恶性疟原虫海南株(FCC1)翻译控制肿瘤蛋白(TCTP)基因的克隆及序列测定

为了获得翻译控制肿瘤蛋白 (TCTP)基因 ,提取恶性疟原虫海南株 (FCC1)总RNA ,直接用总RNA反转录成单链DNA ,再用长距PCR方法扩增出双链cDNA .根据小鼠约氏疟原虫TCTP基因序列设计引物 ,以cDNA为模板合成了恶性疟原虫海南株TCTP基因 .以pMD18 T为载体 ,用大肠杆菌XL1 blue对基因克隆 .测序结果表明 ,此基因序列与小鼠约氏疟原虫TCTP基因序列有 85 %同源性 .由此基因序列推导出的TCTP氨基酸序列 ,与小鼠约氏疟原虫TCTP氨基酸序列有 88%同源性 .进入GenBank国际基因库 (美国 )检索 ,所克隆的基因与基因库中恶性疟原虫基因同源性为 97% ;由所克隆的基因序列推导的TCTP氨基酸序列 ,与国际基因库恶性疟原虫TCTP基因推导的TCTP氨基酸序列仅有 1个氨基酸差异 .恶性疟原虫海南株TCTP基因克隆为进一步研究奠定了基础     

Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue

Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.     

Plasmodium falciparum Rhoptry Proteins of 140/130/110 kd (Rhop-H) Are Located in an Electron Lucent Compartment in the Neck of the Rhoptries

ABSTRACT. To investigate in more detail the structure of the high molecular weight rhoptry protein complex of Plasmodium falciparum, Rhop-H (140/130/110 kd), the complex was affinity purified from parasite extracts using rhoptry protein specific antisera prepared against Rhop-H proteins bound to and eluted from Balb/c mouse erythrocytes, using 0.5 M NaCl. The individual proteins (140 kd/Rhop-1, 130 kd/Rhop-2, and 110 kd/Rhop-3) were separated, electroeluted, and monospecific polyclonal antisera prepared against the individual proteins, and against the affinity purified complex. Immunofluorescence assays and immunoelectron microscopic studies were performed to verify the subcellular localization of the Rhop-H epitopes. Immunoblotting and immunoprecipitation assays were also performed. We report novel findings regarding the localization of the rhoptry proteins to an electron lucent compartment in the neck of the rhoptries. Analysis of the amino acid composition of the individually purified Rhop-H proteins demonstrated a predominance of negatively charged (E, D) as well as hydrophobic residues (L, A, P, S) in the three proteins. The percentage of negatively charged residues was high for all three proteins. Similarities in amino acid composition for the three proteins supports the previous data demonstrating shared properties such as erythrocyte and liposome binding, for the three proteins. Results of antibody characterizations using rhoptry protein specific antisera demonstrate the immunodominance of the Rhop-H complex.     

MAAP: malarial adhesins and adhesin-like proteins predictor

Malaria caused by protozoan parasites belonging to the genus Plasmodium is a dreaded disease, second only to tuberculosis. The emergence of parasites resistant to commonly used drugs and the lack of availability of vaccines aggravates the problem. One of the preventive approaches targets adhesion of parasites to host cells and tissues. Adhesion of parasites is mediated by proteins called adhesins. Abrogation of adhesion by either immunizing the host with adhesins or inhibiting the interaction using structural analogs of host cell receptors holds the potential to develop novel preventive strategies. The availability of complete genome sequence offers new opportunities for identifying adhesin and adhesin-like proteins. Development of computational algorithms can simplify this task and accelerate experimental characterization of the predicted adhesins from complete genomes. A curated positive dataset of experimentally known adhesins from Plasmodium species was prepared by careful examination of literature reports. "Controversial" or "hypothetical" adhesins were excluded. The negative dataset consisted of proteins representing various intracellular functions including information processing, metabolism, and interface (transporters). We did not include proteins likely to be on the surface with unknown adhesin properties or which are linked even indirectly to the adhesion process in either of the training sets. A nonhomology-based approach using 420 compositional properties of amino acid dipeptide and multiplet frequencies was used to develop MAAP Web server with Support Vector Machine (SVM) model classifier as its engine for the prediction of malarial adhesins and adhesin-like proteins. The MAAP engine has six SVM classifier models identified through an exhaustive search from 728 kernel parameters set. These models displayed an efficiency (Mathews correlation coefficient) of 0.860-0.967. The final prediction P(maap) score is the maximum score attained by a given sequence in any of the six models. The results of MAAP runs on complete proteomes of Plasmodium species revealed that in Plasmodium falciparum at P(maap) scores above 0.0, we observed a sensitivity of 100% with two false positives. In P. vivax and P. yoelii an optimal threshold P(maap) score of 0.7 was optimal with very few false positives (upto 5). Several new predictions were obtained. This list includes hypothetical protein PF14_0040, interspersed repeat antigen, STEVOR, liver stage antigen, SURFIN, RIFIN, stevor (3D7-stevorT3-2), mature parasite-infected erythrocyte surface antigen or P. falciparum erythrocyte membrane protein 2, merozoite surface protein 6 in P. falciparum, circumsporozoite proteins, microneme protein-1, Vir18, Vir12-like, Vir12, Vir18-like, Vir18-related and Vir4 in P. vivax, circumsporozoite protein/thrombospondin related anonymous proteins, 28 kDa ookinete surface protein, yir1, and yir4 of P. yoelii. Among these, a few proteins identified by MAAP were matched with those identified by other groups using different experimental and theoretical strategies. Most other interspersed repeat proteins in Plasmodium species had lower P(maap) scores. These new predictions could serve as new leads for further experimental characterization (MAAP webserver: http://maap.igib.res.in).     

Plasmodium yoelii: experimental evidences for the conserved epitopes between mouse and human malaria parasite, Plasmodium falciparum

Bioinformatic analyses of gene homologues have revealed functionally conserved epitopes between human and rodent malaria parasites. Here, we present experimental evidence for the presence of functionally and antigenically conserved domains between Plasmodium falciparum and Plasmodium yoelii asexual blood-stages. Merozoite released soluble proteins (MRSPs) from both P. falciparum and P. yoelii bound to heterologous mouse or human red blood cells, respectively. The presence of conserved antigenic epitopes between the two species of parasites was evident by the inhibitory effect of antibodies, developed against P. yoelii in convalescent mice, on P. falciparum growth and merozoite reinvasion in vitro. Furthermore, mice immunized with P. falciparum MRSPs were protected from infection by a P. yoelii challenge. These data indicate that different species of Plasmodium contain antigenically conserved interspecies domains, which are immunogenic and, thus constitute a potential novel antigen source for vaccine development and testing using a mouse model.     

Comparative kinomics of Plasmodium organisms: unity in diversity

Phosphorylation by protein kinases is a very common and crucial process in many signal transduction pathways in eukaryotes. This review describes comparative protein kinase analysis of two apicomplexa Plasmodium falciparum (3D7 strain) and Plasmodium yoelii yoelii (17XNL strain) which are causative agents of malaria in human and African rat respectively. Sensitive bioinformatics techniques enable identification of 82 and 60 putative protein kinases in P. falciparum and P. yoelii yoelii respectively and these sequences could be classified into known subfamilies of protein kinases. The most populated kinase subfamilies in both the plasmodium species correspond to CAMK and CMGC groups. Analysis of domain architectures enables detection of uncommon domain organization in kinases of both the organisms such as kinase domain tethered to EF hands as well as PH domain. Components of MAPK signaling pathway is not well conserved in plasmodium organisms. Such observations suggest that plasmodium protein kinases are highly divergent from other eukaryotes. A transmembrane kinase with 6 membrane spanning segments in P. falciparum seems to have no orthologue in P. yoelii yoelii. 19 P. falciparum kinases have been found to cluster separately from P. yoelii yoelii kinases and hence these kinases are unique to P. falciparum genome. Only 28 orthologous pairs of kinases seem to be present between these two plasmodium organisms. Comparative kinome analysis of two plasmodium species has thus provided clues to the function of many protein kinases based upon their classification and domain organization and also implicate marked differences even between two plasmodium organisms.     

Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.     

Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species

Plasmodium sporozoites can enter host cells by two distinct pathways, either through disruption of the plasma membrane followed by parasite transmigration through cells, or by formation of a parasitophorous vacuole (PV) where the parasite further differentiates into a replicative exo-erythrocytic form (EEF). We now provide evidence that following invasion without PV formation, transmigrating Plasmodium falciparum and Plasmodium yoelii sporozoites can partially develop into EEFs inside hepatocarcinoma cell nuclei. We also found that rodent P. yoelii sporozoites can infect both mouse and human hepatocytes, while human P. falciparum sporozoites infect human but not mouse hepatocytes. We have previously reported that the host tetraspanin CD81 is required for PV formation by P. falciparum and P. yoelii sporozoites. Here we show that expression of human CD81 in CD81-knockout mouse hepatocytes is sufficient to confer susceptibility to P. yoelii but not P. falciparum sporozoite infection, showing that the narrow P. falciparum host tropism does not rely on CD81 only. Also, expression of CD81 in a human hepatocarcinoma cell line is sufficient to promote the formation of a PV by P. yoelii but not P. falciparum sporozoites. These results highlight critical differences between P. yoelii and P. falciparum sporozoite infection, and suggest that in addition to CD81, other molecules are specifically required for PV formation during infection by the human malaria parasite.     

Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.     

Analysis of DNA from various species and strains of malaria parasites by restriction endonuclease fingerprinting

1. DNA from various rodent Plasmodium species and strains and from P. falciparum, the human parasite, were analysed by agarose gel electrophoresis following digestion with restriction endonucleases EcoRI, Hind III and Bam Hl. Complex patterns of ethidium-stained bands were obtained, which showed similarity but reproducible differences among the various parasite species (P. chabaudi, P. yoelii, P. berghei and P. falciparum). 2. No differences could be discerned among two cloned strains of P. yoelii (33X, and YM) and among pyrimethamine-resistant (pyrimethamine + chloroquine)-resistant and the drug-sensitive P. chabaudi clone from which the resistant clones were derived. 3. From the known complexity of Plasmodium DNA it could be concluded that the visible bands were derived from repetitive DNA fractions.     

Plasmodium falciparum: identification and purification of the phosphoglycerate kinase of the malaria parasite.

Multiplication of the human malaria parasite Plasmodium falciparum within red blood cells is an energy-dependent process and glucose consumption increases dramatically in infected red blood cells (IRBC) versus normal red blood cells (NRBC). The major pathway for glucose metabolism in P. falciparum IRBC is anaerobic glycolysis. Phosphoglycerate kinase (PGK) is one of the key enzymes of this pathway as it generates ATP. We found that the PGK specific activity in P. falciparum IRBC is seven times higher than that in NRBC. The parasitic origin of the increase in PGK activity is confirmed by isoelectric focusing. Indeed, two P. falciparum isoenzymes with neutral isoelectric points were detected. P. falciparum PGK in purified form has a molecular mass of 48 kDa. Antiserum raised against purified P. falciparum PGK specifically recognizes the 48-kDa protein band in P. falciparum and also reacts with P. berghei and P. yoelii IRBC lysates but does not cross-react with PGK associated with NRBC.     

Identification and characterisation of the Plasmodium vivax rhoptry-associated protein 2

Plasmodium vivax is currently the most widespread of the four parasite species causing malaria in humans around the world. It causes more than 75 million clinical episodes per year, mainly on the Asian and American continents. Identifying new antigens to be further tested as anti-P. vivax vaccine candidates has been greatly hampered by the difficulty of maintaining this parasite cultured in vitro. Taking into account that one of the most promising vaccine candidates against Plasmodium falciparum is the rhoptry-associated protein 2, we have identified the P. falciparum rhoptry-associated protein 2 homologue in P. vivax in the present study. This protein has 400 residues, having an N-terminal 21 amino-acid stretch compatible with a signal peptide and, as occurs with its falciparum homologue, it lacks repeat sequences. The protein is expressed in asexual stage P. vivax parasites and polyclonal sera raised against this protein recognised a 46 kDa band in parasite lysate in a Western blot assay.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Comparative histopathology of mice infected with the 17XL and 17XNL strains of Plasmodium yoelii

Plasmodium yoelii 17XL was used to investigate the mechanism of Plasmodium falciparum-caused cerebral malaria, although its histological effect on other mouse organs is still unclear. Here, histological examination was performed on mice infected with P. yoelii 17XL; the effect of P. yoelii 17XL infection on anemia and body weight loss, as well as its lesions in the brain, liver, kidney, lung, and spleen, also was investigated. Plasmodium yoelii 17XL-infected red blood cells were sequestered in the microcirculation of the brain and in the kidney. Compared with the nonlethal P. yoelii 17XNL strain, infection by P. yoelii 17XL caused substantial pulmonary edema, severe anemia, and significant body weight loss. Although P. yoelii 17XNL and 17XL produced a similar focal necrosis in the mouse liver, infection of P. yoelii 17XL induced coalescing of red and white pulp. Mortality caused by P. yoelii 17XL may be due to cerebral malaria, as well as respiratory distress syndrome and severe anemia. Plasmodium yoelii 17XL-infected rodent malaria seems to be a useful model for investigating severe malaria caused by P. falciparum.     

Two Plasmodium falciparum merozoite proteins binding to erythrocyte band 3 form a direct complex

Erythrocyte invasion by malaria parasites requires multiple protein interactions. Our earlier studies showed that erythrocyte band 3 is an invasion receptor binding Plasmodium falciparum merozoite surface protein 1 and 9 (MSP1, MSP9) existing as a co-ligand complex. In this study, we have used biochemical approaches to identify the binding sites within MSP1 and MSP9 involved in the co-ligand complex formation. A major MSP9-binding site is located within the 19kDa C-terminal domain of MSP1 (MSP1(19)). Two specific regions of MSP9 defined as Delta1a and Delta2 interacted with native MSP1(19). The 42 kDa domain of MSP1 (MSP1(42)) bearing MSP1(19) in the C-terminus bound directly to both MSP9/Delta1a and Delta2. Thus, the regions of MSP1 and MSP9 interacting with the erythrocyte band 3 receptor are also responsible for assembling the co-ligand complex. Our evidence suggests a ternary complex is formed between MSP1, MSP9, and band 3 during erythrocyte invasion by P. falciparum.     

Methotrexate and aminopterin lack in vivo antimalarial activity against murine malaria species

The antifolate anticancer drug methotrexate (MTX) has potent activity against Plasmodium falciparum in vitro. Experience of its use in the treatment of rheumatoid arthritis indicates that it could be safe and efficacious for treating malaria. We sought to establish a murine malaria model to study the mechanism of action and resistance of MTX and its analogue aminopterin (AMP). We used Plasmodium berghei, Plasmodium yoelii yoelii, Plasmodium chabaudi and Plasmodium vinckei. None of these species were susceptible to either drug. We have also tested the efficacy of pyrimethamine in combination with folic acid in P. berghei, and data indicate that folic acid does not influence pyrimethamine efficacy, which suggests that P. berghei may not transport folate. Since MTX and AMP utilise folate receptor/transport to gain access to cells, their lack of efficacy against the four tested murine malaria species may be the result of inefficiency of drug transport.     

Plasmodium yoelii yoelii 17XNL constitutively expressing GFP throughout the life cycle

Plasmodium yoelii is a rodent parasite commonly used as a model to study malaria infection. It is the preferred model parasite for liver-stage immunological studies and is also widely used to study hepatocyte, erythrocyte and mosquito infection. We have generated a P. yoelii yoelii 17XNL line that is stably transfected with the green fluorescent protein (gfp) gene. This parasite line constitutively expresses high levels of GFP during the complete parasite life cycle including liver, blood and mosquito stages. These fluorescent parasites can be used in combination with fluorescence activated cell sorting or live microscopy for a wide range of experimental applications.