Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift

<b>Background</b> Understanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated.<p></p> <b>Methods</b> A simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker.<p></p> <b>Results</b> The possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated.<p></p> <b>Conclusion</b> The method allows for the enrichment of red blood cells early after the invasion by P. falciparumparasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.<p></p&gt

Prediction of solubility on recombinant expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: Cellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL (Duffy binding like), CIDR (cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally. METHODS: A computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli. RESULTS: Solubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1α (46.8%) > CIDR2β (42.9%) > DBL2-4γ (31.7%) > DBL2β + C2 (30.6%) > DBL1α (24.9%) > DBL2-7ε (23.1%) > DBL2-5δ (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions. CONCLUSION: The acidic terminal segment (ATS) and CIDR α/β domain types are suitable for recombinant expression in E. coli while all DBL subtypes (α, β, γ, δ, ε) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains

Sub-grouping and sub-functionalization of the RIFIN multi-copy protein family

<p>Abstract</p> <p>Background</p> <p>Parasitic protozoans possess many multicopy gene families which have central roles in parasite survival and virulence. The number and variability of members of these gene families often make it difficult to predict possible functions of the encoded proteins. The families of extra-cellular proteins that are exposed to a host immune response have been driven via immune selection to become antigenically variant, and thereby avoid immune recognition while maintaining protein function to establish a chronic infection.</p> <p>Results</p> <p>We have combined phylogenetic and function shift analyses to study the evolution of the RIFIN proteins, which are antigenically variant and are encoded by the largest multicopy gene family in <it>Plasmodium falciparum</it>. We show that this family can be subdivided into two major groups that we named A- and B-RIFIN proteins. This suggested sub-grouping is supported by a recently published study that showed that, despite the presence of the <it>Plasmodium </it>export (PEXEL) motif in all RIFIN variants, proteins from each group have different cellular localizations during the intraerythrocytic life cycle of the parasite. In the present study we show that function shift analysis, a novel technique to predict functional divergence between sub-groups of a protein family, indicates that RIFINs have undergone neo- or sub-functionalization.</p> <p>Conclusion</p> <p>These results question the general trend of clustering large antigenically variant protein groups into homogenous families. Assigning functions to protein families requires their subdivision into meaningful groups such as we have shown for the RIFIN protein family. Using phylogenetic and function shift analysis methods, we identify new directions for the investigation of this broad and complex group of proteins.</p

A highly conserved segmental duplication in the subtelomeres of Plasmodium falciparum chromosomes varies in copy number

<p>Abstract</p> <p>Background</p> <p>Segmental duplications (SD) have been found in genomes of various organisms, often accumulated at the ends of chromosomes. It has been assumed that the sequence homology in-between the SDs allow for ectopic interactions that may contribute to the emergence of new genes or gene variants through recombinatorial events.</p> <p>Methods</p> <p><it>In silico </it>analysis of the 3D7 <it>Plasmodium falciparum </it>genome, conducted to investigate the subtelomeric compartments, led to the identification of subtelomeric SDs. Sequence variation and copy number polymorphisms of the SDs were studied by DNA sequencing, real-time quantitative PCR (qPCR) and fluorescent <it>in situ </it>hybridization (FISH). The levels of transcription and the developmental expression of copy number variant genes were investigated by qPCR.</p> <p>Results</p> <p>A block of six genes of >10 kilobases in size, including <it>var</it>, <it>rif</it>, <it>pfmc-2tm </it>and three hypothetical genes (<it>n-, o- </it>and <it>q-gene</it>), was found duplicated in the subtelomeric regions of chromosomes 1, 2, 3, 6, 7, 10 and 11 (SD1). The number of SD1 per genome was found to vary from 4 to 8 copies in between different parasites. The intragenic regions of SD1 were found to be highly conserved across ten distinct fresh and long-term cultivated <it>P. falciparum</it>. Sequence variation was detected in a ≈ 23 amino-acid long hypervariable region of a surface-exposed loop of PFMC-2TM. A hypothetical gene within SD1, the <it>n-gene</it>, encoding a PEXEL/VTS-containing two-transmembrane protein was found expressed in ring stage parasites. The <it>n-gene </it>transcription levels were found to correlate to the number of <it>n-gene </it>copies. Fragments of SD1 harbouring two or three of the SD1-genes (<it>o-gene, pfmc-2tm, q-gene</it>) were also found in the 3D7 genome. In addition a related second SD, SD2, of ≈ 55% sequence identity to SD1 was found duplicated in a fresh clinical isolate but was only present in a single copy in 3D7 and in other <it>P. falciparum </it>lines or clones.</p> <p>Conclusion</p> <p><it>Plasmodium falciparum </it>carries multiple sequence conserved SDs in the otherwise highly variable subtelomeres of its chromosomes. The uniqueness of the SDs amongst plasmodium species, and the conserved nature of the genes within, is intriguing and suggests an important role of the SD to <it>P. falciparum</it>.</p

Malaria Burden in Pregnancy at Mulago National Referral Hospital in Kampala, Uganda

Pregnancy-associated malaria is a major global health concern. To assess the Plasmodium falciparum burden in pregnancy we conducted a cross-sectional study at Mulago Hospital in Kampala, Uganda. Malaria prevalence by each of three measures—peripheral smear, placental smear, and placental histology was 9% (35/391), 11.3% (44/389), and 13.9% (53/382) respectively. Together, smear and histology data yielded an infection rate of 15.5% (59/380) of active infections and 4.5% (17/380) of past infections; hence 20% had been or were infected when giving birth. A crude parity dependency was observed with main burden being concentrated in gravidae 1 through gravidae 3. Twenty-two percent were afflicted by anaemia and 12.2% delivered low birthweight babies. Active placental infection and anaemia showed strong association (OR = 2.8) whereas parity and placental infection had an interactive effect on mean birthweight (P = .036). Primigravidae with active infection and multigravidae with past infection delivered on average lighter babies. Use of bednet protected significantly against infection (OR = 0.56) whilst increased haemoglobin level protected against low birthweight (OR = 0.83) irrespective of infection status. Albeit a high attendance at antenatal clinics (96.8%), there was a poor coverage of insecticide-treated nets (32%) and intermittent preventive antimalarial treatment (41.5%)

ARAM: an automated image analysis software to determine rosetting parameters and parasitaemia in Plasmodium samples

Additional file 3: Figure S3. Bland-Altman diagrams. Left Comparison of the cell detection by ARAM and an operator. Right Comparison of the determined rosette size by ARAM and an operator

var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2

<p>Abstract</p> <p>Background</p> <p>The pathogenicity of <it>Plasmodium falciparum </it>is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra-vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by <it>Plasmodium falciparum </it>erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of ≈60 <it>var </it>genes.</p> <p>Methods</p> <p>The study of <it>var </it>gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantitative PCR (qPCR). The expression of the major PfEMP1 in FCR3S1.2 pRBC was analysed with polyclonal sera in rosette disruption assays and immunofluorecence.</p> <p>Results</p> <p>Transcripts from <it>var</it>1 (FCR3S1.2_<it>var</it>₁; IT4<it>var</it>21) and other <it>var </it>genes were detected by semi-quantitative PCR but results from qPCR showed that one <it>var </it>gene transcript dominated over the others (FCR3S1.2_<it>var</it>₂; IT4<it>var</it>60). Antibodies raised in rats to the recombinant NTS-DBL1α of <it>var</it>2 produced in <it>E. coli </it>completely and dose-dependently disrupted rosettes (≈95% at a dilution of 1/5). The sera reacted with the Maurer's clefts in trophozoite stages (IFA) and to the infected erythrocyte surface (FACS) indicating that FCR3S1.2_{<it>var2 </it>}encodes the dominant PfEMP1 expressed in this parasite.</p> <p>Conclusion</p> <p>The major transcript in the rosetting model parasite FCR3S1.2 is FCR3S1.2_<it>var</it>₂(IT4<it>var</it>60). The results suggest that this gene encodes the PfEMP1-species responsible for the rosetting phenotype of this parasite. The activity of previously raised antibodies to the NTS-DBL1α of FCR3S1.2_<it>var</it>₁is likely due to cross-reactivity with NTS-DBL1α of the <it>var</it>2 encoded PfEMP1.</p

SURFIN4.1, a schizont-merozoite associated protein in the SURFIN family of Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>In its effort to survive the human immune system, <it>Plasmodium falciparum </it>uses several parasite-derived antigens most of which are expressed at the surface of the parasitized red blood cells (pRBCs). Recently SURFINs, a new family of antigens encoded by the <it>surf </it>multi-gene family, has been reported. One member of the family, SURFIN_4.2, was found present both at the pRBC-surface and at the merozoite apex.</p> <p>Methods</p> <p>The presence of a second SURFIN member, SURFIN_4.1(PFD0100c, PFD0105c) is reported here. Bioinformatic tools were used to study the structure of the <it>surf</it>_4.1gene. To investigate the expression of <it>surf </it>genes PCR and real-time quantitative PCR (Rt-QPCR) were employed and Northern and Western blots were used to confirm the size of the <it>surf</it>_4.1gene and the SURFIN_4.1protein respectively. Localization of SURFIN_4.1was determined using immunofluorescence assays.</p> <p>Results</p> <p>The <it>surf</it>_4.1gene was found present in one copy by Rt-QPCR in some parasites (3D7AH1, 3D7S8, 7G8) whereas six copies of the gene were identified in FCR3 and FCR3S1.2. <it>surf</it>_4.1was found transcribed in the late asexual stages of the parasite beginning ≈32 hours post invasion and throughout the schizont stages with the level of transcription peaking at late schizogony. The levels of transcript correlated with the number of gene copies in FCR3 and 3D7S8. <it>surf</it>_4.1was found to encode a polypeptide of ≈Mw 258 kDa (SURFIN_4.1) present within the parasitophorous vacuole (PV), around free merozoites as merozoite-associated material, but not at the pRBC-surface. Despite multiple <it>surf</it>_4.1gene copies in some parasites this was not reflected in the levels of SURFIN_4.1polypeptide.</p> <p>Conclusion</p> <p>SURFIN_4.1is a member of the SURFINs, present in the PV and on the released merozoite. The results suggest different SURFINs to be expressed at different locations in the parasite and at distinct time-points during the intra-erythrocytic cycle.</p

Survival of Plasmodium falciparum infected Red Blood Cell Aggregates in Elongational Shear Flow

Rosetting, the formation of red blood cell aggregates, is a life-threatening condition in malaria tropica and not yet fully understood. We study rosette stability using a set of microfluidic stenotic channels, with varied narrowing angle and erythrocytes of blood groups O and A. We find reduced ability of a rosette to pass a stenosis without disruption, the longer the tapered part of the constriction and the narrower the stenosis is. In general, this ability increases with rosette size and is 5-15% higher in blood group A. The experimental results are substantiated by equivalent experiments using lectin-induced red blood cell aggregates and a simulation of the underlying protein binding kinetics.</p