Characterization of a Plasmodium falciparum macrophage-migration inhibitory factor homologue

BACKGROUND: Macrophage-migration inhibitory factor (MIF), one of the first cytokines described, has a broad range of proinflammatory properties. The genome sequencing project of Plasmodium falciparum identified a parasite homologue of MIF. The protein is expressed during the asexual blood stages of the parasite life cycle that cause malarial disease. The identification of a parasite homologue of MIF raised the question of whether it affects monocyte function in a manner similar to its human counterpart. METHODS: Recombinant P. falciparum MIF (PfMIF) was generated and used in vitro to assess its influence on monocyte function. Antibodies generated against PfMIF were used to determine the expression profile and localization of the protein in blood-stage parasites. Antibody responses to PfMIF were determined in Kenyan children with acute malaria and in control subjects. RESULTS: PfMIF protein was expressed in asexual blood-stage parasites, localized to the Maurer's cleft. In vitro treatment of monocytes with PfMIF inhibited random migration and reduced the surface expression of Toll-like receptor (TLR) 2, TLR4, and CD86. CONCLUSIONS: These results indicate that PfMIF is released during blood-stage malaria and potentially modulates the function of monocytes during acute P. falciparum infection.     

Characterization of a Plasmodium falciparum erythrocyte-binding protein paralogous to EBA-175

A member of a Plasmodium receptor family for erythrocyte invasion was identified on chromosome 13 from the Plasmodium falciparum genome sequence of the Sanger Centre (Cambridge, U.K.). The protein (named BAEBL) has homology to EBA-175, a P. falciparum receptor that binds specifically to sialic acid and the peptide backbone of glycophorin A on erythrocytes. Both EBA-175 and BAEBL localize to the micronemes, organelles at the invasive ends of the parasites that contain other members of the family. Like EBA-175, the erythrocyte receptor for BAEBL is destroyed by neuraminidase and trypsin, indicating that the erythrocyte receptor is a sialoglycoprotein. Its specificity, however, differs from that of EBA-175 in that BAEBL can bind to erythrocytes that lack glycophorin A, the receptor for EBA-175. It has reduced binding to erythrocytes with the Gerbich mutation found in another erythrocyte, sialoglycoprotein (glycophorin C/D). The interest in BAEBL's reduced binding to Gerbich erythrocytes derives from the high frequency of the Gerbich phenotype in some regions of Papua New Guinea where P. falciparum is hyperendemic.     

Plasmodium falciparum pyruvate kinase as a novel target for antimalarial drug-screening

BACKGROUND: Global travellers are increasingly at risk of contracting malaria. The increasing occurrence of drug-resistance in many endemic areas emphasizes the need for novel drug targets for antimalarial-screening. In this study, the use of pyruvate kinase as a drug-target is evaluated. The functional validation of a gene encoding pyruvate kinase (designated PK1) has previously been reported. However, alternative copies of this enzyme encoded by Plasmodium falciparum could also circumvent the role of PK1. A survey of genome data revealed a putative ORF seemingly coding for another pyruvate kinase (designated PK2). METHODS: The expression of PK1 and PK2 in in vitro cultures were investigated by RT-PCR. Biocomputational analysis was carried out to identify structural differences between the P. falciparum pyruvate kinases and the corresponding enzymes from its human host. RESULTS: Both PK1 and PK2 were indeed actively transcribed during the intraerythrocytic stages, suggesting the involvement of both enzymes during infection. A comparison of amino acid residues at the effector binding sites of PK1 and PK2, to those of the human pyruvate kinases revealed some significant differences that could serve as targets for selective inhibitors to be designed against parasitic pyruvate kinases. CONCLUSION: Experimental evidence for the expression of both PK1 and PK2 during the blood stages of malaria infection was provided. Interestingly, phylogenetic analysis revealed that the "PK2" type of enzyme appears to be confined to Apicomplexans, an important observation with respect to the assessment of PK2 as a drug-target.     

Characterization of a differential immunoscreen epitope of Plasmodium falciparum using combinatorial agents

A differential serological screening of a lambdagt11 cDNA expression library has identified several clones, which react exclusively to sera samples from persons clinically immune to malaria but not to acute malaria patient sera. One such clone, IPf9, has a 315-bp cDNA insert, which was found to be conserved in different strains of the human and rodent malarial parasite Plasmodium falciparum and Plasmodium berghei, respectively. The induced expression product of IPf9 was used to generate polyclonal sera in rabbits. The IPf9 expression product was also screened with phage surface display combinatorial libraries to isolate reagents that specifically bound to the IPf9 product. The polyclonal antisera and the combinatorial reagents recognized a 50-kDa protein from P. falciparum, and a 53-kDa product from P. berghei. Immunofluorescence studies using asexual and sexual stages of P. falciparum showed the protein to be present within the parasite in each of the asexual and sexual stages. The combinatorial reagents showed a partial inhibition in the growth of P. falciparum in vitro. Mice infected with the P. berghei showed the presence of T-cells that exhibited lymphoproliferation when stimulated with the IPf9 protein. It is suggested that IPf9 protein is a conserved protein epitope, and may be relevant for a protective immune response to malaria.     

Characterization of Plasmodium falciparum sporozoite surface protein 2.

Immunization of mice with Plasmodium yoelii sporozoite surface protein 2 (PySSP2) and circumsporozoite protein protects completely against P. yoelii. The amino acid sequence of PySSP2 suggested that the thrombospondin-related anonymous protein (TRAP) [Robson, K. J. H., Hall, J. R. S., Jennings, M. W., Harris, T. J. R., Marsh, K., Newbold, C. I., Tate, V. E. & Weatherall, D. J. (1988) Nature (London) 335, 79-82] is the Plasmodium falciparum homolog of PySSP2. We report data confirming that TRAP is P. falciparum SSP2 (PfSSP2). Murine antibodies against recombinant PfSSP2 identify a 90-kDa protein in extracts of P. falciparum sporozoites, recognize sporozoites and infected hepatocytes by immunofluorescence, localize PfSSP2 to the sporozoite micronemes by immunoelectron microscopy and to the surface membrane by live immunofluorescence, and inhibit sporozoite invasion and development in hepatocytes in vitro. Human volunteers immunized with irradiated sporozoites and protected against malaria develop antibody and proliferative T-cell responses to PfSSP2, suggesting that, like PySSP2, PfSSP2 is a target of protective immunity, and supporting inclusion of PfSSP2 in a multicomponent malaria vaccine.     

恶性疟原虫乳酸脱氢酶的表达及免疫活性鉴定

目的　在大肠杆菌中表达恶性疟原虫乳酸脱氢酶 (LDHp)与谷胱甘肽S 转移酶 (GST)融合蛋白 ,测定重组蛋白的免疫活性。方法　采用PCR方法特异性扩增恶性疟原虫 (海南株 )乳酸脱氢酶基因 ,经双酶切后克隆入 pGEX 4T 1表达载体中 ,重组蛋白纯化后免疫小鼠制备特异性血清 ,并用琼脂双向扩散法检测效价 ,ELISA、Western bloting检测重组抗原的免疫活性。结果　得到了重组表达的蛋白抗原 ,表达产物能与兔抗恶性疟原虫血清发生反应 ,并能诱导小鼠产生特异性体液免疫应答 ,免疫琼脂扩散法抗体滴度为 1∶16。结论　LDHp在大肠杆菌中获得高效表达且表达产物具有良好的抗原性。     

A Unique Plasmodium falciparum Kelch 13 Gene Mutation in Northwest Ethiopia

Artemisinin combination therapy (ACT) is the first line to treat uncomplicated Plasmodium falciparum malaria worldwide. Artemisinin treatment failures are on the rise in southeast Asia. Delayed parasite clearance after ACT is associated with mutations of the P. falciparum kelch 13 gene. Patients (N = 148) in five districts of northwest Ethiopia were enrolled in a 28-day ACT trial. We identified a unique kelch 13 mutation (R622I) in 3/125 (2.4%) samples. The three isolates with R622I were from Negade-Bahir and Aykel districts close to the Ethiopia–Sudan border. One of three patients with the mutant strain was parasitemic at day 3; however, all patients cleared parasites by day 28. Correlation between kelch 13 mutations and parasite clearance was not possible due to the low frequency of mutations in this study.A report by World Health Organization (WHO) estimated that 3.3 billion people are at risk of contracting malaria. In 2013 alone, there were an estimated 198 million cases of malaria worldwide with 584,000 deaths. Ninety percent of the deaths occurred in sub-Saharan Africa where children are the main victims.¹ According to the WHO, between 2000 and 2013, an expansion of malaria interventions helped to reduce malaria incidence by 30% globally and by 34% in Africa.¹ Many countries have initiated malaria elimination programs.¹ Artemisinin-based combination therapies (ACTs) have been highly effective first-line drugs for the treatment of uncomplicated malaria worldwide.² However, the recent emergence of Plasmodium falciparum strains that are resistant to artemisinin in southeast Asian countries poses a huge challenge to the effectiveness of ACT.^1,3 The possibility of dissemination to or independent emergence of artemisinin-resistant strains in Africa, where the majority of malaria-associated deaths occur, will have potentially catastrophic outcomes.A recent study using genome sequencing of P. falciparum has demonstrated an association between in vivo delayed parasite clearance, in vitro artemisinin “resistance” in a ring-stage assay, and mutations (Y493H, R539T, I543T, and C580Y) in the propeller domain of the parasite kelch 13 gene located on chromosome 13 (PF3D7_1343700 or PF13_0238).⁴ Moreover, an artemisinin-resistant P. falciparum strain that was selected by dose-escalating in vitro culture harbored the mutation, M476I, in the kelch 13 gene. Another study on samples from different southeast Asian countries confirmed the association of kelch 13 mutations and increased parasite clearance half-life. It also demonstrated the independent emergence of these mutations in different geographical areas.⁵ Targeted genetic engineering of kelch 13 using zinc-finger nuclease technology significantly increases the ring-stage survival rate of the parasite in vitro.⁶ Thus, detection of mutations on kelch 13 can be used to track artemisinin resistance in places where clinical treatment failure has not been observed. Kelch 13 mutation screening studies of P. falciparum isolates collected across sub-Saharan African countries have not identified the genotypes associated with treatment failure. However, several other non-synonymous mutations were detected in these countries.^7,8Ethiopia, with a population of more than 90 million, is a malaria-endemic country situated in sub-Saharan Africa. Approximately 68% of the population of Ethiopia live in malarious areas and are at risk of contracting the disease.⁹ There has been a significant reduction in malaria cases in Ethiopia since the introduction of artemether–lumefantrine for the treatment of uncomplicated falciparum malaria in 2004.^9,10 In this study, we aimed to assess the presence of kelch 13 propeller mutations in P. falciparum isolates from northwest Ethiopia as part of a 28-day ACT clinical trial. We identified a mutation in the P. falciparum kelch 13 gene that has not been observed in Asia and Africa before.The study protocol was reviewed and approved by Research and Ethical Review Committee of School of Biomedical and Laboratory Sciences, College of Medicine and Health Sciences, University of Gondar (reference no.: SBMLS/344/05). The study was conducted on P. falciparum DNA samples obtained from malaria patients from five different health centers in Amhara Regional State, northwest Ethiopia, between October 2013 and April 2014. The five sites were Addis Zemen, Aykel, Maksegnet, Negade Bahir, and Sanja. A total of 148 confirmed P. falciparum malaria patients were enrolled in a 28-day ACT efficacy trial. Patients were treated with artemether–lumefantrine (IPCA Laboratories, Mumbai, India). A directly observed therapy model was used for the first and each morning dose thereafter. The evening doses were given to the patient/guardian for self-administration in the presence of health extension workers.Peripheral blood was collected by finger prick and thin and thick blood films were prepared on a microscope slide. Giemsa staining and microscopy was performed as described previously.¹¹ Only those with mono-infection with P. falciparum were included in this study. Finger prick blood samples were collected on Whatman 903 filter paper (GE Healthcare, Mississauga, Canada) at the time of diagnosis, air-dried, individually inserted in a zip-lock bag, and transported to University of Calgary, Canada, for molecular analysis. Genomic DNA extraction from the filter paper blood spots was performed as described previously in a study.¹¹ Plasmodium falciparum kelch 13 was amplified from the genomic DNA samples using nested polymerase chain reaction (PCR) with slight modification.⁴ The annealing temperature was set at 65.5°C for 45 seconds for the second step of the nested PCR to increase specificity.^4,12 Sequencing of the kelch 13 propeller domain gene was performed using Applied Biosystems (Burlington, ON, Canada) 3730XL 96 capillary DNA analyzer. Bidirectional kelch 13 sequences from each patient sample and the reference kelch 13 gene sequence (PF3D7_1343700) (Genbank ID: {"type":"entrez-nucleotide","attrs":{"text":"AL844509.2","term_id":"225631926","term_text":"AL844509.2"}}AL844509.2) were aligned using Clustal Omega software (EMBL-EBI, Hinxton, UK). The sequences in the multiple sequences alignment were edited manually by Jalview software (Dundee, UK) to remove the gaps.¹³ Nested PCR was performed to confirm the species of Plasmodium as described previously in a study.¹¹ Modeling of the predicted structure of the mutant kelch 13 propeller protein from Ethiopian isolates and its putative effect on the function of the protein was performed using Phyre2 (London, UK) (PDB ID: 2WOZ).¹⁴A total of 148 confirmed P. falciparum-infected malaria patients (age = 1–69 years) were enrolled in a 28-day ACT treatment trial. The kelch 13 propeller domain gene was amplified and sequenced from 125 of the 148 blood samples collected on filter paper. As shown in Figure 1 shows the predicted model of kelch 13 propeller domain with the R622I mutation. The model predicts that the substitution from basic-to-aliphatic residue at this position is likely to impact the protein function. Confirmation of the species of the parasite in the samples where mutation was detected was performed using nested PCR to amplify the small subunit ribosomal RNA gene.¹⁵ Patients with mutant kelch 13 were infected with P. falciparum only.Open in a separate windowFigure 1.Predicted model of the Plasmodium falciparum kelch 13 propeller protein with the R622I mutation. (A) Location of the mutation sites relative to the overall model of the protein. The beta-propeller domain of the btb-kelch protein Krp1 (PDB ID: 2WOZ) was used as the modeling template. (B) The colored bars indicate the probability that a mutation to the corresponding residue will have some effect on function of the protein (mutation sensitivity) or on the phenotype of the organism. The tall and red bars indicate that the likelihood is high. Short and blue bars indicate that the likelihood is low. The R622I substitution (vertical arrow) is predicted to impact protein function.

Table 1

Plasmodium falciparum kelch 13 gene nested PCR and mutation results from study sites in northwest Ethiopia

恶性疟原虫MAL13P1.129基因的抗原表位预测及免疫学分析

目的 对恶性疟原虫MAL13P1.129基因的抗原表位进行预测,表达其重组蛋白并进行免疫学鉴定.方法 利用生物信息学分析方法对MAL13P1.129基因的线性抗原表位进行预测,从亲水性、表面可及性、柔韧性及二级结构等方面对抗原表位进行筛选.人工合成经密码子优化的MAL13P1.129基因,构建至PET32a(+)表达载...     

Thrombopoietin in Plasmodium falciparum malaria

Thrombopoietin (TPO) is the key growth factor for platelet production and is elevated in states of platelet depletion. As thrombocytopenia is a common finding in malaria, we analysed TPO regulation before, during and after antimalarial treatment. Before treatment, TPO serum levels were significantly higher in patients with severe malaria (n = 35) than in patients with uncomplicated malaria (n = 44; P = 0.024), normalizing within 14-21 d of therapy. The rapid normalization of TPO levels and increase in low peripheral platelet counts after treatment indicate that the biosynthesis of TPO and its regulation in malaria patients are normal.     

恶性疟原虫DNA探针检测血内恶性疟原虫的初步研究

从培养恶性疟原虫(Fcc-1)中分离纯化基因组DNA,用~(32)P标记,作为探针,按DNA斑点杂交法,检测血样。结果表明:该探针可检出9个恶性疟原虫感染红细胞/10_6红细胞;在现场应用时,与13例恶性疟阳性标本镜检符合率为92％:与1例间日疟原虫病例和正常人血、白细胞之间,未发现非特异性杂交。     

Src-family kinase dependent disruption of endothelial barrier function by Plasmodium falciparum merozoite proteins

Pulmonary complication in severe Plasmodium falciparum malaria is manifested as a prolonged impairment of gas transfer or the more severe acute respiratory distress syndrome (ARDS). In either clinical presentation, vascular permeability is a major component of the pathologic process. In this report, we examined the effect of clinical P falciparum isolates on barrier function of primary dermal and lung microvascular endothelium in vitro. We showed that parasite sonicates but not intact infected erythrocytes disrupted endothelial barrier function in a Src-family kinase-dependent manner. The abnormalities were manifested both as discontinuous immunofluorescence staining of the junctional proteins ZO-1, claudin 5, and VE-cadherin and the formation of interendothelial gaps in monolayers. These changes were associated with a loss in total protein content of claudin 5 and redistribution of ZO-1 from the cytoskeleton to the membrane and the cytosolic and nuclear fractions. There was minimal evidence of a proinflammatory response or direct cellular cytotoxicity or cell death. The active component in sonicates appeared to be a merozoite-associated protein. Increased permeability was also induced by P falciparum glycophosphatidylinositols (GPIs) and food vacuoles. These results demonstrate that parasite components can alter endothelial barrier function and thus contribute to the pathogenesis of severe falciparum malaria.