Theresa L. Coetzer

Real-time quantitative PCR of telomere length

Telomeres cap the ends of chromosomes and are essential for the protection of chromosomes, as well as restricting the replicative potential of a cell. These functions are achieved by the regulation of telomeric repeat length, making the measurement of telomere length a useful aid in the elucidation of the replicative history and potential of cells. Previously published techniques employed either hybridization or flow cytometry methods, which are technically demanding and time-consuming. In 2002, R. M. Cawthon published a real-time polymerase chain reaction (PCR)-based method for telomere length measurement using the Applied Biosystems Prism 7700 sequence detection system. The technique measures the factor by which the ratio of telomere repeat copy number to single-gene copy number differs between a sample and that of a reference deoxyribonucleic acid sample. In many laboratories worldwide, including ours, real-time PCR is carried out using the Roche LightCycler, as opposed to the AB Prism 7700 system. This benchmark details the modifications to Cawthon’s method and describes the parameters and reagents required to measure telomere length using the Roche LightCycler.

Automated malaria detection by depolarization of laser light

Anecdotal experience with full blood count (FBC) technology incorporating analysis of depolarized laser light (DLL) for the enumeration of eosinophils showed that malaria infection generated unusual distributions in the white cell channels. The objective of this study was to identify and define criteria for a diagnosis of malaria using this technology. To determine sensitivity, specificity, and positive and negative predictive values, 224 directed samples referred specifically for malaria were used; true positives were defined as those in which malaria was identified by microscopic and/or immunological methods. For the DLL method, positive was defined as one or more large mononuclear cell(s) for which the 90° depolarized signal exceeded the 90° polarized signal. To determine possible utility in a routine haematology laboratory setting, 220 random undirected FBC samples were evaluated for possible malaria infection by the DLL method. Of the 224 directed samples, 95 were malaria positive as determined by microscopic and/or immunological methods, and 129 were negative. For the DLL method, overall sensitivity was 72% (90% in the case of Black Africans), and specificity 96%. Positive and negative predictive values overall were 93% and 82% respectively. In the utility study a single positive result was identified among the 220 samples studied. This was found to be from a patient with malaria. The detection of unexpected malaria by automated screening FBC analysis could substantially lower the mortality and morbidity from unascertained infection, especially in indigenous African peoples.

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BackgroundThe development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised.MethodsP. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database.ResultsFollowing four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library.ConclusionThe construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.

The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein

BackgroundErythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181) is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R) was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins.Methods4.1R structural domains (30, 16, 10 and 22 kDa domain) and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R.ResultsBlot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R.ConclusionThe interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasites life cycle.

Cross-linking of membrane proteins of metabolically-depleted and calcium-loaded erythrocytes.

Summary. The membranes of erythrocytes undergoing metabolic depletion or an influx of calcium undergo several changes in structure and function. In erythrocytes incubated without substrate we find extensive cross‐linking of membrane proteins by disulphide bonding occurring after 24–48 h, involving all major membrane proteins as well as haemoglobin. Aggregates of mol wt 40 × 106 or greater are formed. These changes are partially reversible by repletion with adenosine. Rapid introduction of calcium (intracellular concentrations approximately 0.6 mm) into metabolically replete erythrocytes with the ionophore A23187 results in transgluta‐minase‐dependent cross‐linking of membrane proteins. Cellular calcium concentrations of approximately 0.3 mm have no cross‐linking effect. Cells undergoing metabolic depletion show a progressive loss of transglutaminase activity to undetec‐table levels at 12 h, so that influx of calcium into such cells cannot cause cross‐linking by a transglutaminase‐mediated reaction. These studies suggest that the metabolic state of the cell and the rate and degree of calcium influx into erythrocytes are critical factors in determining the type of membrane protein cross‐linkage.

Band 3 Cape Town (E90K) causes severe hereditary spherocytosis in combination with band 3 Prague III

Hereditary spherocytosis (HS) is an inherited haemolytic anaemia, characterized by spheroidal, osmotically fragile red blood cells. This disorder exhibits heterogeneity in terms of both clinical severity and underlying molecular defect. We have studied a South African Cape Coloured individual with severe HS owing to a band 3 deficiency caused by two mutations, occurring in trans, in the band 3 gene: a novel variant that we have designated band 3 Cape Town and a previously described mutation, band 3 Prague III. Analysis of erythrocyte membrane proteins indicated a deficiency of both band 3 and protein 4·2, as well as a decreased functional capacity of band 3 to transport anions. Band 3 Cape Town is defined by a GAG→AAG point mutation at codon 90, substituting a glutamic acid with a lysine in the cytoplasmic domain of the molecule, while band 3 Prague III is a codon 870 CGG→TGG point mutation, replacing an arginine with a tryptophan in the transmembrane region of band 3. mRNA is transcribed from both mutant alleles, implying that mutant proteins are synthesized, but are either degraded prior to membrane incorporation or insertion is impaired. We conclude that the combination of these two mutations exacerbated the clinical presentation of the proband.

Plasmodium falciparum exhibits markers of regulated cell death at high population density in vitro

The asexual erythrocytic cycle of the protozoan parasite Plasmodium falciparum is responsible for the pathogenesis of malaria and causes the overwhelming majority of malaria deaths. Rapidly increasing parasitaemia during this 48hour cycle threatens the survival of the human host and the parasite prior to transmission of the slow-maturing sexual stages to the mosquito host. The parasite may utilise regulated cell death (RCD) to control the burden of infection on the host and thus aid its own survival and transmission. The occurrence of RCD in P. falciparum remains a controversial topic. We provide strong evidence for the occurrence of an apoptosis-like phenotype of RCD in P. falciparum under conditions of high parasite density. P. falciparum was maintained in vitro and stressed by allowing growth to an unrestricted peak parasitaemia. Cell death markers, including morphological changes, DNA fragmentation, mitochondrial polarisation and phosphatidylserine externalisation were used to characterise parasite death at the time of peak parasitaemia and 24h later. At peak parasitaemia, mitochondrial depolarisation was observed, together with phosphatidylserine externalisation in both parasitised- and neighbouring non-infected erythrocytes. DNA fragmentation coincided with a decline in parasitaemia. Fewer merozoites were observed in mature schizonts at peak parasitaemia. Growth recovery to near-peak parasitaemia was noted within two intraerythrocytic cycles. The combination and chronological order of the biochemical markers of cell death suggest the occurrence of an apoptosis-like phenotype. The identification of a RCD pathway in P. falciparum may provide novel drug targets, particularly if the pathway differs from the host machinery.

Differential activity of novel gametocytocidal compounds: drug mode-of-action and ex vivo efficacy

As per the 2007 Global Malaria Eradication Plan, malaria transmission blocking is seen as key to malaria elimination strategies. A highlight that has emerged from gametocytocidal assays to identify novel compounds with malaria transmission blocking ability is that, unlike asexual-based assays, greater variability in end-point readout may exist between these assays that interrogate different parasite biological functions. Drug mode-of-action is likely to be an important factor on this outcome. Such variability may be mitigated by screening compounds based on similar pharmacophores in series. One of the major concerns with the current assay platforms is their inability to be robustly used to screen variant pharmacophores accurately as the different assay principles may interrogate different biological functions. As such, compounds targeting a specific biological pathway may in extreme cases either fail in a certain assay, or by contrast, may be flagged as false positives. Taking assay platform differences into account, and relying on good intra-assay variability for each assay optimized in our laboratories, the ATP, pLDH, luciferase reporter and PrestoBlue™ assays were compared in context of a blinded MMV 10-compound set. All the assays were performed in parallel on the same gametocyte population (except for the luciferase reporter lines). The remaining parameters for each assay were all comparable. In each case, the assay was performed for 48 h of continuous drug pressure for at least three replicates. Although direct comparison of absolute inhibition values are difficult between assay platforms, similar trends were observed including comparative performance of the luciferase marker assay and the PrestoBlue™ assay for e.g. DHA and Methylene blue. Interestingly, the ATP assay could not detect any inhibitory activity for some quinoline family members and may therefore be more sensitive in indicating the inability of these compounds to inhibit gametocytes. Data from the luciferase reporter assays for these compounds indicate that the compounds are indeed more active against early stage gametocytes. The signal obtained for these compounds in the PrestoBlue™ and pLDH assays may therefore rather reflect the inability of these assays in discriminating activity of compounds against earlier stages of gametocytes, whereas the ATP assay more accurately reflect these compounds’ activity. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that has to be taken in screening platforms where compounds may be falsely assigned activity (or lack thereof) based on a single assay.