium falciparum</it>. However, Percoll, a hyperosmotic agent, may have harmful effects on the parasites. Magnetic bead column (MBC) separation has been used as an alternative. This is a report of a head-to-head comparison of the <it>in vitro </it>invasive capabilities of parasites isolated by either of the two methods.</p> <p>Methods</p> <p>The <it>P. falciparum </it>laboratory strain isolate 7G8 was grown <it>in vitro </it>using standard procedures and synchronized using 5% sorbitol. On separate days when the schizont parasitaemia was >1%, the culture was split and half was processed by Percoll gradient centrifugation and the other half by magnetic bead column separation. Both processed parasites were placed back in culture and allowed to invade new uninfected erythrocytes.</p> <p>Results</p> <p>In 10 paired assays, the mean efficiency of invasion of 7G8 parasites treated by Percoll gradient centrifugation was 35.8% that of those treated by magnetic bead column separation (95% CI, p = 0.00067) A paired <it>t </it>test with two tails was used for these comparisons.</p> <p>Conclusions</p> <p>In this comparison, magnetic bead column separation of 7G8 schizonts resulted in higher viability and efficiency of invasion than utilizing Percoll gradient centrifugation.</p

Phosphoenolpyruvate carboxylase dentified as a key enzyme in erythrocytic Plasmodium falciparum carbon metabolism

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in thePlasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery

Optimized high gradient magnetic separation for isolation of Plasmodium-infected red blood cells

<p>Abstract</p> <p>Background</p> <p>Highly purified infected red blood cells (irbc), or highly synchronized parasite cultures, are regularly required in malaria research. Conventional isolation and synchronization rely on density and osmotic fragility of irbc, respectively. High gradient magnetic separation (HGMS) offers an alternative based on intrinsic magnetic properties of irbc, avoiding exposure to chemicals and osmotic stress. Successful HGMS concentration in malaria research was previously reported using polymer coated columns, while HGMS depletion has not been described yet. This study presents a new approach to both HGMS concentration and depletion in malaria research, rendering polymer coating unnecessary.</p> <p>Methods</p> <p>A dipole magnet generating a strong homogenous field was custom assembled. Polypropylene syringes were fitted with one-way stopcocks and filled with stainless steel wool. Rbc from <it>Plasmodium falciparum </it>cultures were resuspended in density and viscosity optimized HGMS buffers and HGMS processed. Purification and depletion results were analysed by flow cytometer and light microscopy. Viability was evaluated by calculating the infection rate after re-culturing of isolates.</p> <p>Results</p> <p>In HGMS concentration, purity of irbc isolates from asynchronous cultures consistently ranged from 94.8% to 98.4% (mean 95.7%). With further optimization, over 90% of isolated irbc contained segmented schizonts. Processing time was less than 45 min. Reinfection rates ranged from 21.0% to 56.4%. In HGMS depletion, results were comparable to treatment with sorbitol, as demonstrated by essentially identical development of cultures.</p> <p>Conclusion</p> <p>The novel HGMS concentration procedure achieves high purities of segmented stage irbc from standard asynchronous cultures, and is the first HGMS depletion alternative to sorbitol lysis. It represents a simple and highly efficient alternative to conventional irbc concentration and synchronization methods.</p

An Effective Method to Purify Plasmodium falciparum DNA Directly from Clinical Blood Samples for Whole Genome High-Throughput Sequencing

Highly parallel sequencing technologies permit cost-effective whole genome sequencing of hundreds of Plasmodium parasites. The ability to sequence clinical Plasmodium samples, extracted directly from patient blood without a culture step, presents a unique opportunity to sample the diversity of “natural” parasite populations in high resolution clinical and epidemiological studies. A major challenge to sequencing clinical Plasmodium samples is the abundance of human DNA, which may substantially reduce the yield of Plasmodium sequence. We tested a range of human white blood cell (WBC) depletion methods on P. falciparum-infected patient samples in search of a method displaying an optimal balance of WBC-removal efficacy, cost, simplicity, and applicability to low resource settings. In the first of a two-part study, combinations of three different WBC depletion methods were tested on 43 patient blood samples in Mali. A two-step combination of Lymphoprep plus Plasmodipur best fitted our requirements, although moderate variability was observed in human DNA quantity. This approach was further assessed in a larger sample of 76 patients from Burkina Faso. WBC-removal efficacy remained high (<30% human DNA in >70% samples) and lower variation was observed in human DNA quantities. In order to assess the Plasmodium sequence yield at different human DNA proportions, 59 samples with up to 60% human DNA contamination were sequenced on the Illumina Genome Analyzer platform. An average ∼40-fold coverage of the genome was observed per lane for samples with ≤30% human DNA. Even in low resource settings, using a simple two-step combination of Lymphoprep plus Plasmodipur, over 70% of clinical sample preparations should exhibit sufficiently low human DNA quantities to enable ∼40-fold sequence coverage of the P. falciparum genome using a single lane on the Illumina Genome Analyzer platform. This approach should greatly facilitate large-scale clinical and epidemiologic studies of P. falciparum

Life Cycle-Dependent Cytoskeletal Modifications in Plasmodium falciparum Infected Erythrocytes

10.1371/journal.pone.0061170PLoS ONE84

A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development

Microscopic examination of Giemsa-stained thin blood smears remains the gold standard method used to quantify and stage malaria parasites. However, this technique is tedious, and requires trained microscopists. We have developed a fast and simple flow cytometry method to quantify and stage, various malaria parasites in red blood cells in whole blood or in vitro cultured Plasmodium falciparum. The parasites were stained with dihydroethidium and Hoechst 33342 or SYBR Green I and leukocytes were identified with an antibody against CD45. Depending on the DNA stains used, samples were analyzed using different models of flow cytometers. This protocol, which does not require any washing steps, allows infected red blood cells to be distinguished from leukocytes, as well as allowing non-infected reticulocytes and normocytes to be identified. It also allows assessing the proportion of parasites at different developmental stages. Lastly, we demonstrate how this technique can be applied to antimalarial drug testing

Effect of Sanitation on Soil-Transmitted Helminth Infection: Systematic Review and Meta-Analysis

A systematic review and meta-analysis by Kathrin Ziegelbauer and colleagues finds that sanitation is associated with a reduced risk of transmission of helminthiases to humans

Compartmentation of Redox Metabolism in Malaria Parasites

Malaria, caused by the apicomplexan parasite Plasmodium, still represents a major threat to human health and welfare and leads to about one million human deaths annually. Plasmodium is a rapidly multiplying unicellular organism undergoing a complex developmental cycle in man and mosquito – a life style that requires rapid adaptation to various environments. In order to deal with high fluxes of reactive oxygen species and maintain redox regulatory processes and pathogenicity, Plasmodium depends upon an adequate redox balance. By systematically studying the subcellular localization of the major antioxidant and redox regulatory proteins, we obtained the first complete map of redox compartmentation in Plasmodium falciparum. We demonstrate the targeting of two plasmodial peroxiredoxins and a putative glyoxalase system to the apicoplast, a non-photosynthetic plastid. We furthermore obtained a complete picture of the compartmentation of thioredoxin- and glutaredoxin-like proteins. Notably, for the two major antioxidant redox-enzymes – glutathione reductase and thioredoxin reductase – Plasmodium makes use of alternative-translation-initiation (ATI) to achieve differential targeting. Dual localization of proteins effected by ATI is likely to occur also in other Apicomplexa and might open new avenues for therapeutic intervention